The two-component system (TCS), which comprises histidine kinases (HKs), phosphotransfers (HPs), and response regulator proteins (RRs), plays pivotal roles in regulating plant growth, development, and responses to biotic and abiotic stresses. TCS genes have been comprehensively identified and investigated in various crops but poorly characterized in tomato. In this work, a total of 65 TCS genes consisting of 20 HK(L)s, six HPs, and 39 RRs were identified from tomato genome. The classification, gene structures, conserved domains, chromosome distribution, phylogenetic relationship, gene duplication events, and subcellular localization of the TCS gene family were predicted and analyzed in detail. The amino acid sequences of tomato TCS family members, except those of type-B RRs, are highly conserved. The gene duplication events of the TCS family mainly occurred in the RR family. Furthermore, the expansion of RRs was attributed to both segment and tandem duplication. The subcellular localizations of the selected green fluorescent protein (GFP) fusion proteins exhibited a diverse subcellular targeting, thereby confirming their predicted divergent functionality. The majority of TCS family members showed distinct organ- or development-specific expression patterns. In addition, most of TCS genes were induced by abiotic stresses and exogenous phytohormones. The full elucidation of TCS elements will be helpful for comprehensive analysis of the molecular biology and physiological role of the TCS superfamily. Arabidopsis cytokinin signaling is a typical representative of TCS signal systems. Three transmembrane histidine kinases, namely, AHK2, AHK3, and AHK4 function as cytokinin receptors and negatively respond to stresses in Arabidopsis cytokinin signaling [cis-elements in the promoter of type-A ARRs and further regulate the expression of downstream stress-related genes [A two-component system (TCS) via phosphorylation between histidine and aspartic-acid residues was first identified in bacteria ,2. The Tignaling ,6. Theseed genes ,6.Arabidopsis [Arabidopsis interact with ABA to participate in drought, salt, and low temperature stresses [AHP1, AHP2, and AHP3 in Arabidopsis are significantly repressed by heat stress [OsAHP1/2 knockdown seedlings respond to salt and drought stresses in different patterns [OsHK3 participates in ABA-induced antioxidant defense [never-ripe (Nr) mutant, a histidine kinase mutant, are more sensitive to heat stress via affecting pollen carbohydrate metabolism [Take advantage of available whole genomic sequences, various TCS genes have been successfully identified and investigated in several plant species, including bidopsis , rice [7bidopsis , soybeanbidopsis , and Chibidopsis . Plant Tstresses ,17,18,19t stress . In ricepatterns . OsHK3 p defense . In soyb defense . The roltabolism . Some phtabolism .LeETR4 expression levels enhance flower senescence, and affect fruit set [Tomato, as a model of fleshy fruit plant, is an economically important fruit crop grown worldwide. The reproductive development of tomato is susceptible to various adverse environments, resulting in reduced yield and quality. TCS plays important roles in signal transduction involved in stress responses and plant development. The tomato ethylene receptor and PHY subfamily, which both belong to HK(L) family have been identified and investigated ,27. Someruit set . Moreoveruit set . Howeverhttp://solgenomiSl.net/) to explore putative TCS members in tomato by employing 280 TCS protein sequences from Arabidopsis [Arabidopsis. This nomenclature has also been used in soybean and Chinese cabbage [Glycine max (98), and Brassica rapa (85)  .Twenty HK(L) proteins were identified in tomato and categorized into nine HKs and 11 HK-likes (HKLs) based on the presence of the conserved His-kinase transmitter (HK) domain. Nine HK proteins were further classified into five subgroups: three cytokinin receptor-like SlHKs, three ethylene receptor-like SlHKs, one CKI1-like SlHK, one CKI2/AHK5-like SlHKs, and one AHK1-like SlHK. All of these HKs possess a conserved HK domain that contains five conserved signature motifs, namely, H, N, G1, F, and G2 . AdditioArabidopsis AHK4–AHK6. Gene structure analysis showed that SlHK4, SlHK5, and SlHK6 possess 9–10 introns. These SlHK proteins contain two conserved motifs (motifs 1 and 4), as identified by MEME, as well as four conserved domains, namely, HK, REC, CHASE, and transmembrane (TM) domains, as recognized by Pfam and SMART online tools  with ne tools . Multiplne tools , and CHASlHKL1-SlHKL4 contain one or two introns, similar exon-intron architecture were also found in Arabidopsis homologous genes. However, SlHK7 and SlHK9 have five or six introns  (Arabidopsis.We identified six HP proteins in tomato with four authentic and two pseudo-HPs. Each of the six SlHP genes contains five or six introns except 1 and 2) . HP prot1 and 2) . AlthougArabidopsis. They exhibit quite similar gene structure. All tomato type-A RRs possess four introns, except for SlRR3, which contains only one intron , except SlPRR5, which lose its pseudo-REC domain. SlPRR1–6 contain a CCT domain, which plays an important role in regulating circadian rhythm and controlling flowering time [Arabidopsis with 45% and 46% sequence identities, respectively. SlPRR7 and SlPRR8 are characterized by a pseudo-REC and a Myb domain  were identified in tomato . Tomato e intron . These te intron . Plant te intron . This fie-B PRRs . SlPRR1–ing time . Only twb domain . SlPRR8 ripening .Arabidopsis, rice, maize, soybean, Chinese cabbage, Lotus japonicus, Physcomitrella patens, wheat, and tomato were used to perform multiple alignments and generate phylogenetic trees for exploring the evolutionary relationships of these HK(L)s  proteins from e HK(L)s . The HK, as a tomato-special subgroup. Previous studies suggested that type-C RRs may be the oldest RR genes; type-A RR, as the youngest subgroup, may evolved from type-C RRs by mutations happened in their promoter sequences [Physcomitrella patens exhibit closer relationship with RRs from monocotyledons. Notably, Arabidopsis APRRs are clustered into a distinct subclade in the phylogenetic tree, whereas PRRs from the other species could be further divided into clock and type-B PRR subclades. The type-B PRRs are closely related to type-B RRs, which is consistent with the results of previous phylogenetic analyses [All the RR proteins from the nine species were grouped into type-A, type-B, type-C, and PRR subfamily . Generalequences . Type-C analyses ,10.SlRR29, located on the scaffcold A00, are distributed on 12 tomato chromosomes , non-synonymous rate (Ka), and Ka/Ks of these duplicates were calculated, and duplication time was speculated using the values of Ks -fusion proteins in the epidermal cells of onion. SlHK8, as an ethylene receptor, was predicted to be located in cytoplasm using SubLoc v1.0 website  Table S. But, inytoplasm . Thus, tSlHP4 and SlPHP1, along with their homologous gene AHP4 in Arabidopsis [SlHKL1, SlRR6/7, and SlPRR8 were only detected in flower.The electronic expression profiles of 65 tomato TCS genes in various organs/tissues were downloaded from the tomato eFP browser at bar.utoronto.ca. Among them, the transcripts of 20 TCS genes were quite low in all detected organs/tissues. So we clustered the rest 45 gene expression profiles in various organs with MeV4.8. The heatmap indicated that the expression profiles of tomato TCS genes in leaf, root, flower, and fruit could be divided into four clades A. The geThe expression profiles of tomato TCS genes in six fruit different developmental stage were further summarized and clustered B. They cArabidopsis TCS [The selected 45 tomato TCS genes were subjected to Gene Ontology (GO) enrichment analysis . GO analSlRR1–SlRR5, are generally induced by ZT, particularly at 1 h after treatment. Similarly, the type-A RRs in other species were proven to be obviously upregulated by cytokinin [SlHK8 are obviously downregulated. However, SlHK6–7 could be induced by ZT. Almost all of the detected SlHPs are generally induced, except SlHP2, which is obviously downregulated. Under ABA treatment, tomato HK transcripts are generally induced and their expression levels are maintained at a relatively high level at 8 h. By contrast, most of HPs are induced at the early stages but then decrease to a low level at 8 h except SlHP1. In addition, most of SlRRs positively respond to ABA except SlRR2–3, SlRR23, and SlPRR4–6.Evaluation of the expression levels of 31 randomly selected TCS genes revealed that most of the detected TCS genes in tomato could be induced by exposure to exogenous trans-zeatin and ABA treatment . Howeverytokinin . All theSlPHP1 decrease 2 h after treatment. However, all the tested SlHKs except SlHK4/7 are downregulated after drought treatment. Except SlRR3, the expression of the other tomato type-A RRs are generally downregulated even though the transcript levels of SlRR4–6 increase at 8 h. Except SlPRR4–5, all SlPRRs are obviously upregualted by drought. For salt treatment  are significantly induced by salt stress but repressed at 2 h. The other ethylene receptor like gene, SlHKL2, is obviously repressed by drought. Interestingly, most TCS genes such as SlHP4, SlPRR1, SlPRR2, SlPRR4, and SlPRR5 exhibit similar response patterns to drought and salt. However, some genes including SlHK8, SlHP3, SlHP4, and SlRR23 exhibit an opposite expression patterns under two abiotic stresses.Almost all of the detected SlHPs are upregulated in response to drought stress. All of HPs are evidently induced from 1 h after drought treatment and generally maintain at a higher level, except that the transcript levels of reatment , most ofArabidopsis (56), rice (52), and maize (59), but obviously fewer than that in Glycine max (98), and Brassica rapa (85) (Arabidopsis (17), rice (11), and maize (11), which is only less than that in soybean (36). It was worth mentioning that tomato contains the largest number of type-B RRs (23) in all identified species and is nearly twice as many as that in Arabidopsis (12). In tomato, four pairs of segment duplicates including SlHKL1 and SlHKL4, SlRR12 and SlRR13, SlRR22 and SlRR23, and SlPRR1 and SlPRR2 were found. And a tandem duplicate cluster of SlRR16, SlRR18, and SlRR25–SlRR28 were identified. These tandem duplicates with high similarties exhibit conserved protein and gene structure, which all have a REC domain. Furthermore they occupied a tomato-specific type-B VI subfamily in the phylogenetic tree. Segmental duplication and tandem duplication events both contribute to the expansion of the TCS gene family in tomato. By contrast, in Arabidopsis, Chinese cabbage, and soybean, segmental duplication was the main mechanism contributing to the duplication of TCS genes [Arabidopsis, 10 pairs of segmental duplicates were found which accounted for 35.71% of all Arabidopsis TCS genes [Arabidopsis and soybean. Only one pair of duplicated genes was identified in Chinese cabbage. These results suggested that, unlike that in tomato, segmental duplication might be the main mechanism contributing to the duplication of TCS genes in Arabidopsis, Chinese cabbage, and soybean [Arabidopsis [Ks of segmental duplicates in the tomato TCS genes ranged from 0.6 to 0.79, which corresponded to the divergence time of 46 Mya to 60 Mya, suggesting that the gene duplication events occurred after the split of tomato and Arabidopsis. On the other hand, SlRR16, SlRR18, and SlRR25–28 in the tomato genome clustered together on chromosome A11, forming a tandem duplicate cluster. The calculated divergence time was varied from 5.97 Mya to 26.55 Mya. Tandem duplicates usually occurred more recently than segment duplicates, which probably occurred because tandem duplications