The epilepsies are a broad group of conditions characterized by repeated seizures, and together are one of the most common neurological disorders. Additionally, epilepsy is comorbid with many neurological disorders, including lysosomal storage diseases, syndromic intellectual disability, and autism spectrum disorder. Despite the prevalence, treatments are still unsatisfactory: approximately 30% of epileptic patients do not adequately respond to existing therapeutics, which primarily target ion channels. Therefore, new therapeutic approaches are needed. Disturbed proteostasis is an emerging mechanism in epilepsy, with profound effects on neuronal health and function. Proteostasis, the dynamic balance of protein synthesis and degradation, can be directly disrupted by epilepsy-associated mutations in various components of the ubiquitin-proteasome system (UPS), or impairments can be secondary to seizure activity or misfolded proteins. Endoplasmic reticulum (ER) stress can arise from failed proteostasis and result in neuronal death. In light of this, several treatment modalities that modify components of proteostasis have shown promise in the management of neurological disorders. These include chemical chaperones to assist proper folding of proteins, inhibitors of overly active protein degradation, and enhancers of endogenous proteolytic pathways, such as the UPS. This review summarizes recent work on the pathomechanisms of abnormal protein folding and degradation in epilepsy, as well as treatment developments targeting this area. Proteins are crucial to nearly every function in a cell, including both the general functions found in nearly every cell, such as energy production and DNA replication, as well as neuron-specific processes, such as maintenance of the membrane potential and synaptic vesicle release. Proteins must be folded properly in order to fulfill their functions, but protein folding is incredibly complex, and, thus, misfolding is common . Up to oA large proportion of newly synthesized proteins are folded in the endoplasmic reticulum (ER). The ER is equipped to facilitate proper folding, as its oxidizing environment promotes disulfide bond formation, and it contains many proteins that act as chaperones to further assist folding and act as quality control . ComplexA receptor (GABR) subunits are one example related to epilepsy, but this trend applies to other membrane proteins as well, such as cystic fibrosis transmembrane conductance regulator and peripheral myelin protein [Proteostasis is a delicate balance between protein synthesis and degradation, and disruptions of proteostasis are implicated in numerous diseases, including neurological conditions such as epilepsy. Epilepsy is a neurological disorder characterized by recurrent, unprovoked seizures and involves imbalanced inhibitory and excitatory neuronal circuitry. Neuronal function depends on a vast array of membrane proteins, such as voltage gated ion channels and neurotransmitter receptors and transporters, and membrane proteins in particular, are prone to rapid degradation, as their complex structure results in inefficient folding and assembly . The GAB protein .As mentioned above, GABR subunits are inefficiently trafficked to the cell surface, due to their complex structures, as well as the necessity of assembling with other subunits to form a complete pentameric receptor ,3,4. It When a protein cannot be folded properly, it is degraded. Protein degradation is a crucial function in cells, to properly balance protein synthesis and to eliminate misfolded or mutated proteins ,10. TherUPS mediated protein degradation is a constant and ongoing process that has a profound impact on cell survival and normal functioning. The UPS is critical in maintaining appropriate levels of regulatory proteins, such as those involved in the cell cycle, metabolism, and apoptosis, as well as in degrading misfolded proteins ,12,13. DIn the case of misfolded proteins, the degradation process can begin in the ER with activation of the unfolded protein response (UPR). The UPR is activated in response to ER stress caused by factors such as accumulation of misfolded proteins or oxidative stress ,17,18. TOne of the results of the UPR is enhanced protein degradation, through autophagy or through ER associated degradation (ERAD) ,18. ERADWhen proteins are targeted by ERAD, they are passed to the 26S proteasome. There are multiple types of proteasomes, but the 26S proteasome is found in all cell types and degrades damaged proteins . The 26SThe first step in proteasome-mediated protein degradation is ubiquitination . The proOnce ubiquitinated, the substrate is recognized by the proteasome, either through direct binding of the polyubiquitin chain to 19S subunits or though adaptor proteins that are associated with the proteasome . In ordeEfficient degradation of proteins is especially important for neurons, which, due to their post-mitotic state, have a more difficult time clearing toxic proteins . When pr2+ influx through NMDA receptors activating CaMKII, which phosphorylates the 19S subunit Rpt6 [In order to ensure proper neurotransmission, there is complex feedback between the UPS and synaptic signaling: the UPS both regulates and is regulated by neuronal activity. Induced synaptic activity in cultured neurons results in increased ubiquitination in postsynaptic densities (PSDs), and elevated UPS activity was observed within minutes in dendrites ,25. ConvNMDA receptors, in particular, have been shown to have effects on the UPS. Bingol and Shuman (2006) showed that, in cultured hippocampal neurons, acute NMDA receptor activation resulted in a greater number of proteasomes in dendritic spines, primarily due to a six-fold decrease in the rate at which proteasomes exited the spines, but also aided by a subtler increase in the rate at which proteasomes were trafficked into the spines . This chInterestingly, a decrease in ubiquitinated proteins, which was accompanied by depressed 26S proteasome activity, was found in a study by Tai et al. 2010), after inducing long-term synaptic plasticity in cultured hippocampal neurons via NMDA exposure . The dec, after iWhile the UPS is influenced by neuronal activity, the UPS can also exert control over synaptic signaling, which has clear applicability to diseases of dysregulated synaptic transmission, such as epilepsy. The turnover of several crucial synaptic proteins has been demonstrated to be regulated by the UPS, including PSD-95, Shank, GKAP, AKAP, SPAR, and GRIP1 ,25. ActiFbxo2−/− mice were shown to have altered NMDAR expression, with elevated GluN1 and GluN2A [Various parts of the UPS, besides the proteasome itself, can influence signaling. FBXO2 is a substrate-recognizing component of an E3 ligase complex known to regulate NMDARs, and d GluN2A . These md GluN2A . This drd GluN2A . The Glud GluN2A . Nedd4 ad GluN2A . Additiod GluN2A . In combCa2 family are responsible for the medium afterhyperpolarizing potentials (mAHP) that play a role in the regulation of neuronal excitability [Ca2.2 was transcriptionally downregulated; likewise, in hippocampal slices incubated with the GABA receptor blocker gabazine, KCa2.2 was reduced [Ca2.2 is a substrate of the E3 ligase UBE3A, so the decreased protein level was hypothesized to be due to UPS proteolysis [Proper proteasome balance is also necessary for other ion channels. The calcium-activated potassium channels in the Ktability . In chro reduced . KCa2.2 teolysis . Indeed,teolysis .FBXL20) [Scrapper−/− mice, there is a greater density of synaptic vesicles in the active zone [Scrapper−/− mice have reduced paired-pulse facilitation (PPF) and an increased mEPSC frequency [Scrapper−/− mice have elevated glutamate and GABA concentrations throughout the brain [Another E3 ligase that regulates components of neurotransmission is SCRAPPER (encoded by FBXL20) ,40,41. SFBXL20) ,41. RIM1FBXL20) ,41. Unsuive zone . Brain srequency . AdditioThe epilepsies are a broad group of neurological disorders characterized by repeated unprovoked seizures, and together are one of the most common neurological disorders ,42. ApprER stress is seen in multiple animal models of epilepsy, as well as human brain tissue. In rats treated with kainic acid (KA) to induce seizures, multiple signs of ER stress were observed . After KThis induction of ER stress is detrimental, as suggested by the electron micrographs of rat hippocampal tissue after KA administration, revealing swollen mitochondria and swollen rough ER, which is indicative of neuronal injury . This waGABRG2(Q390X), results in intracellular accumulation of misfolded protein [+/Q390XGabrg2 mice [+/Q390XGabrg2 mice, a decrease in the number of cells positive for NeuN  is observed in the somatosensory cortex [GABRG2 truncation mutations, W429X and W461X, have been shown to mimic the effect of Q390X on ER stress, as expression of these mutant γ2 resulted in more GADD153, relative to wildtype γ2, and the induction of GADD153 corresponded to the amount of cDNA transfected into the cells [Genetic epilepsy can be directly linked to ER stress, independent of seizures. A Dravet syndrome-associated mutation,  protein ,44. This protein ,44. In t90X mice . Elevate90X mice . Additio90X mice . Increasy cortex . Finallyy cortex . Furtherhe cells ,44.Additional evidence for a connection between genetic epilepsy and ER stress comes from mutations in RNF13 . RNF13 iThe UPS is known to be impaired by the protein aggregates of neurodegenerative