in plants were more likely to participate in stress responses and these tandem duplicates were not retained as long as nontandem duplicates [In this study, a total of 65 TCS genes, including 20 HK(L)s, six HPs and 39 RRs, were identified from tomato genome. The number of TCS family members in tomato is slightly bigger than that of apa (85) . In detaCS genes . In Arabbidopsis . In thisplicates   enhanced flower senescence and failed to fruit set [phyA, phyB1, and phyB2 single, double, or triple mutants indicated that they participated in modulating the carotenoid formation and the time required for phase transitions during fruit ripening [SlPRR8 was verified to regulate plastid development and fruit ripening [Expression analysis indicated that 45 TCS genes in tomato are predominantly expressed in the root, fruit, or flower, whereas the transcripts of other genes could not be detected in any tomato organs . Twelve  studied ,29,35. Truit set . AnalysiArabidopsis, soybean and Chinese cabbage [Arabidopsis negatively responded to drought [SlRR9, SlRR22, and SlRR23 are upregulated by drought but their homologous genes ARR1 and ARR12 negatively responded to drought [SlHK4/5/7, SlHP1, SlRR1/21/22, and SlPRR1/3/4/5/8 were analyzed by qRT-PCR. In detail, nine genes including SlHK4/7, SlHP1, SlRR21/22, and SlPRR1/3/8 are generally induced by drought, which are consistent with the promoter analyses, but the other three genes  are downregulated. The inconsistent results of SlHK5, SlRR1, and SlPRR5 between expression profiles and promoter analyses were also observed in soybean and Chinese cabbage [Arabidopsis, rice, tomato, and soybean [LE-ETR3 (Nr) participated in salt and heat stresses, and reducing expression of LE-ETR4 led to an enhanced hypersensitive response [PHYA, PHYB1, and PHYB2 were verified to modulate drought stress responses [Increasing evidence verified that TCS proteins are involved in responses to various abiotic stresses ,18,19,23 cabbage ,18,19,23 drought . In addi cabbage ,23. Plan soybean ,25,42,43response ,42,43. MArabidopsis, rice, maize, soybean, Lotus japonicus, Physcomitrella patens, wheat, and Chinese cabbage, respectively, were downloaded from Phytozome [http://solgenomics.net/) with E-value of 1 × 10−5 as the threshold [http://pfam.janelia.org/). Then we searched for TCS genes with HMMER 3.0 using the global HMM profile of these TCS characteristic domains with expected values less than 0.1. After removing redundant sequences, a total of 118 putative elements were identified. As a final quality check, each identified sequence using the two strategies above was subsequently confirmed using Pfam (http://pfam.janelia.org/) and SMART (http://smart.embl-heidelberg.de/) databases according to whether or not it possessed the structural characteristics and conserved domains of TCS elements, i.e., HisK, HATPase, REC, CHASE domain for cytokinin binding (CHASE), ethylene-binding domain (C2H4), and HPt domains. Tomato TCS homolog proteins in Arabidopsis were identified using BLASTP search against Arabidopsis databases of TAIR website (http://www.arabidopsis.org/) with default expected values. ExPASy [Protein sequences of all known plant TCS genes, particularly 56, 52, 51, 98, 62, and 85 members in the genome of hytozome  and thenhreshold . Meanwhi. ExPASy  was used. ExPASy .Arabidopsis, rice, and tomato genome was calculated by DNAStar . Phylogenetic analysis was performed using MEGA 5.0 program by neighbor-joining (NJ) method with 1000 replicates of the bootstrap based on the full-length protein sequences [The exon–intron organizations of all tomato TCS genes were mapped using Gene Structure Display Server . Each faequences .Ks and Ka were calculated using the Codeml procedure of the PAML program [T = Ks/2x (x = 6.56 × 10−9) [All the TCS genes were assigned to the corresponding tomato chromosomes based on the SGN database. A pair of genes were identified as tandem duplicates if the genes both shared ≥40% amino acid sequence similarity and separated by fewer than five intervening genes . PGDD 5 was adop program . Diverge × 10−9) .cis-regulatory elements in the promoter regions were identified using PlantCARE website [The upstream sequences (1.5 kb) of the initiation codon in TCS genomic DNA were obtained from Phytozome  as the p website .Xba I and BamH I restriction sites under the control of the 35S cauliflower mosaic virus promoter (35S CaMV). The pFGC:GFP empty vector was used as control. The recombinant vectors were transformed into onion epidermal cells by particle bombardment using the Biolistic PDS-1000/He gene gun system  [−1 sucrose for 5 min and the fluorescence of GFP was photographed by a Leica DMLE camera .The randomly selected TCS genes were amplified using gene-specific primers  and clonCA, USA) . After 1S. lycopersicum L.) cv. Micro-Tom from Tomato Genetics Resource Center  was used for expression analysis. The seedlings were grown in a growth chamber in temperature-controlled greenhouses of Zhejiang University under day/night temperatures of 28/20 ± 1 °C and light intensity of 250 μmol·m−2·s−1 with 16-h day length. Three-week-old tomato seedlings were used for abiotic stresses and exogenous hormone treatments. For cytokinin and ABA treatment, the seedlings were sprayed with 100 μM ZT and 100 μM ABA, respectively. The second true leaf on each plant was sampled at 0 (control), 1, 2, 4, and 8 h after spraying. To induce drought stress, the seedlings were transferred to filter paper and the leaves were collected at 0, 1, 2, 4, and 8 h. For high salt treatment, the nutrient solution was supplemented with 100 mM NaCl and the leaves were separately collected at 0, 1, 2, 4, and 8 h after treatment. All phytohormone and abiotic treatments were repeated three times and each treatment contained at least 20 seedlings. All materials were frozen at −75 °C until RNA isolation.Tomato (http://bar.utoronto.ca [http://geneontology.org/) and KEGG pathway enrichment analysis [The electronic expression data of tomato TCS genes in various organs were obtained by gene locus from the tomato eFP browser at ronto.ca . The eleronto.ca . Tomato analysis  with defSlUbi3 gene (GenBank accession number X58253) was selected as an internal control [Ct−ΔΔ method. Heatmap was generated by Multiple Array Viewer using the relative expression data of each gene [The total RNA was extracted from the collected materials using TRIZOL reagent  according to the manufacturer-recommended protocol. The first cDNA strand was generated from 1 μg of total RNA using the PrimerScript RT reagent kit  according to the manufacturer’s instructions. Specific primers used in the qRT-PCR were designed by Primer 5 Software, and each primer was searched in the tomato database to ensure its specificity. The qRT-PCR reactions were performed on the CFX96 Real Time System machine , programmed to heat for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 45 s at 55 °C, and at the end, 1 cycle of 1 min at 95 °C, 30 s at 50 °C and 30 s at 95 °C. Two biological and three technical replicates for each sample were performed with 15 μL of reaction volume using the SYBR Premix Ex Taq kit . The tomato  control . The relIn our study, 20 HK(L)s, six HPs and 39 RRs were identified from tomato genome. Gene classification, gene structures, conserved domains, chromosome distribution, phylogenetic relationship, synteny relationship, gene duplication events, and subcellular localizations of the TCS genes were predicted and analyzed in detail. The tomato TCS elements showed significant sequence and domain conservation except type-B RRs. Gene duplication events mainly occurred in the RR family of tomato TCS genes. Both segment duplication and tandem duplication contributed to gene expansion. The subcellular localization of selected proteins displayed a diverse subcellular targeting and probably played divergent roles. Most TCS genes are predominantly expressed in tomato reproductive organs particularly in fruit development. Meanwhile, promoter analyses and qRT-PCR results indicated that almost all of TCSs could respond to various stresses and exogenous hormone treatments. The identification of tomato TCS elements would provide a more comprehensive sight and solid foundation to elucidate their roles in mediating hormone cross-talk and stress responses in further.


The TT genotype was significantly associated with hallucinations, aberrant motor behavior, and psychosis in AD . No association was observed between T102C alleles or genotypes and delusions, agitation/aggression, depression, and apathy (p > 0.05). Thus, the 5HT2A T102C might be a susceptible factor for hallucinations, aberrant motor behavior, and psychosis in AD. The potential mechanism of this polymorphism in BPSD in AD requires further exploration.The serotonin receptor gene (5-HT2A) has been reported to be a susceptible factor in behavioral and psychological symptoms of dementia (BPSD) in Alzheimer's disease (AD). However, previous results were conflicting. We aim to investigate the association of 5-HT2A T102C with BPSD in AD using a meta-analysis. Studies were collected using PubMed, Web of Science, the Cochrane Library databases, Chinese National Knowledge Infrastructure (CNKI), and Embase. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess associations. Nine studies with 1899 AD patients with/without BPSD were included in this meta-analysis. The 102C and CC genotypes were associated with psychosis in AD (102C:  Cognitive decline is one of the major neuropsychiatric features in Alzheimer's disease (AD) . HoweverSerotonin  is a key neurotransmitter involved in many aspects of human and animal behavior, including aggression, hallucinations, delusions, depression, anxious behavior, and the regulation of appetite –8. The aMicheli et al.  = 3.19 [2.12–4.79]) was found to be associated with 5HT2A C102 under the allelic model. No significant association was found between 5HT2A C102 and delusions, hallucinations, agitation/aggression, depression, apathy, and aberrant motor behavior susceptibility in the analysis as a whole . No association was observed between 5HT2A T102C and delusions, agitation/aggression, depression, and apathy susceptibility under the TT versus CT/CC model  under the CC versus TT/CT model. No other evident associations between 5HT2A T102C and delusions, hallucinations, agitation/aggression, depression, apathy, and aberrant motor behavior susceptibility under the CC versus TT/CT model were observed . This heterogeneity was contributed mainly by one positive study [p = 0.48) heterogeneity and showed that it had the highest effect on 5HT2A T102C allelic association with the effect of depression in AD.Significant heterogeneity was observed between 5HT2A 102C and depression  . The shape of funnel plot did not reveal any obvious asymmetry , hallucinations (390 cases and 768 controls), agitation/aggression (497 cases and 267 controls), depression (603 cases and 537 controls), apathy (439 cases and 88 controls), aberrant motor behavior (404 cases and 124 controls), and psychosis (110 cases and 99 controls), separately. Secondly, AD is a multifactorial disease. Gene-gene interactions may play important roles in the pathology of BPSD in AD, but most studies lack information about gene-gene interactions. Thirdly, most of the patients in the present study were Caucasians, which may limit the general application of the results to other populations.The current meta-analysis suggests an increased risk of psychological symptoms of psychosis in AD for the 5HT2A C102 allele and CC genotype and a decreased risk of hallucinations, aberrant motor behavior, and psychosis in AD for the 5HT2A TT genotype. To confirm these results, further study with larger sample size and multiple ethnicities is necessary.


Chronic obstructive pulmonary disease (COPD) has been recently characterized as a disease of accelerated lung aging, but the mechanism remains unclear. Tetraspanins have emerged as key players in malignancy and inflammatory diseases. Here, we found that CD9/CD81 double knockout (DKO) mice with a COPD-like phenotype progressively developed a syndrome resembling human aging, including cataracts, hair loss, and atrophy of various organs, including thymus, muscle, and testis, resulting in shorter survival than wild-type (WT) mice. Consistent with this, DNA microarray analysis of DKO mouse lungs revealed differential expression of genes involved in cell death, inflammation, and the sirtuin-1 (SIRT1) pathway. Accordingly, expression of SIRT1 was reduced in DKO mouse lungs. Importantly, siRNA knockdown of CD9 and CD81 in lung epithelial cells additively decreased SIRT1 and Foxo3a expression, but reciprocally upregulated the expression of p21 and p53, leading to reduced cell proliferation and elevated apoptosis. Furthermore, deletion of these tetraspanins increased the expression of pro-inflammatory genes and IL-8. Hence, CD9 and CD81 might coordinately prevent senescence and inflammation, partly by maintaining SIRT1 expression. Altogether, CD9/CD81 DKO mice represent a novel model for both COPD and accelerated senescence. COPD is a growing cause of mortality and morbidity worldwide, and is expected to be the third leading cause of death by 20202. In light of the considerable attention paid to the comorbidities of COPD, such as cardiovascular disease, diabetes mellitus, and osteoporosis, it is increasingly regarded as a systemic inflammatory lung disease4. Although the mechanisms underlying the relationship between COPD and these comorbidities remain unclear, the prevailing hypothesis is that a spill-over effect from the lung causes the extra-pulmonary comorbidities5: according to this theory, various inflammatory molecules such as CRP, IL-1β, and IL-6 secreted in the lung, spill out from the lung and induce systemic inflammation, as well as multi-organ disease. However, very few correlations between lung and serum markers have been observed, implying that a simple spill-over of mediators from the lung is not necessarily responsible for the systemic inflammation observed in COPD6. Given that the prevalence of COPD increases with age, that the abundance of alveolar senescent cells is elevated in the lungs of patients with COPD, and that COPD and aging share common mechanisms, COPD is considered to be a model for accelerated senescence of the lung, similar to other lifestyle-related diseases9. However, due to the complex nature of the mechanisms underlying COPD and aging, their precise interrelationship remains unclear.Chronic obstructive pulmonary disorder (COPD) is a progressive disease state characterized by poorly reversible airflow limitation and an abnormal inflammatory response of the lungs to noxious particles, particularly cigarette smoke (CS)10. The hallmarks of aging include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication11. Importantly, these mechanisms contribute to the pathogenesis of a variety of chronic diseases, including atherosclerosis, osteoporosis, cataracts, cancer, neurological diseases, and respiratory diseases13. Despite remarkable progress in the biology of aging over the past quarter century, the molecular mechanisms linking aging with age-related diseases have not yet been elucidated. However, the discovery of several aging models, such as Klotho, SAM, ATR, and SMP-30, has provided us with considerable new information regarding the pathogenesis of age-related diseases and potential therapeutic targets17. Among the key players in mammalian ageing, the sirtuins (SIRT1-SIRT7) are NAD+ dependent deacetylases that control a wide range of processes implicated in the regulation of homeostasis18. SIRT1, the best-characterized sirtuin in mammals, undoubtedly plays a key role in governing management of cellular stress management and ensuring a healthy lifespan19. SIRT1 expression is reportedly reduced in chronic inflammatory conditions, including aging20. Moreover, the activation or overexpression of SIRT1 increases lifespan in fly, yeast, worm, and mouse21. Importantly, SIRT1, whose expression is reduced in the lung of COPD patients, also plays pivotal roles in humans23. Because SIRT1 has critical effects in chronic inflammatory diseases, including cardiovascular disease and diabetes mellitus, considerable effort has been devoted to discovering pharmaceutical activators of SIRT1 for use in therapeutic applications24.Aging is a natural process characterized by progressive functional impairment and reduced capacity to respond appropriately to environmental stimuli and injury27. A unique feature of tetraspanins is their propensity to interact with one another and with various other transmembrane molecules, including integrins and growth factor receptors, thereby acting as molecular organizers in tetraspanin-enriched microdomains. By organizing various functional molecules, tetraspanins are involved in a wide variety of biological processes, including cell migration, proliferation, survival, and morphogenesis, and thus influence immune diseases, infection, angiogenesis, and cancer metastasis28. CD9 and CD81, closely related tetraspanins, are expressed abundantly in the lung, and both CD9 knockout (KO) and CD81 KO mice exhibit quite similar phenotypes, such as infertility. Unexpectedly, younger CD9/CD81 double KO (DKO) mice develop COPD-like phenotypes30. Macrophages from DKO mice express elevated levels of MMP-9 production, probably due to disorganization of integrin-tetraspanin complexes in tetraspanin-enriched microdomains30. CD9 forms a complex with CD14, thereby stabilizing CD14/TLR4 complexes; consequently, CD9 KO mice exhibit enhanced macrophage-dominant inflammation and TNF-α production in the lungs after lipopolysaccharide stimulation31. Notably, CD9/CD81 DKO mice are more susceptible to cigarette-smoke-induced emphysema (manuscript in preparation). Our preliminary data suggest that levels of CD9 and CD81 are reduced in blood monocytes from COPD patients (paper in preparation). Considering that CD9/CD81 DKO mice develop not only emphysema but also some of the extra-pulmonary diseases seen in COPD patients, such as body weight loss and osteoporosis, we regard these DKO mice as a novel model for human COPD32. However, given that COPD is increasingly viewed as accelerated senescence of the lungs8, and that these tetraspanins have pleiotropic functions as molecular facilitators in a wide range of cells and tissues, we sought to explore the aging-like phenotype and its underlying mechanisms in this mouse model of COPD.Tetraspanins are cell-surface proteins that span the membrane four times and are ubiquitously expressed in multiple organsWe found that CD9/CD81 DKO mice with the COPD-like phenotype progressively develop an accelerated aging syndrome, with symptoms including cataracts, osteoporosis, emphysema, and atrophy of the skin, muscle, and adipose tissue. In mechanistic terms, our findings revealed that double deletion of CD9/CD81 in epithelial cells downregulates the expression of SIRT1, thereby decreasing cell proliferation and augmenting inflammation. Thus, CD9/CD81 DKO mice could represent a unique model for COPD leading to accelerated senescence. Given that SIRT1 is considered a key molecule that protects against various lifestyle-related diseases and aging, the molecular organizers CD9 and CD81, which may maintain the expression of SIRT1 and other aging-related pathways, could serve as novel therapeutic targets, not only for COPD but also for aging more generally.See the Supporting information for an expanded METHODS section.34. These mice were backcrossed into the C57BL/6 J background for more than seven generations in a barrier facility, and all animal procedures were performed in accordance with the Osaka University guidelines on animal care. Genotyping of all breeding pairs was confirmed by PCR analysis. Eight- to 80-week (wk)-old DKO mice and wild-type (WT) littermates matched for age and sex were used.All animal experiments were approved by the Animal Care and Use Committee of Osaka University, and all of the animal procedures were performed in accordance with the Osaka University guidelines on animal care. CD9-KO, CD81-KO, and CD9/CD81-DKO mice were described previouslyA549 cells  were cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. For siRNA transfection, the cells were transfected with an siRNA mixture against human CD9, CD81, or control random RNAs  using Lipofectamine RNAiMAX (Invitrogen). The cells were cultured for 2 days, and the gene-silencing effect of the siRNAs was assessed by immunoblotting with anti-CD9 and anti-CD81 monoclonal antibodies (Abs). In some experiments, the cells were trypsinized 2 days after transfection, re-cultured in DMEM overnight, and then stimulated with 50 ng/mL TNF-α. The cells were harvested after a further 24-h incubation. Alternatively, 5 µM SRT1720  was added to the culture at 4 h after CD9 and CD81 siRNA transfection, and the cells were harvested 2 days after transfection. Concentrations of IL-8 in culture supernatants were measured by enzyme-linked immunosorbent assay using Quantikine (R&D Systems).4), transfected with siRNA against human CD9 and CD81 or control RNAs, were seeded on 96-well plates and incubated overnight. After further incubation for 2 days, the kit reagent WST-8 was added to the medium, and the cells were incubated for 1 h. The absorbance of samples (450 nm) was determined using a scanning multi-well spectrophotometer. For the apoptosis assay, 3.0 × 104 cells were seeded on 96-well plates and incubated overnight. After further incubation for 2 days, quantities of histone-associated DNA fragments in cell lysates were measured using Cell Death Detection ELISA PLUS (Roche).The number of viable cells was determined using a Cell Counting Kit-8 (Dojindo). Cells (3.0 × 10Tissues or cells were lysed in a lysis buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS supplemented with or without Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Lysates containing equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary Abs followed by peroxidase-conjugated secondary Abs. The following primary Abs were used: mouse anti-human CD9 , mouse anti-human CD81 , rat anti-mouse CD9 , hamster anti-mouse CD81 , mouse anti-human SIRT1 , mouse anti-mouse SIRT1 , rabbit anti-human SIRT6 (EPR5079(N); Abcam), rabbit anti-mouse SIRT6 , rabbit anti-FOXO3a (CST), mouse anti-p53 , rabbit anti-p21 , rabbit anti-p21 , mouse anti-p16 , rabbit anti-Klotho (Abcam), mouse anti-WRN , rabbit anti-ATR (CST), NF-κB , rabbit anti-phospho-NF-κB p65 , rabbit anti-acetyl-NF-κB p65 , rabbit anti-IκB (CST), and rabbit anti-β-actin . Immunoreactive signals were visualized using SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific). For densitometry, blots were analyzed on a LAS-3000 Imager (Fujifilm) or Amersham Imager 600 .Isolated cells from mouse spleen were incubated with anti-CD16/CD32 mAb  to block the Fc-receptor and labeled with the following Abs (purchased from BD-Pharmingen) for flow cytometry analysis: rat anti-mouse CD4-FITC (GK1.5), rat anti-mouse CD8-PE (53-6.7), rat anti-CD11b-PE (M1/70), rat anti-mouse CD19-APC (1D3), rat anti-mouse CD21-FITC (7G6), rat anti-mouse CD23-PE (B3B4), mouse anti-mouse IgD-FITC (AMS9.1), goat anti-Ig-PE, rat anti-mouse B220-APC or B220-FITC (RA3-6B2), and anti-DX5 (Ha1/29). Stained cells were analyzed on a BD FACSCanto II (BD Biosciences).Tissue samples were excised and fixed in 10% buffered neutral formalin and embedded in paraffin. The sections were stained with hematoxylin-eosin (HE), periodic acid-Schiff (PAS), or toluidine blue. In some experiments, tissues were fixed by perfusion of the fixative. Lungs were inflated to 25 cm of water pressure with 10% buffered neutral formalin via an intratracheal cannula and embedded in paraffin.Cells were cultured in glass-bottom plates, fixed in 3% paraformaldehyde, and then permeabilized with Tris-buffered saline containing 0.3% Triton X-100. Nonspecific recognition was blocked with Blocking One . The permeabilized cells were incubated with 1 µg/mL primary Ab overnight at 4 °C and subsequently incubated with Alexa Fluor 488-conjugated goat anti-mouse, goat anti-rabbit, or goat anti-rat Abs (Molecular Probes). Nuclei were visualized using DAPI (Molecular Probes). For actin filament staining, rhodamine phalloidin (Molecular Probes) was used. Immunofluorescence images were obtained using the Leica TCS SPE microscope system (Leica Microsystems). The following primary Abs were used: mouse anti-human CD9 , mouse anti-human CD81 , mouse anti-human SIRT1 , rabbit anti-cleaved caspase-3 (CST), and rabbit anti-Ki-67 .5 or 5.0 × 105), transfected with siRNA against human CD9 and CD81 or control RNAs, were seeded on 24-well plates. After incubation for 2 days, the cells were fixed, and a staining solution containing X-Gal was added to the fixed cells and incubated for 20 h at 37 °C. The proportion of cells positive for SA-β-Gal activity was determined by counting the number of blue cells in four independent microscope fields.Cytochemical staining for SA-β-Gal activity was performed using a Senescence Detection Kit (BioVision). Cells  were immersed in RNAlater solution (Invitrogen) and stored at −80 °C until RNA extraction. The lungs were transferred into TRIzol reagent (Invitrogen) and homogenized. Cells transfected with siRNA against human CD9 and CD81 (n = 3) or control RNAs (n = 3) were harvested and lysed in RLT reagent (Invitrogen). Total RNA was extracted from the lysates using the RNeasy mini kit (QIAGEN). After reverse transcription, cDNA was hybridized using Agilent SurePrint G3 Mouse or Human GE Microarrays (Agilent Technologies) according to the protocol specified in the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Microarray signal intensity was normalized using the RMA algorithm implemented in GeneSpring GX ver.12 (Agilent Technologies). Alternatively, gene expression profiles of inflammatory cytokines in RNA extracted from A549 cells treated or not treated with TNF-α were obtained by RT-PCR using TaqMan Array Plates; Human cytokine network (Applied Biosystems) on a 7900HT Fast Real Time PCR System (Applied Biosystems).35, TargetMine36, and KeyMolnet37.To identify biologically relevant molecular networks and pathways in gene expression data from mice lungs or epithelial cells, three distinct bioinformatic pathway analysis tools were used: Ingenuity Pathways Analysis (IPA)All numerical results are expressed as means ± standard deviation. Separate animals were examined in experiments containing multiple time points in Fig. At 80 weeks of age, CD9/CD81 DKO mice were smaller and had less hair of a brownish color than WT mice, although DKO and WT mice could not be distinguished at 3 wk of age Fig. . In addiHistological examination revealed that DKO mice developed emphysema and osteoporosis at 80 wk Fig. . Consist15. Although younger DKO mice at 1 month of age did not exhibit any differences, mice at 18 months of age had an elevated aging score compared to WT mice, which was true even at 6 months of age , and ATR (ataxia telangiectasia and Rad3-related protein) were unaltered . Our DNA microarray analysis indicated that the expression profile of genes related to cellular movement and immune response of CD9/CD81 DKD epithelial cells was similar to that of DKO lungs  double deletion of the related tetraspanins CD9 and CD81 in mice caused progressive multiple aging phenotypes and a shortened life span, and (2) CD9 and CD81 play coordinate roles in maintaining SIRT1 expression, thereby facilitating anti-aging and anti-inflammatory activity.7, COPD has increasingly been viewed as an accelerated senescence of the lung. Given this background, CD9/CD81 DKO mice could be regarded as a novel model for both COPD and aging.Based on our previous work, we regarded CD9/CD81 DKO mice as a COPD-like model. Surprisingly, aged CD9/CD81 DKO mice developed not only emphysema but also non-pulmonary comorbidities such as osteoporosis and body weight loss, eventually leading to multiple aging phenotypes. Given that senescence in epithelial and endothelial cells is elevated in patients with COPD39. Among them, Klotho-deficient mice exhibit multiple aging phenotypes including osteoporosis, emphysema, arteriosclerosis, and atrophy in major organs including the sex organs, skin, and pituitary, whereas overexpression of Klotho extends the lifespan of mice40. Notably, the discovery of the Klotho gene has not only advanced our understanding of the aging process, but also the development of therapeutic applications. Although CD9/CD81 DKO mice have a similar phenotype to Klotho mice, a few major differences should be noted. First, although Klotho is specifically expressed in the kidney and brain, both CD9 and CD81 are ubiquitously expressed throughout whole body, albeit to varying degrees26. Second, α-Klotho is a multifunctional protein similar to a hormone, and regulates the metabolism of phosphates, calcium, and vitamin D. Consistent with this hormone-like role, Klotho mice develop aging phenotypes such as emphysema and osteoporosis in tissues, such as lung and bone, in which the Klotho protein is not expressed14. Meanwhile, CD9 and CD81, which are ubiquitously expressed in aged organs, function as molecular facilitators, or modulators of signal transduction pathways. These differences in localization and function could explain some of the phenotypic differences between these models, e.g., atherosclerosis in Klotho mice and cataracts in CD9/CD81 DKO mice. Because tetraspanins have never been reported to play a role in cataracts or atrophy of organs such as the testis, adipose tissue, thymus, pancreas, and submandibular glands, further studies are warranted. Neither decreased Klotho protein nor hypercalcemia was observed in the lung or epithelial cells of CD9/CD81 DKO mice; therefore, it is unlikely that the Klotho protein actively participates in the pathogenesis of DKO mice. Thirdly, while Klotho mice live for less than 2 months14, CD9/CD81 DKO mice live for approximately 1.5 years, indicating that the DKO mice should be viewed as undergoing accelerated senescence, rather than as manifesting a progeroid syndrome.To date, several aging models with distinct features have been reported41. Moreover, elastin/collagen staining and ultrastructural studies of histological sections suggested that the alveolar destruction and remodelling process were ongoing in the DKO lung30. Therefore, all of these findings in our previous papers could indicate that abnormal development had little, if any, effect in the lung pathology. Given that senile or aged lung is characterized by alveolar enlargement without wall destruction, a pathologic hallmark of emphysema42, our DKO mice could be regarded as a model of senile emphysema rather than merely a model of aged lung.Importantly, it is unlikely that the emphysema in DKO mice resulted from defective alveolarization, because the lung phenotype in DKO mice is normal at the age of 3 weeks, by which time mouse alveolarization is complete23.Both COPD and aging are complex and highly heterogeneous conditions. Consequently, the molecular mechanisms that govern these processes remain incompletely understood. Importantly, Sirt1 overexpression attenuates the upregulation of the senescence markers p21, p16, and p53 in the lungs of SIRT1 heterozygous mice, thereby protecting against lung senescence in epithelial cells, but not in macrophages43. For example, deletion of CD9 in epithelial cells moderately up-regulated expression of p21, but deletion of CD81 had a much more dramatic effect 46. This process is characterized by activation of several signaling molecules, including NF-κB, Forkhead box O, and Klotho. A reduction in SIRT1 expression is associated with elevated activation of RelA/p65 NF-κB, the master regulator of inflammation47 Importantly, CD9/CD81 DKO mice exhibited increased inflammation in vitro and in vivo at both the protein and mRNA levels els Figs . Indeed,Although the spill-over effect has been considered to be the major mechanism underlying the comorbidities of COPD, this theory is not sufficient to explain this complex systemic disease. Given their pleiotropic functions as molecular organizers and their ubiquitous expression, tetraspanins could affect not only the spill-over effect, but also exert local effects on several pathways, as seen in our bioinformatic analysis Fig.  and 6. Ain vitro and in vivo48. Therefore, the osteopenia and sarcopenia seen in DKO mice cannot simply be attributed to the spill-over effect from the lung, but must involve disorganization of the fusion machinery after CD9/CD81 depletion. Second, both CD9 and CD81, which are also abundantly expressed in vascular endothelial cells and lymphatic endothelial