disorders, as well as by brain ischemia and traumatic brain injury ,10,14. SIn two mouse models of induced status epilepticus (SE) (kainic acid and pilocarpine administration models), there was an accumulation of polyubiquitinated proteins in the hippocampus . This waFBXL20, in mice [FBXL20 and epilepsy [The E3 ligase Nedd4-2, which regulates ion channels, was also downregulated in both the acute and chronic stages, but not in the latent period in the pilocarpine-administered mouse model . Pilocar in mice . Similarepilepsy . E3 ligaepilepsy .In line with the correlation of dampened UPS activity in areas with the least cell death, that study found proteasomal inhibition to be neuroprotective . When cuA complementary study found that inhibition of a DUB exasperated neuronal injury after KA-evoked SE in mice . DownregGenetic epilepsy has an especially complex relationship to the UPS and proteostasis. In addition to the mechanisms discussed in the prior three sections—whereby elevated synaptic transmission can impact the UPS and therefore profoundly change the expression profile of synaptic proteins, and seizure activity can cause proapoptotic ER stress—the mutated proteins can exert their own effect on the UPS. Many mutations directly dysregulate components of the UPS, such as E3 ligases, while other epilepsy-causing mutations alter the proteostatic network with their rate of degradation .GABRA1, GABRG2, and SLC6A1 , γ2(R82Q) and γ2(P83S), are also retained with the ER . When muSLC6A1, encoding GABA transporter 1 (GAT-1), is associated with ER retention of epilepsy-causing mutations. Several studies from the Kang lab found that the GAT-1 mutations manifesting with clinically diverse symptoms have similar cellular phenotypes [Another gene in the GABAergic signaling pathway, enotypes ,53,54. Tenotypes ,53,54. Aenotypes ,53,54. Ienotypes . Furtherenotypes .A number of epilepsies and neurological disorders with comorbid seizures are due to genetic abnormalities that affect E3 ligases . As E3 lEPM2A or EPM2B, although, recently, mutations in the related gene PRMD8 have been implicated in LD [EPM2A encodes the phosphatase laforin, and EPM2B  encodes malin, an E3 ubiquitin ligase. Lafora disease is characterized by the presence of Lafora bodies, intracellular accumulations of insoluble glycogen-like carbohydrates, in the brain and other tissues [Lafora disease (LD) is a fatal, neurodegenerative disease belonging to the group of progressive myoclonic epilepsies ,58,59,60ed in LD ,56,57,58 tissues ,56,58,59 tissues ,56,59.The exact mechanisms by which laforin or malin mutations result in epilepsy and neurodegeneration are unknown, but hypotheses include altered energy homeostasis and impairments of the UPS ,57,58,59Epmb−/− mice with no malin, elevations in ubiquinitated proteins is also seen, suggesting this phenotype is due to the loss of E3 ligase function, and not due to the presence of mutant protein [Involvement of proteostasis pathways in LD is supported by the finding that Lafora bodies contain 6–28% protein, in addition to polyglusans ,60. The UBE3A) result in the neurodevelopment disorder Angelman syndrome, while duplication of the maternal allele of the chromosomal region that spans the UBE3A gene, 15q11.2–q13.1, results in another neurodevelopmental disorder, Duplication 15q syndrome (Dup15q) [Deletion or loss-of-function mutations in maternally-imprinted Ubiquitin Protein Ligase E3A ((Dup15q) ,61. Both(Dup15q) . UBE3A i(Dup15q) ,61. The (Dup15q) . For Ang(Dup15q) ,61.Cullin 3 (CUL3) is a scaffold for the E3 ligase class Cullin-RING ligases (CRL) ,74. Two Mutations in another CUL3-associated protein, rho-related BTB domain-containing protein 2 (RHOBTB2), have been found in several patients with epileptic encephalopathy ,75. RHOBAdditionally, a CUL3 substrate, potassium channel tetramerization domain-containing protein 7 (KCTD7), has been implicated in epilepsy . Two sibHECW2) in patients with severe epilepsy, intellectual disability, and hypotonia [Hecw2 knockout mice do not display CNS abnormalities, zebrafish with knockdown of the HECW2 ortholog hecw2a had severe morphological abnormalities of the brain and spinal cord, as well as cell death [Multiple reports have identified mutations in the HECT, C2, and WW domain containing E3 ubiquitin protein ligase 2 gene  [Fmr1 knockout mouse has neuronal hyperexcitability [Fmr1−/− neurons failed to show the ubiquitination of the AMPA receptor subunits GluA1 and GluA2 that was seen in wildtype neurons [Fmr1−/− neurons [Fmr1−/− mouse have shown an absence of E3 ligase Mdm2-mediated ubiquitination of PSD95, leading to an excess of excitatory synapses [Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and is caused by loss of function of the n (FMRP) . In additability ,62. This neurons . Other ssynapses .Alterations to the ubiquitination process beyond the substrate recognition and ubiquitination steps mediated by E3 ligases can also result in seizures . DeubiquCHRNA7  and OTUD7A (ovarian tumor deubiquitinase 7A) [Otud7a, a DUB, in mice replicates much of the human phenotype and, thus, is believed to be the causative gene of 15q13.3 microdeletion syndrome [15q13.3 microdeletion syndrome is characterized by a wide range of neuropsychiatric phenotypes, including epilepsy, developmental delay, and/or autism spectrum disorder ,63. The nase 7A) ,63. Knocsyndrome .OTUD7A in epilepsy came when a patient with epileptic encephalopathy and severe developmental delay was found to have a homozygous missense mutation in OTUD7A, L233F [Further support for the involvement of A, L233F . FibroblA, L233F . The patA, L233F . The 19SA, L233F . AdditioA, L233F .OTUD7A mutation, a homozygous de novo frameshift E375Dfs*11, in addition to a 15q13.3 microdeletion spanning part of OTUD7A [Caenorhabditis elegans model was created with a frameshift corresponding to the human mutation, in the OTUD7A homolog otub-2 [otub-2(ta112) worms showed impaired locomotion, supporting the link between patient hypotonia and the E375Dfs*11 OTUD7A mutation [In 2021, another patient with epilepsy and developmental delay, as well as severe hypotonia, was reported to have a f OTUD7A . To inveg otub-2 . The hommutation .Drosophila, loss-of-function mutations in the ortholog of USP9X, faf, were shown to confer resistance to seizures when crossed with the prickle model of epilepsy, splepk/+ [splepk flies with the USP9X inhibitor Degrasyn/WP1130 was also seizure-protective [Drosophila mutant alleles generating no protein product, while the human mutations do produce mutant protein, implicating a possible dominant-negative effect [Another deubiquitinase, Ubiquitin Specific Peptidase 9 X-Linked (USP9X), has been connected to genetic epilepsy. A patient with epileptic encephalopathy was found to have a de novo missense mutation (S1012P) at a conserved residue, and another patient with infantile spasms was found to have the missense mutations G1890E . Interespksple/+ . In the otective . The aute effect .In addition to the seizure-related disorders with direct connections to the UPS discussed above, a number of other epilepsies have misfolded proteins that are prone to aggregation . While aSyntaxin binding protein 1 (STXBP1), or Munc18-1, is part of the machinery involved in the release of synaptic vesicles ,65. MutaAlthough the aggregates do not appear to be cytotoxic, they do represent a dominant-negative feature of mutant STXBP1: wildtype STXBP1 forms dimers, and the mutant proteins retain the ability to assemble with the wildtype protein ,65. ConsGABRG2 cause epilepsy, including GABRG2(Q390X) found in Dravet syndrome. The truncation of the γ2 protein results in a hydrophilic domain in place of the hydrophobic TM4, and this hydrophilic domain fails to stably insert into the membrane [Several mutations in membrane . As a remembrane . Wildtypmembrane ,66. γ2 is amongst the most severe epilepsies, other disease-associated mutations in GABR subunits share some of the molecular pathomechanisms. Several other truncation mutations in γ2 have been studied, and many form dimers and multimers, although not to the extent of γ2(Q390X). For instance, while γ2(R136X) is not prone to aggregation, W429X, which is linked to febrile seizures and generalized tonic-clonic seizures, does display moderate dimerization tendencies [Although the clinical phenotype of Dravet syndrome connected to ndencies ,66. Yet,ndencies . Non-trundencies . All of ndencies .Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a neurodegenerative disorder with a range of symptom severity ,68,69,71Progressive myoclonus epilepsy type 1 (EPM1), also known as Unverricht-Lundborg disease, is a neurodegenerative disease caused by mutations in Stefin B (StB) ,82. StB,KCNQ2 encodes the KV7.2 subunit of the neuronal KV7 potassium channel, which is responsible for the M-current that suppresses neuronal excitability [V7 channel cause epilepsies of varying severity [V7.2(M518V) mutation, identified in a patient with early-onset epileptic encephalopathy, was found to have poorer trafficking to the axon initial segment (AIS) and greater ubiquitination compared to wildtype KV7.2 protein [V7.2(M518V) and resulted in aggregation of the mutant protein, demonstrating the involvement of the UPS in this disease [V7.3 also stabilized the mutant KV7.2 and enhanced aggregation [V7.2(M518V) caused cell shrinkage and nuclei condensation (features of apoptosis) in HEK293T cells [V7.2(M518V) also increased cell death [tability . Mutatioseverity . The KV7 protein . Treatme disease . Interesregation . The int3T cells . In tranll death . These ill death . A lack GRIN1-related neurodevelopmental disorder is characterized predominantly by intellectual disability, epilepsy, and other neurological symptoms like hypotonia [GRIN1 do not generally cause clinical phenotypes [ypotonia ,84. Missenotypes ,84. InstSPTAN1 encodes non-erythrocyte αII spectrin, and several patients with epileptic encephalopathies have been reported with SPTAN1 mutations [utations . The speutations . Imagingutations . Interesutations .GABRA1 and the mutant neuroserpin responsible for FENIB [A few existing, common AEDs have been found to have secondary effects on proteostasis and the UPS, in addition to their manipulation of neurotransmission . Zonisamor FENIB ,3,23,68.The AED valproic acid (VPA) is one of the most commonly used AEDs and has a complex mechanism of action . In regaCarbamazepine (CBZ) is an AED and mood stabilizer that also has been shown to affect protein degradation. CBZ has been shown in vitro to reduce both mutant huntingtin and mutant α-synuclein, via autophagy enhancement . The livTo relieve the burden on the UPS and restore proteostasis, chaperones that aid in protein folding may be of use in seizure-associated diseases, such as those discussed above . MoleculPBA is sold as the FDA approved drug Buphenyl for the treatment of urea cycle disorders, but has shown promise for a variety of neurological disorders with misfolded proteins . PBA hasC. elegans expressing the various mutants, and localization of the protein within the ventral nerve cord was restored [For aggregation-prone STXBP1 mutants, PBA not only increased the total levels of STXBP1, including wildtype, but it also decreased the fraction of detergent-insoluble protein, indicating that less of it was in aggregates and, thus, even more STXBP1 protein was functional . Importarestored . Furtherrestored .Epm2b−/− mice [PBA improved motor and neurobehavioral phenotypes in Lafora disease model −/− mice . This wa−/− mice . For ATP−/− mice . PBA als−/− mice . When th−/− mice . Creatin−/− mice . These m−/− mice . In a no−/− mice . Additio−/− mice .Ambroxol is a common mucolytic agent that crosses the blood–brain barrier and also acts as a pharmacological chaperone for acid β-glucosidase (GCase), the causative enzyme in Gaucher disease . GaucherA receptors, SAHA application substantially upregulated α1, increasing protein levels 5-fold, and the half-life of a1(A322D) was tripled from 40 min to 125 min [A receptors on the cell surface [Suberanilohydroxamic acid (SAHA), also called Vorinostat, is an FDA-approved drug used for the treatment of cutaneous T-cell lymphoma . Additio 125 min . Partner 125 min . Additio 125 min . Further surface .SAHA also rescues γ2 mutations that have reduced surface expression due to ER retention, such as N79S, R82Q, P83S, and R177G . GABAergBiP inducer X (BIX), or 1--2-thiocyanate-ethanone, induces expression of the molecular chaperone BiP and prevents neuronal death from ER stress . BIX is 2α that crosses the blood–brain barrier [A receptors [Dinoprost (DNP) is a synthetic analogue of prostaglandin F barrier . Dihydro barrier . Both co barrier . DNP and barrier . The hig barrier . Additio barrier . Two epieceptors . Of noteeceptors . Finallyeceptors . The moseceptors .Multiple other chemical chaperones have shown promise for neurological disorders with epilepsy. A class of oxysterol derivatives were demonstrated to act as pharmacological chaperones for NPC1, the protein causing Niemann-Pick type C1, a lysosomal storage disorder with many neurological manifestations, including seizures . The oxyValosin-containing protein (VCP) is part of the retrotranslocation machinery that transports polyubiquitinated proteins from the ER to the cytosol for degradation via the UPS ,96. Eer1GFAPTsc1 CKO model of tuberous sclerosis complex, 17AAG decreased seizure frequency, although drug administration began weeks before normal seizure onset [The heat shock chaperone Hsp90β recruits the glutamate transporter GLT-1  to the 20S proteasome . In tissre onset . HSP990 A technology recently receiving a lot of attention is proteolysis targeting chimerics (PROTACs). PROTACs enhance the endogenous interactions between E3 ligases and their substrates, by utilizing a heterobifunctional molecule that consists of an E3 ligase ligand and a ligand to bind the substrate ,30,100. Several PROTACs targeting tau have been discovered, including TH006, which reduced tau levels in vitro, and in a mouse model of Alzheimer’s disease, lessened the neurotoxicity of Aβ . The firCa2.2 [The interrelationship between neuronal transmission, epilepsy, protein quality control, and the ubiquitin proteasome system is complex and multidirectional. For example, excitatory synaptic activity, such as after NMDA receptor activation, can result in complex changes to proteasome localization and activity and to ubiquitination patterns, thereby modifying the entire profile of proteins found in postsynaptic densities ,13,25,32Ca2.2 .Unsurprisingly, given the effects even normal neuronal activity has on the UPS, status epilepticus can cause dramatic changes to the UPS, including downregulation of UPS activity and deubiquitinase expression, which vary in magnitude across brain regions and cell types ,46. The As in the UPS, ER stress is also interlinked with neuronal loss and epilepsy. Epilepsy can cause impairments to the protein folding machinery in the ER, resulting in ER stress that can progress to apoptosis. Multiple animal models have demonstrated that seizures can cause elevations in ER stress-related factors, such as BiP and CHOP, and human epileptic brain tissue also shows increases in both ER chaperones and proapoptotic caspases ,42,45. EGABRA1, GABRG2, and SLC6A1 mutations [Epileptic disorders can be due to misfolded proteins that are degraded too quickly, or to misfolded proteins that accumulate in aggregates. Accelerated degradation of misfolded proteins that still maintain at least partial function is seen in utations ,53,54,55utations . It is lDysregulated degradation can also arise from mutations that alter the function of E3 ligases or their partnering proteins, or even via alterations to a substrate protein that impairs the ability of its associated E3 ligase to degrade it, as is the case for the CUL3 substrates, RHOBTB2 and KCTD7 ,75,76. BV7.2(M518V) [Further connections between epilepsies and compromised proteostasis can be found in the variety of genetic epilepsies that have intracellular protein aggregates. Proteins such as STXBP1 and Stefin B are prone to aggregation, even in wildtype forms, and epilepsy-associated mutations exacerbate aggregation ,65,72. U2(M518V) ,43,44.In terms of treatment, then, multiple modulations of proteostasis show promise for these many epilepsies. For proteins with partial function that are subject to accelerated ERAD, selectively inhibiting their associated components of the degradation pathway may be beneficial, such as VCP inhibition via Eer1 for α1(A322D) . ConversImpaired proteostasis is a common feature in many other diseases, both neurological and not. Neurodegenerative disorders, including amyotrophic lateral sclerosis, Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, and spinocerebellar ataxias are known to involve the accumulation of misfolded proteins and impairments of the UPS ,64. Decr


Polypyrimidine tract binding protein 3 (PTBP3) plays a critical role in post-transcriptional regulation. The role of PTBP3 in various human tumours was explored and analysed in this study based on the Cancer Genome Atlas and Gene Expression Omnibus datasets. PTBP3 was highly expressed in most tumours, such as breast invasive carcinoma, colon adenocarcinoma and hepatocellular carcinoma. PTBP3 overexpression generally predicts poor overall survival and disease-free survival in patients with adrenocortical carcinoma, lung squamous cell carcinoma, and pancreatic adenocarcinoma. However, low PTBP3 expression predicts poor prognosis in kidney renal clear cell carcinoma. We also explored PTBP3 genetic alterations in different tumour tissues. The result found that the frequency of PTBP3 alteration (>4%) was the highest in uterine tumours with “mutation” as the primary type. Furthermore, we found a significant correlation between PTBP3 expression and tumour mutational burden and microsatellite instability in various human tumours, and found that PTBP3 expression was positively correlated with TMB in ACC, STAD, PAAD, LUAD, and SARC. Two enhanced phosphorylation levels of S30 and S426 in colon cancer, ovarian cancer, and uterine corpus endometrial carcinoma were found. Further analysis indicated that PTBP3 expression was positively correlated with the cancer-associated fibroblasts for most tumour types. This study also found a relationship between immune checkpoints and N6-methyladenosine-related markers and PTBP3 expression. Moreover, the “mRNA surveillance pathway” and “RNA degradation” were involved in the functional mechanisms of PTBP3. These results provide new insights for molecular studies, and integrative analysis provided a framework for determining the predictive, prognostic, and therapeutic relevance of PTBP3 in cancer patients. Polypyrimidine tract binding protein 3 (PTBP3), also known as the regulator of differentiation (ROD) 1, is located on human chromosome 9q32. PTBP3 is a member of the polypyrimidine tract binding (PTB) protein family, which also