cells, additively promote angiogenesis and lymphangiogenesis in vitro and in vivo49. Because VEGF signaling plays critical roles in vivo during the development of the early cardiovasculature and several organ systems50, a reduction of angiogenesis or possibly lymphangiogenesis might contribute to the atrophy of organs such as muscle and adipose tissue in DKO mice. Thus, CD9 and CD81 might regulate inflammation/COPD/aging not only through the spill-over effect, but also by organizing molecular complexes in tetraspanin-enriched microdomains. However, given their pleiotropic functions and their ubiquitous expression of tetraspanins, further study using a lung specific DKO mouse would be intriguing in order to verify the contribution of tetraspanins in the lung on extrapulmonary effect.First, both CD9 and CD81, which are abundantly expressed in macrophages and myoblasts, regulate fusion and differentiation into osteoclast and myoblasts, respectively, both 52. Using a drug-repositioning strategy, we identified 72 drugs that upregulate CD9 expression from a library of 1,165 compounds. Among them, the statins exerted anti-inflammatory effects by upregulating tetraspanin CD9 and CD81 expression in macrophages53. Although statins possess potent anti-inflammatory properties that positively affect COPD and cardiovascular disease54, these beneficial effects might be attributable to the upregulated expression of CD9 and CD81. Recently, a structural study of full-length CD81 revealed that this protein has a cone-like architecture and undergoes a conformational change after cholesterol binding, suggesting a potential mechanism for regulation of tetraspanin function55.Because COPD is increasingly regarded as a major health problem around the world, there is an urgent need to develop novel therapies for this disease. Given that anti-aging molecules have been developed with some efficacy in mouse and human, and that COPD has been viewed as a form of accelerated senescence, anti-aging molecules could be used to treat COPD. Given that SIRT1 functions as a hub protein among the complex networks associated with aging, and that tetraspanins protect against senescence through both direct and indirect interactions with SIRT1, pharmacological modulation of tetraspanins represents a novel therapeutic strategy for both COPD and aging56.Therefore, CD9/CD81 DKO mice represent a unique model for COPD leading to accelerated senescence. Furthermore, the molecular organizers CD9 and CD81, which might maintain the expression of SIRT1 as well as other aging pathways, are promising novel therapeutic targets, not only for COPD, but also for agingSupporting informationSupplemental Table 3Supplemental Table 4


To our knowledge, this is the first study on molecular effect of chronic low dose radiation exposure on human population from high background radiation areas at transcriptome level using high throughput approach. These findings have tremendous implications in understanding low dose radiation biology especially, the effect of low dose radiation exposure in humans.The high level natural radiation areas (HLNRA) of Kerala coast in south west India is unique for its wide variation in the background radiation dose (<1.0mGy to 45mGy/year) and vast population size. Several biological studies conducted in this area did not reveal any adverse effects of chronic low dose and low dose rate radiation on human population. In the present study, global transcriptome analysis was carried out in peripheral blood mono-nuclear cells of 36 individuals belonging to different background dose groups  to find out differentially expressed genes and their biological significance in response to chronic low dose radiation exposure. Our results revealed a dose dependent increase in the number of differentially expressed genes with respect to different background dose levels. Gene ontology analysis revealed majority of these differentially expressed genes are involved in DNA damage response (DDR) signaling, DNA repair, cell cycle arrest, apoptosis, histone/chromatin modification and immune response. In the present study, 64 background dose responsive genes have been identified as possible chronic low dose radiation signatures. Validation of 30 differentially expressed genes was carried out using fluorescent based universal probe library. Abundance of DDR and DNA repair genes along with pathways such as MAPK, p53 and JNK in higher background dose groups (> 5.0mGy/year) indicated a possible threshold dose for DDR signaling and are plausible reason of observing  Studying the biological effects of low dose and low dose-rate ionizing radiation (IR) in humans has important implications to human health and radiation protection science. Increasing evidences suggest that the effect of IR at low and high doses of exposures is qualitatively and quantitatively different ,2. At hiThe linear no threshold (LNT) hypothesis is based on the fact that even the smallest doses of radiation exposure have the potential to cause an increase in health risk to humans. However, the extrapolation of data from high dose exposures to low dose region of the dose response curve is one of the criticisms about LNT. Also, defence mechanisms such as DNA repair processes and elimination of damaged cells have not been taken into consideration for LNT hypothesis . DNA repIn daily life, humans are exposed to low dose and low dose-rate ionizing radiation from various sources such as natural background, occupational, accidental, medical applications (diagnostic and therapeutic) etc. Natural chronic low dose exposure prevailing in high level natural radiation areas (HLNRA) in the world offers unique opportunity to study the biological effects of low dose radiation directly on humans. The HLNRA of Kerala is a narrow coastal line extending from Neendakara panchayat (Kollam district) in the south to Purakkad panchayat (Allapuzzha district) in the north. It has patchy distribution of monazite in the beach sand containing 8–10% of thorium-232 and therefore the human population  is exposed continuously to natural chronic low dose radiation. Because of varied level of background radiation dose (<1.0 to 45.0 mGy/year), this area is suitable for dose response studies. Studying the biological effect of radiation exposure in humans at low dose and low dose-rates especially below 100mSv is highly challenging, but extremely essential for risk estimation. Human population residing in high background radiation areas provide the most appropriate opportunities to study the effect of IR at such low doses as they have been exposed continuously at all stages of development/organogenesis from birth to death.in vivo radio-adaptive response among HLNRA group of individuals after a challenge dose of 1.0 and 2.0Gy of gamma radiation from Kerala coast using affymetrix Human Genome U133 Plus 2.0 Gene Chip. The objective was to find out differentially expressed genes in different HLNRA dose groups  with respect to NLNRA (control group) and their biological significance in response to chronic low dose radiation. The NLNRA (group I) group of individuals were considered as control population and the expression profile observed in this group was considered as background level changes in gene expression at a particular gene. Analysis was carried out to identify differentially expressed genes at a threshold fold change value of 1.3, 1.5 and 2.0 fold with adjusted p-value < 0.05. The differentially expressed genes (up and down-regulated) in each HLNRA dose group  as compared to NLNRA (Group I) is given in In order to get a large number of biologically significant genes from various molecular and cellular pathways that are differentially expressed in different background dose groups, analysis was carried out at a fold change threshold of 1.3 and adjusted p-values < 0.05. Our data revealed a total of 138 (39 up and 99 down), 1361 (611 up and 750 down) and 2427 (889 up and 1538 down) genes to be differentially expressed between Group I vs. Group II, Group I vs. Group III and Group I vs. Group IV, respectively  and down-regulated  common genes among the three groups. These common genes are involved in RNA processing, cell cycle regulation, apoptosis, microtubule formation, nucleotide metabolism, transcription initiation and regulation, cell growth, signal transduction, protein folding, cytokine activity and ion transport etc.As shown in the Venn diagram , a totalJUN, NR4A2), apoptosis , regulation of cell cycle , response to DNA damage , metabolic processes , RNA processing , Immune response , signal transduction , DNA repair , protein transport , histone/chromatin modification , response to oxidative stress , protein ubiquitination  are over-represented in higher dose groups (Group III and Group IV) as compared to Group I  as compared to NLNRA to find out relevant cellular and molecular pathways activated in response to chronic low dose exposure in HLNRA individuals. Our analysis showed that in higher dose groups (Group III and IV), various important pathways such as MAPK signaling, Jak-STAT signaling, p53 signaling, T cell receptor signaling, B cell receptor signaling, insulin signaling, purine metabolism, apoptosis, cell cycle, DNA repair, ubiquitin mediated proteolysis, focal adhesion, gap junction etc. were found to be over-represented . RepreseIL1A, IGHG1), apoptosis , RNA splicing  and translational initiation  etc. However, high dose groups (>5.0 mGy/year) have higher representation/abundance of genes involved in DNA damage response that include DNA repair, response to oxidative stress, immune response, chromatin modification (methylation and histone modification), mRNA processing, cell cycle, and apoptosis , base excision repair , nucleotide excision repair , homologous recombination repair , mismatch repair (PMS1) and other repair genes . DDR regulated pathways such as MAPK signaling, p53 signaling, JAK-STAT signaling were also activated in higher dose groups (Group III and IV). Although expression levels of p53 gene did not show any change, but p53 regulated genes such as CDKN1A, TRAF4, ATF3, TNFRSF10B, APAF1, DUSP1, PMAIP1, GADD45B, BCL6, PLK3 etc were differentially expressed in our data. Some of the genes involved in MAPK pathway were DUSP1, DUSP10, AKT2, ATF2, BCL10, CD38, CDC42, CSNK1A1, DDIT3, DUSP5, GFRAL, GNAS, HIPK3, IL8 etc. Several transcription factors and signaling molecules, which are involved in regulating DNA damage response and repair such as