includes PTBP1 and PTBP2 . The PTBin vitro and tumour growth and metastasis in vivo by binding to the 5′UTR HIF-1α mRNA to enhance HIF-1α protein expression  Project and Gene Expression Omnibus (GEO) databases were used to explore the expression profile of PTBP3 across various tumour types. The main investigated contents are as follows: gene expression profile, survival status, genetic alteration, protein phosphorylation, immune infiltration, N6-methyladenosine, and relevant cellular pathways. Comprehensive pan-cancer analysis aims to investigate the potential molecular mechanism of PTBP3 in the pathogenesis, clinical prognosis, and immunotherapy of various human tumours.http://timer.cistrome.org/) to analyse the differences in PTBP3 expression in different tumour types and adjacent normal tissues. However, lymphoid neoplasm diffuse large B-cell (DLBC) lymphoma, brain lower grade glioma (LGG), ovarian serous cystadenocarcinoma (OV), testicular germ cell tumours (TGCT), and thymoma (THYM) had only tumour tissue but no adjacent normal tissue. The Gene Expression Profiling Interactive Analysis, version 2 (GEPIA2) website was used to analyse PTBP3 expression in DLBC, LGG, OV, TGCT, and THYM  to analyse the protein expression of PTBP3 in breast cancer, colon cancer, uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma, and ovarian cancer. The GEPIA2 website was also used to analyse PTBP3 expression in different pathological stages of all TCGA cancers.We used Tumour Immune Estimation Resource, Version 2  and disease-free survival (DFS) significance map data across all TCGA tumours . The medThe cBioPortal tool was used to analyse the alteration frequency, mutation type, copy number alteration, and mutated site information across all TCGA tumours . After l6A)-related markers  using the TIMER2 tool. The relationship between PTBP3 expression and immune checkpoints  was explored using the HOME FOR RESEARCHERS website tool (https://www.aclbi.com/).We analysed the association between PTBP3 expression and immune infiltrates  across all TCGA tumours and the relationship between PTBP3 expression and N6-methyladenosine (mhttps://string-db.org/) was used to construct the protein-protein interaction network  was used in this study (https://www.r-project.org/), and statistical significance was set at p < 0.05  were higher than those in the corresponding adjacent normal tissues. Only a few tumour types showed no differential expression . In contrast, PTBP3 showed lower expression in the tumours of kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and thyroid carcinoma (THCA)  relative to the corresponding adjacent normal tissues.Next, we used TIMER2 to explore the expression of PTBP3 in different tumour types in the TCGA repository. As shown in p < 0.05)   . There w < 0.05) . In genep < 0.05) than in normal tissues . DFS analysis data showed that high PTBP3 expression was associated with poor prognosis for ACC, LUSC, and PAAD (p < 0.01).To understand the relationship between PTBP3 expression and prognosis and OS, we divided tumour cases into high- and low-expression groups according to the median expression of PTBP3. TCGA and GEO datasets were used to analyse the differences in survival rates between the two groups. As shown in and PAAD . Howeverp < 0.05). The RFS rate of the high PTBP3 expression group was also worse in the PAAD, LIHC, and BRCA groups . However, the OS rate of the high PTBP3 expression group was better in the KIRC and STAD groups .We also used the Kaplan–Meier plotter tool to analyse the relationship between PTBP3 expression and OS, progression-free survival (PFS), first progression and post-progression survival, DFS, disease-specific survival (DSS), distant metastasis-free survival, and relapse-free survival (RFS). As shown in p = 0.0415) and DFS (p = 0.0069), but not PFS (p = 0.0792) and DSS (p = 0.499)  was the highest in uterine tumours with “mutation” as the primary type. The “amplification” type was the primary type in the ACC, with a frequency of approximately 3% . Mutatio= 0.499) .We also analysed the relationship between PTBP3 expression and tumour mutational burden (TMB) and microsatellite instability (MSI) in some tumours. PTBP3 expression was positively correlated with TMB in ACC, STAD, PAAD, LUAD, and SARC, but negatively correlated with THYM, UVM, KICH, THCA, and KIRC. Moreover, we observed a positive correlation between PTBP3 expression and MSI for READ, UCEC, STAD, CHOL, and MESO, and a negative correlation for DLBC, HNSC, UCS, PRAD, and ESCA .Phosphorylation-dephosphorylation cascades are ubiquitous in tumour development. Thus, we compared PTBP3 phosphorylation levels between normal and tumour tissues. As shown in PTBP3 mediates alternative splicing regulation of pre-mRNA and plays a role in the regulation of cell proliferation, differentiation, and migration. Tumor-infiltrating immune cells are linked to the initiation, progression, or metastasis of cancer. Thus, the TIMER, CIBERSORT, CIBERSORT-ABS, TIDE, XCELL, MCPCOUNTER, QUANTISEQ, and EPIC algorithms were used to analyse the relationship between PTBP3 expression and tumour-infiltrating immune cells. This study found a positive correlation between PTBP3 expression and neutrophil cell count in most tumour types, especially in BLCA, CESC, DLBC, KIRC, LIHC, PRAD, and STAD. . The relWe also analysed the relationship between PTBP3 expression and immune checkpoints . In 29 of 33 tumours, the expression of PTBP3 was positively correlated with the expression of immune checkpoint CD274. In 27 of 33 tumours, the expression of PTBP3 was positively correlated with the expression of immune checkpoints PDCD1LG2, while a negative correlation was found in the tumour of THYM. A positive relationship between PTBP3 expression and all immune checkpoints was found in the tumours of UVM, STAD, SKCM, OV, LIHC, and LGG .6A) modification. The m6A-related genes include ZC3H13, YTHDF1/2/3, YTHDC1/2, RBM15, METTL3/14, KIAA1429, IGF2BP1/2/3, WTAP, HNRNPD, HNRNPC, HNRNPA2B1, FTO, FMR1, ALKBH5 and so on. As shown in We also analysed the relationship between PTBP3 expression and N6-methyladenosine (mp < 0.001), and we used these five genes to compute a heatmap that showed their positive association with PTBP3  and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The GO data showed that “single-stranded RNA binding” and “mRNA 3′-UTR binding” were among the top hits. This might be involved in the effect of PTBP3 on tumour pathogenesis . Figure PTBP3, a member of the PTB family, has been reported to be involved in tumorigenesis and progression, such as in lung cancer , pancreaThis study showed that PTBP3 is highly expressed in most tumours. The expression levels of PTBP3 in tumours of BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, LIHC, LUAD, LUSC, READ, STAD, and UCEC were higher than those in the corresponding adjacent normal tissues, whereas low expression was observed in KICH, KIRC, KIRP, and THCA. Furthermore, we found that PTBP3 overexpression generally predicted poor OS in patients with ACC, LGG, LUSC, and PAAD, in which ACC and LGG were reported for the first time. However, the OS and DFS rates of the high-expression group were better only in the KIRC group. These results suggest that PTBP3 is a potential biomarker for predicting the prognosis of multiple cancers.6A markers in almost all human tumours, such as DLBC, PRAD, PAAD, LIHC, KIRP, THYM, and UVM. We speculated that PTBP3 protein is involved in N6-methyladenosine modification, or that it is an important component of methylase. In the immune infiltration analysis, we found that high PTBP3 expression was positively correlated with the cancer-associated fibroblast and neutrophil infiltration levels in most tumours, such as ACC, GBM, LIHC, and SARC. However, its expression was negatively correlated with cancer-associated fibroblasts in TGCT and STAD. In addition, the expression of PTBP3 was positively correlated with the expression of immune checkpoints PDCD1LG2 in most tumours, while a negative correlation was only found in THYM tumours. Our findings may provide a novel clinical biomarker for predicting response to immunotherapy.Our analysis yielded interesting findings. Especially in N6-methyladenosine analysis, we found that PTBP3 expression is related to all mn = 515) and LUSC  and found a correlation between the high PTBP3 expression and poor OS prognosis (p = 0.0025) and poor DFS (P-0.0048) specific for LUSC but not for LUAD. This was an important discovery, and further research is needed.A recent study suggested that PTBP3 promotes migration of non-small cell lung cancer (NSCLC) cells by regulating E-cadherin in the epithelial–mesenchymal transition signalling pathway and that there was a significant association between high PTBP3 expression in NSCLC tissues with poor OS . MoreoveBased on the TCGA database, the survival analysis results indicated a correlation between high PTBP3 expression and poor OS and DFS for PAAD. This study is the first to report a comprehensive pan-cancer analysis of PTBP3 in human tumours. PTBP3 was highly expressed in most tumours, and predicts poor survival. PTBP3 expression was significant correlated with the immune cell infiltration, TMB, MSI, PDCD1 and m6A-related markers. PTBP3 may be a new and promising prognostic and immunotherapy biomarker in human tumors. We believe that these findings may lay the groundwork for prospective functional experiments and may eventually have a positive impact in clinical practice.


Recently, it was reported that calcineurin is activated in a variety of cancers. Given that abnormalities in calcineurin signaling can lead to malignant growth and cancer, the calcineurin signaling pathway could be a potential target for cancer treatment. For example, NFAT, a typical substrate of calcineurin, activates the genes that promote cell proliferation. Furthermore, cyclin D1 and estrogen receptors are dephosphorylated and stabilized by calcineurin, leading to cell proliferation. In this review, we focus on the cell proliferative functions and regulatory mechanisms of calcineurin and summarize the various substrates of calcineurin. We also describe recent advances regarding dysregulation of the calcineurin activity in cancer cells. We hope that this review will provide new insights into the potential role of calcineurin in cancer development.Calcineurin, a calcium-dependent serine/threonine phosphatase, integrates the alterations in intracellular calcium levels into downstream signaling pathways by regulating the phosphorylation states of several targets. Intracellular Ca While the extracellular Ca2+ concentration is 11.5 mM, the steady-state intracellular Ca2+ concentration is kept very low at several tens of nanomoles. It is well known that the concentration of Ca2+ varies greatly in different cellular compartments, for example, intracellular calcium, 100 nM; endoplasmic reticulum (ER), 0.5–1 mM; and mitochondria, 100–200 nM [Intracellular calcium ions , which cleaves phosphatidylinositol 4,5-biphosphate (PIP2) to produce diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (InsP3). InsP3 then binds to the InsP3 receptor (InsP3R) and stimulates the release of Ca2+ from intracellular stores, such as ER [2+ [2+ release via InsP3R is regulated by complex feedback mechanisms [The binding of a hormone or growth factor to a G protein coupled receptor (GPR) or a tyrosine kinase receptor (RTK) initiates the Cahate InsP. InsP3 t2+ concentration trigger signal transduction, which regulates a wide range of cellular events such as gene expression, cell cycle, cell motility, autophagy, and apoptosis [2+ diffuse through the cell and have effects on the distal sites [2+ increases in the mitochondria and causes the release of cytochrome c and subsequently triggers apoptosis [2+, the dysregulation of calcium homeostasis can impair cell function and trigger cell death, and by doing so, may contribute to heart disease, cancer, and mental disorders. As a result, Ca2+ signals must be tightly regulated during the cell cycle. Indeed, previous studies have characterized Ca2+ transients during the cell cycle [2+ in rat fibroblasts and found that complex changes occurred in calcium levels during the cell cycle [2+ were the lowest at the beginning of the G1 phase but were subsequently increased at the G1/S border.Changes in Capoptosis ,5. Localal sites . Long-tepoptosis . Due to ll cycle ,9. For ell cycle . They sh2+ oscillations are narrow in the sense of periodic rise and fall, while Ca2+ concentration alterations are broad in the sense of simple Ca2+ concentration changes. To facilitate different cellular processes, intracellular downstream effectors decode the frequency, amplitude, and duration of intracellular Ca2+ oscillations [2+ binding sites. CaM is involved in the regulation of cell proliferation and cell cycle through CaM-dependent phosphorylation/dephosphorylation events [2+ binds to CaM, the Ca2+/CaM complex can interact with and control target proteins such as serine/threonine phosphatase calcineurin (CaN) and the family of Ca2+/CaM-dependent protein kinases (CaMK).Callations . The mosn events . When Ca2+/CaM regulates cell cycle progression, especially through the activation state of the cyclin-dependent kinase (CDK) complex, is of great interest, and therefore has been studied intensively. It has been shown that although all eukaryotic cells require Ca2+ signaling for cell proliferation, some transformed cells and tumor cell lines are less dependent on Ca2+ for cell growth [2+-mediated signaling pathways, therefore, play important roles in tumorigenesis, tumor progression, metastasis, and invasion [The molecular mechanisms through which Cal growth ,14. Ca2+invasion . In thisCell division is precisely regulated by a series of CDKs, whose activity is dependent on the binding to different cell cycle-specific cyclins . Cyclins1 phase is the retinoblastoma protein (Rb). In the early G1 phase, CDK4/6-cyclin D initiates the phosphorylation of Rb, and, subsequently, CDK2-cyclin E phosphorylates Rb [1/S boundary and to commence the S phase. CDK2-cyclin A plays an important role in S phase progression through the phosphorylation of proteins involved in DNA replication [2 phase [2 phase, CDK1-cyclin B is activated, allowing for the entry of cells into mitosis [2/M transition, CDK1-cyclin A activity is necessary for prophase initiation [One of the critical targets of the CDK-cyclin complex in the Glates Rb ,18,19,20lates Rb ,22. Thislication ,24. As c[2 phase ,26. In t mitosis . During itiation .2+ is required at several different points during the cell cycle. Cells are most sensitive to the depletion of extracellular Ca2+ at early G1 and near the G1/S boundary [1, Ca2+ regulates the expression of immediate-early genes, such as FOS, JUN, and MYC. Ca2+ is also required for Rb phosphorylation at the G1/S boundary. Spontaneous Ca2+ oscillations at the G1/S phase transition have been described in synchronized immortalized cell lines. These Ca2+ oscillations are accompanied by DNA replication [In mammalian cells, Caboundary ,29. In elication .2+ levels increase. Ca2+ regulates certain targets directly and others indirectly through Ca2+-binding proteins, such as CaM. Ca2+/CaM activates various pathways involved in the regulation of cellular processes, such as secretion, cell motility, ion homeostasis, and gene transcription. CaM also modulates a large number of intracellular enzymes, including protein kinases, protein phosphatases, phosphodiesterases, adenylyl cyclases, and ion channels [Once hormone receptors are activated, intracellular Cachannels .1, specifically the G1/S transition. Consistent with this notion, CaM antagonists block cell cycle progression in early or late G1 phases [2+, CaM undergoes a major conformational change, and forms a Ca2+/CaM complex to interact with a variety of target proteins such as cellular kinases [2+/CaM signaling are the CaMKs [2 phases, is also activated by the CaN/NFAT pathway [1 to S phase [1/S [2/M transition [CaM is required for cell cycle progression through G1 phases . Further1 phases . Upon bi kinases . The beshe CaMKs ,36. CaN he CaMKs  and CDK4he CaMKs . The samhe CaMKs . In conthe CaMKs . In addi pathway . CaMK re S phase ,43, and  S phase . In the  S phase . As a rease [1/S ,47,48 oransition , with deansition ,51. Not ansition ,52,53.2+-binding regulatory subunit, calcineurin B (CnB). In mammals, three independent genes, PPP3CA, PPP3CB, and PPP3CC, encode CnAα, CnAβ, and CnAγ, respectively. CnAα and CnAβ exhibit ubiquitous expression patterns, whereas the CnAγ expression is restricted to the testis and brain [PPP3R1 and PPP3R2, respectively). The CnB1 protein is expressed ubiquitously, whereas the CnB2 protein is specifically expressed in the testes. CnA contains an amino-terminal catalytic domain followed by a CnB-binding domain, a CaM-binding domain, and an autoinhibitory domain. CaN also contains a nuclear localization signal (NLS) in the catalytic domain and a nuclear export signal (NES) in the carboxyl terminus. The autoinhibitory domain of CnA blocks the catalytic site and the NLS [2+-binding motifs and an amino-terminal myristylation site. CaN is activated by the increased intracellular Ca2+ concentration in the cell and plays essential roles in multiple signaling processes [2+/CaM to the regulatory domain leads to the attenuation of autoinhibition, followed by dramatic enzymatic activation. Of note, although there are multiple kinases that are regulated by CaM, and CaN is the only phosphatase directly regulated by Ca2+/CaM.CaN, also known as protein phosphatase 2 B (PP2B), is a serine/threonine protein phosphatase that is conserved in all eukaryotes ,55,56,57nd brain ,59,60,61 the NLS . CnB conrocesses . The bin2+ concentrations, and a small portion of CaN can translocate to the nucleus and interact with the target substrates [As mentioned above, CaN is widely distributed in various mammalian tissues and is particularly abundant in neural tissues . Howeverbstrates .Several proteins have been reported to inhibit CaN, including AKAP79 (A-kinase anchoring protein-79) , PMCA2  transcription factors ,103. Fou S phase ,117. NFApromoter . In addipromoter  or inducpromoter . Thus, NGiven the diverse expression and function of each NFAT isoform, its dysregulation is known to be associated with tumorigenesis, Alzheimer’s disease, and the development of autoimmune and inflammatory diseases. A novel therapeutic strategy for treating NFAT-related diseases is to develop new ways to selectively regulate specific NFAT isoforms .Approximately 600 proteins with conserved sequences that bind to CaN have been identified . Many of2+ increase caused by mitochondrial depolarization. Activated CaN dephosphorylates DRP1, which then relocates to the mitochondria and promotes mitochondrial fission [Key