cJUN, FOSB, JUND, CREBZF, FOXO3, ATF2, HEY1, NR4A2 etc were found to be activated in higher dose groups. A representative list of important genes involved in DDR processes is given in Several genes involved in different DNA repair pathways were present in higher dose groups (Group III and Group IV: > 5.0 mGy/year). These includes genes involved in non-homologous end joining repair , immune system , stimulus to DNA damage , apoptosis , mRNA processing , transcriptional regulation  and signal transduction  and ubiquitin dependent proteolysis  etc. A representative scatter plot showing the microarray intensity values of selected genes in individuals of different background dose groups is given in We have also identified 64 genes (36 up-regulated and 28 down-regulated) which were highly expressed (≥ 2.0 fold) in Group IV and showed background radiation dose dependent increase or decrease (up and down regulation) in their expression profile. PMAIP1, PAPD4, DDIT3, GIMAP8, PPIF, CSRNP1, BTG1, CDKN1A, DUSP10, GADD45B, TSC22D2, METTL13, cJUN, DUSP1, KIR3DS1, JUND, EIF1, ATXN1, SNRPA1, DHFRL1, NAMPT, ZNF167, THAP2, KLF6, BBS10, PLK3, PDK4, SETDB2, KDM6B, CCR2.Thirty differentially expressed genes (22 up-regulated and 8 down-regulated) were validated using RT q-PCR. Validation of these genes was carried out in 54 individuals, which include 30 individuals used for microarray analysis and another independent set of 24 individuals from the same dose groups of Kerala population. These genes were selected from different dose group comparisons involved in various biological processes, having different expression levels in order to get a good representation from microarray data. These include As shown in ex-vivo/ in-vivo effect of low dose and dose-rate exposure 55. Human population living in high level natural radiation areas (HLNRA) is exposed to chronic low dose background radiation since generations and provides unique opportunity to understand the in-vivo effects of low dose radiation exposure directly on humans. Most importantly, the unique feature of HLNRA of Kerala coast is the non-uniform distribution of monazite in the beach sand which leads to varying level of background radiation (<1.0 to 45mGy/year) along the 55 km long stretch allowing us to investigate in-vivo dose response, if any at various biological end points. Several epidemiological  and biological studies carried out in this population have not revealed any significant difference between NLNRA and HLNRA population [The present study is focused on the cellular responses to chronic low dose and low dose-rate radiation in humans. The baseline gene expression is very important to understand the pulation ,18,20,22pulation ,23.In the present study, global gene expression profile was carried out to understand biological response of human cells exposed to chronic low dose and low dose-rate radiation. The individuals included in the study are classified into four different background dose groups based on the individual dose received annually by them. The first group receives a dose ≤ 1.5mGy/year and are considered as control population (NLNRA) and the other three groups are from HLNRA receiving a dose >1.5mGy/year. Our results have shown that a large number of genes are differentially expressed in HLNRA individuals as compared to NLNRA. Interestingly, a dose dependent increase in the number of differentially expressed genes with respect to background radiation levels was observed in HLNRA groups. The plausible explanation could be that individuals belonging to higher background dose groups (> 5.0mGy/year) have accumulated larger doses and thus stimulating many genes from different cellular and molecular pathways to maintain genome integrity. Further, detailed gene ontology analysis revealed an over-representation of genes involved in DNA damage response (DDR), DNA repair, cell cycle regulation, mRNA processing, protein transport, stress response, histone/chromatin modification, methylation, apoptosis, transcriptional regulation, signal transduction, and immune response in individuals belonging to higher dose groups (Group III and Group IV) of HLNRA. The above findings are supportive of the results obtained from DNA damage and repair study in HLNRA, where significantly lower induction of DNA damage and efficient repair of DNA strand breaks observed in individuals belonging to higher dose groups (>5.0 mGy/year) of HLNRA ,21,23.UNG and APEX2 involved in base excision repair, RAD23B and ERCC4 genes which play important role in nucleotide excision repair, PMS1 from mismatch repair pathway and DCLRE1C (Artemis), XRCC4, LIG4 genes involved in NHEJ pathway. The differentially expressed genes clearly indicate the active involvement of all the molecular pathways of DNA repair in HLNRA individuals (>5.0 mGy/year). For instance, APEX2 is the regulatory gene of BER which controls both short patch and long patch pathways. Uracil DNA glycosylase (UNG) is the major glycosylase for the removal of uracil from DNA and initiates base excision repair [DCLRE1C (Artemis), XRCC4, LIG4 indicates that DSB repair is occurring in HLNRA and perhaps mediated through artemis-dependent pathway. Additionally, differential expression of ERCC4 and RAD23B suggests possible involvement of nucleotide excision repair pathway. Activation of genes involved in DDR response , cell cycle check points , apoptosis  and signal transduction pathways such as mitogen activated protein kinase (MAPK) pathway, Jun amino-terminal kinases (JNK) pathway, p53 pathway, T cell receptor signaling, B-cell receptor signaling, Jak-stat signaling, purine metabolism, ubiquitin mediated proteolysis, and gap junction etc are suggestive of DDR to be active in this population exposed to chronic low dose radiation.Our results have also shown that, Group II (≤ 5.0mGy/year) has very few significantly differentially expressed genes as compared to Group III and Group IV of HLNRA (>5.0mGy/year). Most of these genes are involved in transcriptional regulation, protein transport and RNA processing. Interestingly, an abundance or over-representation of DDR and DNA repair genes was observed in higher dose groups (>5.0 mGy/year). These included DNA repair genes such as n repair  indicatiOur observation that more number of DNA damage or stress response genes in higher background dose groups suggests that perhaps a certain level of minimum damage is required for the cells to sense and activate DDR signaling ,59. Our DUSP1, DUSP10, AKT2, ATF2, DDIT3, DUSP5, GNAS, HIPK3, IL8 etc.) and p53 pathway  in higher dose groups of HLNRA (>5.0 mGy/year). Several known radiation responsive genes such as CDKN1A and GADD45B etc involved in cell cycle check point activation were upregulated in HLNRA individuals.The signal transduction pathways such as mitogen activated protein kinase (MAPK) pathway, Jun amino-terminal kinases (JNK) pathway and p53 pathway have been shown to play an important role in low dose radiation induced adaptive response ,61,62. O0/G1 exposed to chronic low dose exposure is limited. In the present study, representation of genes involved in modulation of immune response , cell-cell communication and gap junction  in HLNRA population suggest their possible role in radio-adaptive response.Immune response, cell to cell communication and gap junction genes and proteins are known to play important role in radio-adaptive response ,64,65. Hc-JUN, JUND, FOS, ATF2, NR4A2, Sp1, FOXO3, CREBZF, which are known to play important role in DDR signaling and repair. Genes such as cJUN, JUND, FOS, ATF2, ATF3 and CREBZF belong to activating protein 1 (AP1) family of transcription factors (TFs). AP1 family TFs are stress responsive transcription activators and has been implicated in DNA repair by its ability to regulate a large set of genes functioning in DNA repair [Another interesting finding is the transcriptional induction of a number of transcription factors such as A repair . RecentlA repair ,67. NR4AA repair ,69 IncreHIST1H1E, H3F3B, HIST1H2BC, HP1BP3, KDM6B, ING3, MYST4, SETDB2, MECP2, MLL, SUV420H1 etc) involved in epigenetic processes such as histone/chromatin modification, DNA packaging, methylation, and DNA metabolism to be over-represented. It indicates the alteration of chromatin structure is one of the important cellular response to chronic low dose exposure. Further, Fachin et al. (2009) observed several biological processes such as ubiquitin cycle (UHRF2 and PIAS1), DNA repair , cell cycle regulation/proliferation , and stress response  to be active chronic low level radiation exposures[Studies on alteration of gene expression profile in human cells in response to low dose ionizing radiation are limited. Few studies have been carried out where transcriptional response in occupational workers exposed to very low dose ionizing radiation have been reported ,50. Moraexposures. InteresDDIT3, GADD45B, JUN, PMAIP1, DUSP1, PAPD4, DUSP10, BTG1, PPIF, TSC22D2, TMEM132C, GIMAP8, METTL13, CSRNP1, KIR3DS1, TNFSF10 etc. which have potential to be used as possible low dose radiation signatures in humans.We have also identified a set of dose responsive genes In summary, our findings clearly indicated that individuals exposed to background doses of >5mGy/year have shown alteration in expression of many genes involved in important functions or pathways. These included DDR signaling, DNA repair, RNA metabolism, epigenetic changes (histone/chromatin modifications), cell cycle regulation, immune response, apoptosis etc. These may be the primary reason of not getting any detectable change at phenotype or DNA damage levels in HLNRA individuals exposed to chronic low level background dose exposures.in vivo radio-adaptive response and non-carcinogenesis in HLNRA population. These findings have tremendous implications in understanding the molecular effect of low dose radiation biology, especially the effect of low dose radiation in humans at low dose region.Sixty four dose responsive genes were identified, which are the possible radiation signatures for chronic low dose radiation exposure. In conclusion, global gene expression profile in response to natural chronic low dose radiation revealed active DDR and repair processes and their interaction with various cellular and molecular pathways in HLNRA individuals belonging to higher dose groups >5mGy/year. These findings indicated a possible threshold dose of 5mGy/year for signaling DDR and a plausible reason of observing S1 Table(DOCX)Click here for additional data file.