proteins associated with tumor development such as myocyte enhancer factor 2 (MEF2), kinase suppressor of ras 2 (KSR2), DAXX, c-Jun, and nuclear factor I (NFI), are known CaN substrates ,136,138. fission . In neur fission . CaN-dep fission , while d fission .1/S progression mediated by the degradation of cyclin D1. FK506 also decreases the protein level of CDK4 via a yet unknown mechanism. Taken together, CaN activates CDK4-cyclin D1 through multiple mechanisms to promote cell cycle progression [Recently, we found that CaN inhibits cyclin D1 degradation by dephosphorylation of the Thr286 residue  Figure a. Treatmgression ,38. The gression . It is agression . This ingression  , a Ca2+-selective ion channel, is highly expressed in prostate cancer and is a prognostic marker [2+ to enter the cell, which in turn promotes NFAT activation [The mechanisms that drive CaN activation in cancer are not well understood. In some cancers, CaN hyperactivation has been associated with mutations. For example, the EL4 murine T-cell lymphoma cell line expresses a mutant form of CaN, in which the aspartic acid at position 477 is mutated to asparagine and the negative regulation of the phosphatase activity by the autoinhibitory domain is impaired by this mutation . In contc marker ,170. Inctivation . It is ativation ,173,174.tivation . Moreovetivation . Recentltivation , which iHighly activated CaN is supposed to activate its substrates, including NFAT, to promote proliferation. Similar to CaN, its target NFAT is also constitutively activated or overexpressed in numerous cancers and may contribute to cancer development and progression . ConversOwing to the high frequency of CaN/NFAT activation in cancer and the contribution of these molecules in cancer progression, the CaN/NFAT pathway could be a potential therapeutic target. Indeed, the anticancer effects of CaN inhibitors have been studied extensively in the past. For instance, cyclosporine A or FK506 induced apoptosis and rapid tumor clearance, resulting in the regression of leukemia . Cyclosp2+ channels/transporters/pumps can provide promising potential for the treatment of cancer patients. [2+ concentrations alters the expression levels of the genes involved in cell proliferation, resulting in an antitumor effect [2+ reduces the growth rate of tumor cells without affecting the cells in normal tissues [2+ levels.There is growing acceptance that targeting dysregulated Caatients. . Bufferir effect . Moreove tissues . Further2+ is involved in tumor growth and alters the expression of the genes involved in cell proliferation. Furthermore, previous studies have suggested that the modulation of nuclear Ca2+ signaling may be effective in cancer therapy. Activation of CaN and its downstream dephosphorylation has been identified as a mechanism by which nuclear Ca2+ regulates cell proliferation and cell cycle progression. As discussed above, dephosphorylation of proteins by CaN plays an important role in tumor formation and progression. Therefore, in the future, it will be necessary to identify the molecular mechanism of CaN activation and the substrates that promote cancer cell growth. Targeting the interactions of activated CaN with specific substrates in cancer cells, without affecting the normal immune function of CaN, may effectively inhibit the growth of cancer cells, leading to the establishment of new tumor-specific therapies.Nuclear Ca


NK cells are considered an important component of innate immunity, which is the first line of defensing against tumors and viral infections in the absence of prior sensitization. NK cells express an array of germline-encoded receptors, which allow them to eliminate abnormal cells and were previously considered a homogenous population of innate lymphocytes, with limited phenotypic and functional diversity. Although their characteristics are related to their developmental origins, other factors, such as tumors and viral infections, can influence their phenotype. Here, we provide an overview of NK cells in the context of the tumor microenvironment, with a primary focus on their phenotypes, functions, and roles in tumor micro-environment. A comprehensive understanding of NK cells in the tumor microenvironment will provide a theoretical basis for the development of NK cell immunotherapy. CD56bright NK cells usually express CD122, NKp46, and NKp80, while CD56dim NK cells express more markers, including CD16, CD57, and PEN5. NK cells can be found in peripheral, lymph node, spleen, liver, lung, and bone marrow  cells, which originate in the bone marrow, were first identified in 1975 . NK celle marrow –6. More eristics .The NK cell cytotoxic attack is immediate, and does not require prior antigen-priming or MHC-restriction, and NK cell status depends on the balance of activating and inhibitory signals among the various receptors interacting with their ligands . ActivatCarrega et al. showed that, in patients with non-small cell lung cancer, tumor-infiltrating NK cells express several activation markers, including NKp44, CD69, and HLA-DR, yet showed profoundly impaired cytotoxic potential . Furtherbright and CD16- NK cells were observed in the tumor stroma. Although these NK cells displayed some activation markers, such as NKp44, CD69, and HLA-DR, their cytolytic potential was lower than that of NK cells in the peripheral blood and normal lung tissue  include of NKp44, NKp46 and NKp30 and they can play an important role in most functions exerted by NK cells. NKp44 is a transmembrane glycoprotein and it has three mRNA splice variants which display different signaling capability. NKp44-1has the ITIM in their cytoplasmic tail but NKp44-2 and 3 are not. NKp44 ligands which expressed by tumor cells comprises cellular and cell-released forms. Mixed-lineage leukemia protein-5 (MLL5), termed 21spe-MLL5, Cell surface-associated heparan sulfate (HS) proteoglycans (HSPGs) and Proliferating Cell Nuclear Antigen (PCNA) are expressed on the surface of tumor cells, while Platelet-Derived Growth Factor (PDGF)-DD and Nidogen-1 (NID1) glycoprotein are secreted by tumor cells as soluble molecules to interact with NKp44. Among these NKp44 ligands, 21spe-MLL5, HSPG and (PDGF)-DD interact with NKp44 result in activation of NK cells. However, PCNA and NID1 inhibit the cytolytic function of NK cells. Tumor-infiltrated NK cells expressed higher level of KLRC1(NKG2A) gene and KLRD1(CD94). Also, KLRB1 gene (CD161) was expressed on tumor-infiltrated NK cells, which could bind to the CLE2D ligand on tumor cells to inhibit NK cell-mediated cytotoxicity –40. Smalvia secreted and membrane bound forms of IFN-γ. Thus, NK cells played an important and unique role in targeting stem-like tumors or poorly differentiated tumors  in response to cytokine-TGFβ signaling; however, intermediate group 1 innate lymphoid cells and group 1 innate lymphoid cells could not control local tumor growth and metastasis, and TGF-β signaling in NKp46+ cells suppress NK cell-mediated tumor immunosurveillance  accumulate at the tumor site, as well as circulating monocytes, recruited by the tumor-derived chemotactic factor, CCL2. Initially, these monocytes polarize into M1 cells, which exhibit cytotoxicity against tumor cells. M1 cells secrete cytokines, including IFN-γ and IL-12, which activate NK cells; however, with carcinoma progression and metastasis, TAMs polarize into cells and secrete large amounts of IL-10 and TGF-β, which suppress NK cell cytotoxicity. In contrast, M2 cells stimulate regulatory T cells (Tregs) and Th2 cells, which generate an immunosuppressive environment for NK cells . Other ileukemia . In addileukemia , 56, whieillance Figure 1Cong et al. investigated the role of NK cells in the tumor microenvironment by using credibly induced KrasG12D(KRAS) knockout into mouse lung cancer models. Further, they applied anti-NK1.1 monoclonal antibody, PK136to depletion of number of NK cells, or Kras mice with an Nfil3-/- mouse knockout model, and showed that depletion of NK cells significantly accelerated tumor development during tumor initiation, while depletion of NK cells during promotion and development had no effect on tumor development. The author hypothesized that NK cells could effectively prevent the occurrence of tumor, but could not control the occurrence and development of lung cancer. In addition, they found that the quantity of NK cells, T cells, B cells, and MDSCs in the lungs declined progressively, while the number the macrophages were increased, particularly the quantity of M2 cells, which function as immune suppressive cells. Further, tumor-infiltrated NK cells showed significantly attenuated cytotoxicity, and the expression levels of granzyme B, perforin, CD107a, IFN-γ, and TNF-α were gradually reduced in lung NK cells during lung cancer development. In contrast, the expression of molecules associated with activation and cytotoxicity, including NKp46, CD69, CD44, CD226, CD16/32, FasL, TRAIL, and CD122, and the inhibitory molecules, CTLA4, CD96, CD94, PD-1, PD-L1, Tim3, CD276, LAG3, and CD244, were unchanged in lung NK cells during lung cancer development.As established, glucose metabolism is essential for the function of human and mouse NK cells; hence, dysregulation of glucose metabolism can lead to NK cell dysfunction. FBP1 is important in glucose metabolism and inhibits glycolysis in human tumor