Glucose is the key source for most organisms to provide energy, as well as the key source for metabolites to generate building blocks in cells. The deregulation of glucose homeostasis occurs in various diseases, including the enhanced aerobic glycolysis that is observed in cancers, and insulin resistance in diabetes. Although p53 is thought to suppress tumorigenesis primarily by inducing cell cycle arrest, apoptosis, and senescence in response to stress, the non-canonical functions of p53 in cellular energy homeostasis and metabolism are also emerging as critical factors for tumor suppression. Increasing evidence suggests that p53 plays a significant role in regulating glucose homeostasis. Furthermore, the p53 family members p63 and p73, as well as gain-of-function p53 mutants, are also involved in glucose metabolism. Indeed, how this protein family regulates cellular energy levels is complicated and difficult to disentangle. This review discusses the roles of the p53 family in multiple metabolic processes, such as glycolysis, gluconeogenesis, aerobic respiration, and autophagy. We also discuss how the dysregulation of the p53 family in these processes leads to diseases such as cancer and diabetes. Elucidating the complexities of the p53 family members in glucose homeostasis will improve our understanding of these diseases. L25Q;W26S lacks the ability to induce cell cycle arrest and apoptosis triggered by DNA damage, but retains the ability to induce oncogene-induced senescence; knock-in mice expressing p53L25Q;W26S display significant tumor suppression against oncogenic rat sarcoma viral oncogene homolog (RAS)-induced tumor formation . Ph. PhTP53-ycolysis . F2,6P2 roblasts  19]. Ho. HoTP53-myocytes , suggestmyocytes . Recipromyocytes . p53-null mice showed that p53 can induce gluconeogenesis and cell cycle arrest in the liver upon starvation, although prolonged starvation causes p53-mediated apoptosis, leading to severe liver atrophy [p53-null mouse model, and showed that p53 promotes gluconeogenesis by directly inducing Pantothenate kinases-1 (PANK1), whose product catalyzes the first and rate-limiting step of coenzyme A (CoA) synthesis, which is critical for gluconeogenesis in the liver [PANK1-knockout mice display impaired gluconeogenesis after starvation [Contrary to glycolysis, which breaks down glucose and produces pyruvate, gluconeogenesis is the metabolic pathway that generates glucose from pyruvate . Emergin atrophy . Mechani atrophy . Anotherhe liver . As expearvation . Similararvation . p53 actarvation . TIGAR is induced by p53, leading to reduced F2,6P2 and decreased PFK1 activity, therefore preventing the conversion of F6P to F1,6P2 during glycolysis. The TIGAR-mediated accumulation of F6P promotes glucose flux toward PPP [TIGAR-null mice are developmentally normal, but display defects in scavenging ROS and intestinal regeneration [6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) [2. Since the kinase activity of PFKFB3 is higher than its phosphatase activity, the p53-mediated inhibition of PFKFB3 results in decreased F2,6P2, which promotes PPP and inhibits glycolysis. In contrast to TIGAR and PFKFB3, cytoplasmic p53 has been shown to inhibit PPP and NADPH production by binding to and preventing the formation of the active dimer of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP [PFKFB4 [2. However, the phosphatase activity of PFKFB4 was shown to be slightly higher than the kinase activity, and the p53-mediated inhibition of PFKFB4 resulted in increased F2,6P2, leading to reduced PPP and enhanced glycolysis [p53 is also involved in the pentose phosphate pathway (PPP), a metabolic pathway that branches off from glycolysis . For exaward PPP ,29,30,31ward PPP ,32. Consneration . Further(PFKFB3) . PFKFB3  the PPP . p21-act the PPP . It has  [PFKFB4 . PFKFB4 ycolysis ,38. The Pyruvate produced by glycolysis can enter the TCA cycle, which is coupled to oxidative phosphorylation to generate ATP in mitochondria. ATP is generated by the ATP synthase FoF1 complex, through using a proton gradient produced by the electron transport chain at the inner mitochondrial membrane . Mitochop53-knockout HCT116 cells compared with p53 wild-type HCT116 cells [cytochrome c oxidase assembly protein (SCO2), which is required for complex IV assembly [p53-null cells is rescued by reintroducing SCO2 expression to physiological levels [p53 is thought to promote oxidative phosphorylation and maintain mitochondrial integrity, either directly or indirectly . The act16 cells . In addi16 cells . Mechaniassembly , and thel levels ,42.p53R2 (RRM2B), which encodes p53-controlled ribonucleotide reductase, cause the depletion of mitochondrial DNA (mtDNA) [mitochondrial transcription factor A (TFAM) in order to increase the content of mtDNA [apoptosis-inducing factor (AIF) [AIF-deficient cells exhibit reduced complex I and III expression in the mitochondria, and reduced AIF expression leads to reduced oxidative phosphorylation in mice [ferredoxin reductase (FDXR) [mitochondria-eating protein (MIEAP), which promotes the elimination of oxidized proteins in mitochondria, as well as damaged mitochondria themselves, to support mitochondrial activity [Several other p53 target genes potentially promote oxidative phosphorylation. For instance, mutations in the p53 target gene  (mtDNA) ,44. Furt (mtDNA) ,46. p53 of mtDNA . p53 alsor (AIF) . AIF-def in mice . p53 alse (FDXR) , which ie (FDXR) . p53 alsactivity .pyruvate dehydrogenase kinase 2 (PDK2) [Parkin. Parkin increases the protein expression of PDHA1, which is a component of PDC, leading to the increased mitochondrial respiration that indirectly limits glycolysis [p53 also regulates the TCA cycle. p53 represses the promoter of ycolysis . Malic eycolysis . p53 alsycolysis ,57.p53 promotes oxidative phosphorylation not only in the nucleus, functioning as a transcription factor, but also in the mitochondria. Besides the well-known function of p53 in enhancing apoptosis by translocating to the mitochondrial membrane , the conperoxisome proliferator-activated receptor gamma coactivator 1 alpha and beta (PGC-1α and PGC-1β), which are positive master regulators of mitochondrial biogenesis [p53-null cells is easily compensated by enhancing glycolysis to maintain the cellular ATP amounts [Although there is substantial evidence that suggests p53 enhances oxidative phosphorylation and maintains mitochondrial integrity, some reports challenge these concepts. For example, telomerase knockout mice show severe telomere dysfunction and p53 activation, leading to aging phenotypes, mitochondrial dysfunction, and reduced oxidative phosphorylation . These cogenesis . These d amounts . The phyOverall, p53 seems to have contradictory roles in glucose metabolism. p53 inhibits glycolysis, which prevents the production of ATP and precursors for biosynthesis, but it also promotes oxidative phosphorylation and the TCA cycle, which produce ATP and precursors for biosynthesis, respectively. In addition, p53 enhances PPP flux and reduces ROS by inducing TIGAR. p53 is also known to reduce ROS by inducing GLUT9, which transports uric acid that is the one of the strongest antioxidants in cells . Howeverp53 controls glycolysis and mitochondrial metabolism to maintain glucose homeostasis in cells. If glucose is not available, cells can utilize other fuels, such as glutamine and lipid as energy sources, but the surrounding cellular environments may limit their availability. To compensate for this limitation, cells break down and utilize their own components as energy sources, such as proteins and organelles. This process is called “autophagy”, and consists of highly controlled catabolic steps that digest cytoplasmic components and organelles in lysosomes. Autophagy is also a tumor suppressive mechanism that removes unfolded proteins, damaged cellular components, and damaged organelles to maintain cellular homeostasis ,69. As aThe AMP-activated protein kinase (AMPK) plays an essential role in cellular energy homeostasis, and is activated by decreased levels of intracellular ATP. AMPK inhibits mechanistic target of rapamycin (mTOR) kinase, which is a critical repressor of autophagy. It was reported that p53 enhances autophagy by inhibiting mTOR pathways via the activation of AMPK . The gendamage-regulated autophagy modulator (DRAM), a lysosomal protein [death-associated protein kinase 1 (DAPK1), a kinase acting in the early steps of autophagy [unc-51-like kinase 1 and 2 (ULK1 and ULK2), autophagy-initiation kinases downstream of mTOR [BCL2 associated agonist of cell death (BAD) [BCL-2-associated X protein (BAX) [PUMA [BCL2 interacting protein 3 (BNIP3) [transglutaminase 2 (TGM2) to prevent oncogenic transformation [autophagy protein 5 (ATG5), ATG7, and UV radiation resistance associated gene (UVRAG) [p53 also promotes autophagy by inducing various autophagy-related genes, including  protein ; death-autophagy ,80; unc- of mTOR ; and sevth (BAD) , BCL-2-ain (BAX) , PUMA [8X) [PUMA , and BCL (BNIP3) ,85. In aormation . p53 alsormation ,88. Protormation . p53-medormation . Full-leormation . TAp73 i (UVRAG) . Consistent with the ability of p53 to induce autophagy, alternative reading frame protein (ARF), an upstream positive regulator of p53, also induces autophagy. Although full-length ARF is localized in both the nucleolus and mitochondria ,95,96,97cep-1 in C. elegans promotes autophagy, leading to an increase of its lifespan [TIGAR inhibits autophagy by decreasing ROS upon nutrient deprivation [Although many reports suggest that p53 promotes autophagy, several reports have challenged this concept. The inhibition of p53 can promote autophagy in human, mouse, and nematode cells, and the repression of autophagy is mediated by cytoplasmic, but not nuclear, p53 . The p53lifespan . In addirivation , suggestrivation . ATG7, arivation . These fDiabetes is defined as the dysregulation of glucose homeostasis, resulting in hyperglycemia. There are two types of diabetes, type 1 and type 2. Type 1 diabetes develops primarily in children and young adults, and is caused by an inability to produce enough insulin due to β-cell death in the pancreas, which is triggered mainly by autoimmune diseases. Type 2 diabetes generally develops in adults, and is caused by a failure to properly respond to insulin, called insulin resistance, and/or by impaired insulin secretion due to β-cell dysfunction/loss. Diabetes can increase the risk of cancer, and the common diabetes drug metformin is an anti-cancer drug . As mentObesity caused by a high-fat diet increases metabolic stress and ROS, which can activate p53 and thus trigger adipose tissue inflammation, senescence, and insulin resistance in type 2 diabetes. Specifically, p53 induces semaphorin 3E (Sema3E), which promotes macrophages infiltration and the inflammation of adipose tissue, leading to insulin resistance ,108. p53R172P that lacks apoptotic function but retains the ability to induce senescence can rescue lymphomagenesis, but also induces severe diabetes accompanied with the senescence of pancreatic β cells [p53 gene encoding Δ40p53 exhibit increased p53 activity in β cells, and develop diabetes with reduced pancreatic β cell mass [ARF-Binding Protein 1 (ARF-BP1), an E3 ligase for p53, display p53 stabilization and diabetic phenotypes [Impaired insulin secretion due to the dysfunction/loss of pancreatic β cells significantly contributes to the development of both type 1 and type 2 diabetes. Many reports suggest that the induction of p53 in β cells triggered by metabolic stress enhances β cell failure and diabetes. For example, NADPH oxidase 2 (NOX2)-derived oxidative stress induces p53 and the apoptosis of β cell lines . Co-trea β cells . Transgeell mass . Mice wienotypes . The β cenotypes . Cytosolenotypes . Hyperglenotypes . miR-200enotypes . Finallyenotypes . These denotypes , and alsp53-knockout mice that are fed a high fat diet exhibit marked obesity and hepatic lipid accumulation compared with wild-type mice [p53 gene, “super p53” mice, exhibit improved glucose tolerance [Although many reports suggest that p53 activity can contribute to diabetes, there are several studies showing that p53 can inhibit diabetes. For example, ype mice . The actype mice . Knock-iype mice . Mice caolerance . These colerance . Consistolerance .p53 is mutated in about half of the existing cancers. Many mutations are missense mutations in the DNA binding domain that not only abrogate DNA binding, but also act as dominant negatives that inhibit wild-type p53. Some mutant p53 proteins also show gain-of-functions to promote malignancy ,132, sucG103S/E256G, which retains DNA binding ability, is overexpressed in several liver cancer cell lines, and binds and activates the promoter of hexokinase 2 (HK2) [Several reports showed that mutant p53 may have a role in glucose metabolism in cancers. When ample glucose is available, mutant p53 stimulates glycolysis by promoting GLUT1 translocation to the plasma membrane, which supports energy production and provides building blocks in cancer . Mutant  2 (HK2) . HK2 is  2 (HK2) . It was  2 (HK2) . However 2 (HK2) , and the 2 (HK2) . Therefop53-knockout mice, p63- and p73-knockout mice exhibit severe developmental abnormalities, suggesting substantial functional diversity among the homologues [TAp63-null mice exhibit insulin resistance, obesity, and glucose intolerance, all of which are associated with defects in glucose uptake in cells [SIRT1, AMPKα2, and liver kinase B1 (LKB1), resulting in increased fatty acid synthesis and decreased fatty acid oxidation [TAp73-null mice show improved insulin sensitivity and glucose tolerance in mice fed a high-fat diet [p63  and p73 mologues . Althougmologues ,147,148.in cells . TAp63 ixidation . In contfat diet . Althougfat diet ,152. p53-null mice [TAp73-null mice show decreased oxygen consumption and mitochondrial complex IV activity [On the other hand, there are some similarities between p53, TAp63, and TAp73. Similar to p53, TAp63 and TAp73 inhibit glycolysis, but by different mechanisms. TAp63 and TAp73 can induce islet amyloid polypeptide (IAPP), which is a peptide hormone from pancreatic β cells that can inhibit glycolysis through the inhibition of HK2 . Furtherull mice . Contrarull mice ,155. ΔNpull mice  or repreull mice ,158. Simull mice . TAp73-nactivity , suggestactivity , and TApactivity . Taken tGlucose is the central energy source for most organisms, from single cell organisms such as bacteria and yeast, to multicellular organisms such as worms and humans. During the evolution from sea anemones to Homo sapiens, p53 family members acquired many cellular functions , includip53 induces TIGAR to promote PPP and generate GSH for reducing ROS, thus protecting cells from the genomic instability that is caused by oxidative stress. p53 induces autophagy to protect cells from damaged organelles and proteins. These p53 functions can prevent the initiation of tumor development by maintaining genomic stability. However, these functions can also promote cancer. The antioxidant function of p53 can protect cancers from apoptosis that is triggered by oxidative stress, drugs, and oncogenic activation. Autophagy also supports cancer survival in response to nutrient stress and chemotherapeutic drugs. Therefore, even the same functions of p53, such as autophagy and antioxidant functions, can lead to different outcomes for the human body. These complexities must be kept in mind in order to understand the effects of p53, depending on the stage of cancer and the tissues from which they are derived.