cells and hematopoietic progenitor cells. Levels of FBP1 showed a 69-fold increase in tumor-infiltrated NK cells, in which glycolysis was inhibited; however, when FBP1 was inhibited, NK cell glycolysis function was restored. Thus, FBP1 weakens the cytotoxicity of tumor-infiltrated NK cells by inhibiting glycolysis; however, FBP1 can also impair the viability of tumor-infiltrated NK cells directly and independently of glycolysis. Lactic acid accumulated in the tumor microenvironment is a potent inhibitor of NK cell effector function and viability. Intracellular acidification and decreased ATP synthesis caused by lactic acid may be related to impaired IFN-γ production by NK cells .INF2, MIEF2, FIS1, and GDAP1; high expression of fission genes drives mitochondrial fragmentation. Hypoxia is a key feature of the tumor microenvironment and tumor infiltrated NK cells are enriched for hypoxia signatures, including expression of HK2, SLC7A5, SLC2A3, and KDM3A. In addition, NK cells cultured under hypoxia show reduced expression of granzyme B, IFN-γ, and CD107a after activation, suggesting impaired functionality. Drp1 is the main regulator of mitochondrial fission, and drives division at specific points along mitochondria. Compared with paired tumor-adjacent normal liver NK cells, tumor-infiltrated NK cells upregulate Drp1 pro-fission activity through phosphorylation of its Ser616 residue. Restoration of mitochondrial morphology by knocking down Drp1, and in response to mdivi-1 treatment, enabled tumor-infiltrated NK cells to kill tumor cells. Therefore, mitochondrial fragmentation is correlated with decreased NK cell antitumor capacity  in NK cells, resulting in complete “paralysis” of cell metabolism and transport, which reduces the anti-tumor response of NK cells and fails to reduce tumor growth  obesity . Frank C obesity . In addi obesity . Studies obesity .Róisín M. Loftus et al. found that NK cells isolated from human solid tumors were deficient in their pro-inflammatory functions, including production of IFN-γ and tumor cytotoxicity. Tumor cells are known to have a high demand for glutamine in addition to glucose, so it is likely that low levels of glutamine are also present in the tumor microenvironment. Glutamine limited tumor microenvironment can inhibit the expression of cMyc in NK cells, resulting in reduced NK cell metabolism and inhibition of anti-tumor NK cell function , 62.bright cells may be preferentially recruited tumor sites -bearing lungs. Further, these Tregs had upregulated CCR4, which can bind chemokines to attract Tregs into LLC-bearing lungs. They also found that LLC-bearing lung tissue secreted elevated levels of CCL22, which can also attract many Tregs to the tumor microenvironment. Surprisingly, the CCL22 was secreted by NK cells, with the phenotype, NK1.1ruitment –71; howeruitment –75.+ T cells. When NK cells and DCs are co-cultured, DC maturation is reduced; however, this can be partially reversed by the addition of CTLA4 .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


VDR), observed as associated with spine pathologies. The present study was aimed at investigating—through high-throughput gene and protein analysis—the response of human disc cells to vitamin D, depending on the VDR FokI variants. The presence of FokI VDR polymorphism was determined in disc cells from patients with discopathy. 1,25(OH)2D3 was administered to the cells with or without interleukin 1 beta (IL-1β). Microarray, protein arrays, and multiplex protein analysis were performed. In both FokI genotypes (FF and Ff), vitamin D upregulated metabolic genes of collagen. In FF cells, the hormone promoted the matrix proteins synthesis and a downregulation of enzymes involved in matrix catabolism, whereas Ff cells behaved oppositely. In FF cells, inflammation seems to hamper the synthetic activity mediated by vitamin D. Angiogenic markers were upregulated in FF cells, along with hypertrophic markers, some of them upregulated also in Ff cells after vitamin D treatment. Higher inflammatory protein modulation after vitamin D treatment was observed in inflammatory condition. These findings would help to clarify the clinical potential of vitamin D supplementation in patients affected by IDD.Vitamin D showed a protective effect on intervertebral disc degeneration (IDD) although conflicting evidence is reported. An explanation could be due to the presence of the FokI functional variant in the vitamin D receptor ( Vitamin D supplementation has been proposed in several pathological contexts to improve skeletal and non-skeletal health, but a considerable body of clinical trials has failed to establish clear evidence of benefit .In general, to establish the clinical success of a hormonal supplementation, the mechanism of action of the hormone should be firstly in vitro defined and then the safety and effectiveness of its administration should be validated in vivo.VDR) inside the disc, which are able to respond to the hormone by modulating proliferative and metabolic pathways [Considering the intervertebral disc degeneration (IDD)-related pathologies, few in vitro studies reported the presence of human cells expressing the vitamin D receptor  couples more efficiently with the transcription factor II B than the longer peptides  and leads to a higher transcriptional rate of vitamin D-dependent genes [The presence of the FokI variant rs2228570) in the nt genes ,7. This 0 in the nt genes ,11,12,13nt genes .In addition to the aforementioned genetic considerations, there are conflicting views concerning the potential of vitamin D in IDD and low back pain treatment. Age-dependent IDD, often associated with osteoporosis—whose treatment aimed at increasing the bone mineral density includes vitamin D supplementation—have been suggested as being responsible of endplate calcification by blocking nutrient and oxygen supply in the disc . NeverthVDR polymorphism. Vitamin D showed an inhibitory effect on the proliferation and metabolic activity of the disc cells and a pro-apoptotic induction, regardless to the VDR genotype, while Ff bearing cells showed the most anti-inflammatory and catabolic behavior.In a recent preliminary study on a selected, small panel of markers , the regVDR FokI variants. The findings of the study could help to clarify the clinical potential of vitamin D supplementation in patients affected by IDD.Based on these results, the present study was aimed at investigating through large-scale, high-throughput microarray and protein analysis, the response of disc cells to vitamin D in basal or inflamed condition, depending on the 2D3 treatment determined an upregulation of 160 genes in homozygous FF disc cells and of 113 genes in heterozygous Ff cells. In basal condition, 25 genes of those upregulated in FF cells were also upregulated in Ff. The most relevant upregulated genes in the context of intervertebral disc (IVD) pathophysiology in homozygous FF disc cells were ADAMTS15, ACAN, BGN, KRT18, KRT19, SOX9, IGFBP2, IGFBP5, IL7 and TIMP3, COL11A2 along with COL10A1, COMP, CCR3, CCR5, and VEGFD nd VEGFD , that weFF disc cells were downregulated in Ff cells, among which ACVRIC, CCL13, CCR2, COL15A1, COL2A1, COL9A3, CXCR2, BMP7, DCN, FGF18, FRZB, INFG, IL10, IL17A, IL1R2, IL2, IL31RA, IL5, IHH, IDO2, NODAL, TGFB1, and TNMD, together with other 46 genes that were exclusively downregulated in Ff such as KRT14, TERT, and VEGFR  remodeling in FF cells, inflammation seems to hamper the synthetic activity mediated by vitamin D, whereas it does not affect much the trend of Ff cells observed in basal condition. In fact, in Ff cells, both in basal and inflamed conditions, there is a general upregulation of genes encoding for enzymes involved in ECM remodeling/catabolism. Conversely, these genes were downregulated in basal and remained substantially unchanged in inflamed condition in vitamin D-treated FF cells.In FF cells and oppositely downregulated, with a few exceptions, in Ff cells in basal condition, whereas, in both inflamed FF and Ff cells almost all the angiogenic markers were downregulated.Angiogenic markers appeared upregulated by vitamin D treatment in Interestingly, some of the above-mentioned markers are also involved in the cross-talk between TGFβ/BMP and WNT signaling, playing important roles in the formation of bone and cartilage, and in chondrocytes hypertrophy and appeared modulated by vitamin D in disc cells. FF and an upregulation in Ff cells of both pro- and anti-apoptotic mediators as well as of chemokines and their receptors was observed in basal condition, with non-relevant modifications upon inflammation.After vitamin D treatment, a clear downregulation in FF and Ff cells which did not allow to identify a clear trend. As a general consideration, IL-1β stimulus seems to switch off the vitamin D activity on all the analyzed genes, particularly in FF cells.Finally, considering the modulation of inflammation by vitamin D, there is a general up- or downregulation of inflammatory markers in the two groups of Regardless of the treatment, I-309 (CCL1), IL-1α, IL-4, IL-5, IL-7, IL-10, IL12-p40 (IL12B), IL-13, IL-15, MCP-2 (CCL8), MCP-3, M-CSF, MDC (CCL22), MIG (CXCL9), MIP-1-δ (CCL15), RANTES, SCF (KITLG), SDF-1 (CXCL12), TARC (CCL17), TNFβ, EGF, IGF-1, angiogenin (ANG), and oncostatin M (OSM) were undetected in all the samples. Among these