IgG4-related disease (IgG4-RD) is recognized as an immune-mediated condition with pathology features of lymphoplasmacytic infiltrate, storiform fibrosis, and obliterative phlebitis, accompanied with or without elevated serum IgG4 concentrations. However, few of pulmonary artery IgG4-RD causing pulmonary hypertension (PH) was reported.The medical records of 3 patients with pulmonary artery IgG4-RD inducing PH were analyzed retrospectively.Imaging findings demonstrated that the lesions of 3 patients located in pulmonary artery, which were initially diagnosed as pulmonary thrombus or malignant tumor. Computed tomography pulmonary angiography (CTPA), ultrasonic cardiogram, and positron emission tomography/computed tomography (PET/CT) didn’t support the diagnosis of pulmonary thrombus or malignant tumor. Right heart catheterization (RHC) showed definite PH. Biopsy by right heart catheterization in 2 patients or pneumonectomy in 1 patient confirmed the diagnosis as IgG4-RD. Treated with glucocorticoids and cyclophosphamide or rituximab, 2 patients’ IgG4 concentrations declined sharply and the lesions shrunk gradually. Another patient treated with glucocorticoids died of heart failure.IgG4-RD involved pulmonary artery causing PH was rare. A high index of awareness of this disease is required for early diagnosis and treatment. PET/CT might be a valuable approach to distinguish pulmonary artery IgG4-RD from pulmonary thrombus and malignant tumor. However, published literature on pulmonary artery IgG4-RD causing pulmonary hypertension (PH) is limited. In this study, we aim to improve awareness of the clinical and imaging manifestations of pulmonary artery IgG4-RD causing PH to avoid a delay in diagnosis or misdiagnosis.IgG4-related disease (IgG4-RD), involved single or multiple organs, is known as an immune-mediated disorder with typical histopathologic features, with or without elevated serum IgG4.1.1Three patients were diagnosed as pulmonary artery IgG4-RD which initially was misdiagnosed as pulmonary thrombus or malignant tumor. The diagnosis of this disease was based on biopsy by right heart catheterizationin in 2 patients and pneumonectomy in 1 patient. We retrospectively analyzed these cases by reviewing these patients’ medical records, laboratory findings, radiological findings and pathological changes, therapeutical strategies, and following times. The Ethics Board in our hospital  approved our study and all patients provided written informed consent.2The major clinical and auxiliary examination data of 3 patients (men) with pulmonary artery IgG4-RD were shown in Table Our initial management for these patients was to use glucocorticoids and cyclophosphamide for patient 3, glucocorticoids for patient 2, and glucocorticoids and rituximab for patient 1 as described in Table Before treatment, expression levels of serum cytokines in patient 1 and 3, including interleukin-4, interleukin-5, interleukin-10, interleukin-13, transforming growth factor-β (TGF-β), immunoglobulin E, IgG4, and endothelin were substantially higher than that in healthy person (except for IL-5 in patient 3 and IL-13 in patient 1). After treatment, the serum IgG4 and ILs returned to normal. Before treatment, flow cytometry showed that CD3+/CD4+(Th) and CD4+CD25+/CD127+(Treg) were obviously high, and after treatment, these lymphocytes reduced obviously.3Pulmonary artery IgG4-RD inducing PH is a rare form of IgG4-RD and might become another unnegligible cause of PH. IgG4-RD can involve virtually any organ. Besides the most frequently involved pancreas, biliary trees, and kidneys, it attacks large to medium-sized arteries extensively, such as abdominal aorta, thoracic aorta, iliac artery, coronary artery, renal artery. IgG4-RD involving pulmonary artery is rarely reported in published literature. From another angle, the lack of familiarity with its clinical and imaging manifestations may limit its diagnostic rate.In this study, these 3 patients’ symptoms and physical examination were unremarkable, and all of them have no multi-organ involvement, which can be a vital clue for differential diagnosis. Besides that, elevated serum IgG4 concentrations can be a valuable hint leading us to a quick diagnosis. However, the serum IgG4 level is not a routine item of blood tests in current clinical practices. Furthermore, as a general conclusion, pathological diagnosis is the gold standard for diagnosing IgG4-RD, but it may be difficult to obtain biopsy specimen in IgG4-RD with pulmonary artery involvement in some cases, and this main method for definite diagnosis make little sense in early diagnostic procedures. So the delay in diagnosis or even misdiagnosis is hardly avoidable. Back to this study, we find that imaging examination played a crucial role in the diagnosis process, and may be an alternative method for the diagnosis.–15 In our study, PET/CT is also considered as an effective method to distinguish IgG4-RD from malignant tumors and pulmonary thrombus. The malignant tumor showed higher SUV intake while pulmonary thrombus showed no SUV intake. Proven useful in diagnosing the pulmonary artery IgG4-RD, these image characteristics of PET/CT should be explored and validated in further study and may be updated into the consensus diagnostic criteria for IgG4-RD in the future.CTPA and ultrasonic cardiogram are important diagnostic approaches to pulmonary vascular lesion. However, it is difficult to identify pulmonary artery IgG4-related lesions from malignant tumors and pulmonary thrombus. Recently, several case reports have indicated PET/CT scan might be a valuable approach to diagnosis of some IgG4-RD. The guideline didn’t involve pulmonary artery IgG4-RD causing PH. In our study, we show 3 patients with pulmonary artery IgG4-RD causing PH, which presents one new contributory factor of the cause of PH.PH from the 5th World Symposium at Nice, France is divided to 5 classifications, including pulmonary arterial hypertension, pulmonary hypertension due to left heart disease, pulmonary hypertension due to lung diseases and/or hypoxemia, chronic thromboembolic pulmonary hypertension and pulmonary hypertension with unclear and/or multifactorial mechanisms.,18 A Japanese study showed complete remissions in only 61% of IgG4-RD patients in 1 year following up. The response to glucocorticoids is different according to affected organs and the degree of fibrosis. Retroperitoneal fibrosis, sclerosing mesenteritis, and fibrosing mediastinitis are less effective to glucocorticoids. In our study, early treatment of glucocoricoids combined application conventional steroid-sparing agents or rituximab lead to good clinical outcomes, which may generate clues for further medication study. In addition, the serum IgG4 and cytokines concentration may become some good biomarkers for IgG4-RD improvement, which should be explored and validated in further study. The radiological assessment to pulmonary artery lesions by CTPA, MRI, and ultrasonic cardiogram is also an appropriate evaluation method for disease process.The glucocorticoids is the first-line, standard approach for most patients with IgG4-RD.,10,20–24 some immune cells and cytokines were measured in our study, and it suggested that the lesion of pulmonary artery was formed in multiple processes. To be specific, the changes of Th2 cell, Treg cell, and cytokines support the probable mechanisms mentioned in previous studies (Fig.  The presentation of ultrasonic cardiogram also approved this probable IgG4-RD causing PH mechanism (Fig. With the progression of diagnostic techniques, this disease has been recognized gradually, but the pathogenesis is not fully clear. According to previous study,ies Fig. .[2] The ism Fig. .In conclusion, to the best of our knowledge, these patients with IgG4-RD presenting pulmonary artery obstruction causing PH is rarely reported in previous studies. These diseases might be delayed or mistakenly diagnosed as pulmonary thrombus or malignant tumor. Therefore, a high index of awareness is required for the early diagnosis and treatment of this disease, which can prevent disabling tissue fibrosis, severe organ failure, and even death. The image characteristics of PET/CT might be valuable clues for the early diagnostic accuracy of this disease.Conceptualization: Yong Wang.Data curation: Hui Deng, Sheng Zhao, Yunlong Yue, Yong Liu, Yali Xu, Jin Qian, Xiaorong Ma, Peiliang Gao, Xiaoyan Yao, Xin Jiang, Xiqi Xu, Zhicheng Jing, Yong Wang, Lei Pan.Formal analysis: Xinying Xue.Funding acquisition: Xinying Xue.Investigation: Sheng Zhao, Zhicheng Jing, Lei Pan.Methodology: Sheng Zhao, Yong Liu, Zhicheng Jing, Lei Pan.Project administration: Yunlong Yue, Yong Liu.Resources: Yunlong Yue, Yong Liu, Yali Xu, Jin Qian, Xiaorong Ma, Xiaoyan Yao, Xin Jiang, Yong Wang.Software: Yunlong Yue, Yong Liu.Supervision: Zhicheng Jing, Yong Wang, Lei Pan.Writing – original draft: Peiliang Gao, Lei Pan, Xinying Xue.Writing – review & editing: Sheng Zhao, Lei Pan, Xinying Xue.