proteins, IL12-p40, MCP-2, MDC, MIG, MIP-1, SCF, SDF-1, TARC, TNFβ, EGF, angiogenin, and oncostatin M showed no changes of the correspondent gene expression.There was a substantial agreement between gene and protein patterns, as confirmed especially by the high correspondence between undetected/unmodulated genes and proteins. FF cells the vitamin D treatment determined an activation of VEGF and upregulation of PDGF-BB secretion, whereas in Ff cells an activation of IL-2 (downregulation of the gene), IL-3, and IL-8 (upregulation of CXCL8) secretion. Moreover, in Ff cells upregulation of MCP-1 (CCL2) secretion and gene expression and inhibition of TGFB1 and IFNγ secretion, along with a downregulation of the correspondent genes, and of leptin were observed.In basal condition, in FF cells secreted higher amount of TNFα, although the related gene expression appeared downregulated, as well as IL-3. Conversely, PDGF-BB was downregulated and GM-CSF (CSF2), IL-1β, TPO, and VEGF secretion were inhibited.Upon inflammation in presence of vitamin D, Ff cells showed an activation of the PDGF-BB and leptin, with an inhibition of G-CSF (CSF3), IL-2 (confirming the gene downregulation), and IL-3 secretion. Moreover, the secretion of IL-6, IL-8, GRO/GROα (CXCL1), ENA-78 (CXCL5), GM-CSF, and MCP-1 was downregulated too, albeit for all these markers an opposite upregulation of the correspondent genes was observed.In the same conditions, Among the 24 evaluated proteins, IL-17A was the only one undetected in all the samples, both in basal and inflamed conditions. p = 0.004), NP (p= 0.03), and EP (p = 0.03) cells; this increase was also observed for MMP-3 in pooled cells (p = 0.03) and AF (p = 0.03) cells, whereas for MMP-13 in pooled cells (p = 0.005) only. A clear increase, although not significant, was also observed in term of BMP-2 realease in pooled (p = 0.08) and in AF (p = 0.09) cells  cells .IL-3 showed no changes both in basal and inflamed conditions after vitamin D treatment.p = 0.008) and CCL20 (p = 0.05). Although not statistically significant, IL-1β, IL-5, and MMP-13 also showed a clear tendency to decrease .In inflamed conditions after vitamin treatment, no change in the secretion of MMP-1, MMP-3, BMP-2, and BMP-7 were observed in none of the samples. The secretion of IL-1β, IL-5, TNFα, and CCL20 was observed at single cell population level, whereas in pooled cells a significant decrease was observed for TNFα (p = 0.0003), as well as in all the three single cell populations . Similarly, IL-12p70 was decreased in pooled cells (p = 0.001), as well as in NP (p = 0.005) and AF cells (p = 0.03), whereas IL-4 was decreased in pooled cells (p < 0.0001) and in NP (p = 0.007) and EP (p = 0.03). IL-33, IFNγ, CD40L, and IL-13 showed a decrease in pooled cells , and in AF cells only , whereas IL-10 was decreased in pooled cells (p = 0.006) and in NP cells only (p = 0.009). Finally, IL-17E showed a decrease mediated by vitamin D in pooled cells (p = 0.009) as well as in EP cells only (0.04).A significant general decrease in the secretion of several other cytokines and inflammatory factors was observed after vitamin D treatment in inflamed condition. Interestingly, this decrease was seen in the pooled cells as well as confirmed in all or some of the single cell populations. GM-CSF appeared decreased in presence of vitamin D in pooled cells . The decrease was also confirmed at single cell population level, although not statistically significant, but showing a clear trend —having a protective role in cartilage ECM degradation and inflammation [Ff cells.Vitamin D promoted the synthesis of matrix proteins in D tissue ,26. Ff cammation ,28—was hFF cells there were also those related to angiogenesis, along with hypertrophic markers such as type X collagen, alkaline phosphatase and BMPs, the latter three upregulated in Ff cells after vitamin D treatment too. This suggests an involvement of vitamin D in the control of some pathways leading to hypertrophy in disc cells especially bearing the FF genotype. Moreover, since IVD is avascular, vessels located near the disc are fundamental for its nutrition, but the presence of neo-vascularization inside the disc is considered associated to the development of pain and degenerative processes. Therefore, it could be interesting to better understand the role of vitamin D in mediating angiogenesis-related processes in the disc through functional studies.Among the upregulated markers in Interestingly, some of the markers modulated by vitamin D treatment in disc cells are also involved in the TGFβ/BMP and WNT signaling pathways which jointly regulate each other and mediate the formation of bone and cartilage by progenitor cells, particularly by skeletal progenitor cells, and are also involved in chondrocytes hypertrophy ,30,31.Ff cells in basal condition, but inflammation determined a downregulation of both gene expression and protein secretion. A very similar behavior was observed for IL-3, with an increased secretion in Ff cells in basal condition and in inflamed FF cells, but it is downregulated in inflamed Ff cells. Interestingly, the secretion of IL-6, a cytokine known to be highly upregulated by IL-1β treatment [Ff cells after vitamin D treatment, confirming previously published data [Ff cells. In basal condition, vitamin D promoted the upregulation of CXCL8  as well as the secretion of CCL2 in Ff cells. On the contrary, both these chemokines were downregulated in inflamed conditions, likewise CXCL1, CXCL5, CSF2, and CSF3. CCL20, a chemokine involved in Th17 response [Ff cells, but a concomitant downregulation of its receptor CCR6 and of the pro-inflammatory IL17A was observed. Since the expression of CCR6 and IL-17A was also increased in PBMCs derived from IDD patients and in pathologic IVD tissues [Ff cells to limit self-detrimental effects of CCL20-mediated signaling. In addition, IL-17A was undetectable in the supernatant of all Ff samples, suggesting that it was not released in a biologically relevant amount.Inflammation is another peculiar aspect to consider during data interpretation. Although the expression of many inflammatory genes was modulated by vitamin D, the number of proteins detectable in the cell culture supernatant was low, suggesting that a low number of inflammatory mediators have a relevant role in this context. Nevertheless, these proteins allowed to explain better the gene modulations and to identify putative feedback mechanisms activated by disc cells. After vitamin D treatment, the pro-inflammatory IL-2 was more secreted by reatment ,32, was hed data . A comparesponse , produceresponse , was als tissues ,35, theiFf cells after vitamin D treatment was suggested also by the downregulation of IFNγ at both gene and protein level and by the upregulation of IL1RN, in agreement with the increase of IL-1Ra secretion previously observed in basal condition [IL1RN in inflamed cells did not correspond to an increase of IL-1Ra secretion after vitamin D treatment.A general anti-inflammatory attitude of ondition . On the Ff cells appeared more responsive in the control of inflammation than FF cells. In fact, inflamed FF cells downregulated the secretion of IL-1β and CSF2, but increased that of TNFα.As a general conclusion concerning inflammatory markers, VDR variants and in the specie/specificity of the responses. Moreover, the cross-talk with many other pathways and inflammatory cells in vivo, the different cell models , and inflammatory stimulus (10 fold higher in the mice study) might have led to these differences too.Considering TNFα pathway, our data were different in comparison with those reported by an in vivo and in vitro study on mice and their NP cells and showing that vitamin D acts through the inhibition of NF-κB pathway to retard IVD degeneration, modulating inflammatory responses, oxidative stress, apoptosis, and senescence . The incff genotype were collected, due to the low frequencies of this genotype in the Italian population (about 13%), as already published [Ff cells was performed in NP, AF, and EP cells obtained from seven donors.The main limitation of this study is that no cells bearing the on about %, as alrFf bearing cells treated with vitamin D demonstrated a general catabolic behavior, with increase of MMPs release, despite an increase of the anabolic BMP2 in basal condition. An anti-inflammatory attitude of these cells after vitamin D treatment was also confirmed in inflamed environment, with a general decrease of cytokines and inflammatory factors.This validation allowed to better explain the results obtained with omics on pooled cells. In general, the trend obtained by pooled cells of seven donors were confirmed in the single NP, AF, and EP cell populations, in particular AF cells showed to be the most responsive. Briefly, the results confirmed previously published  and omicThe study was approved by the Ethic Committee of the San Raffaele Hospital  and specimens were collected after obtaining the patient’s informed consent. NP, AF, and EP were collected from L5-S1 discs, Pfirrmann grade IV, of four patients  with diagnosis of discopathy and who underwent discectomy. Six cell populations, three for each donor, were used for the experiments for each genotype.g for 5 min. The cells were plated at 104 cells/cm2 and cultured up to 80–90% of confluence in low glucose (1 mg/mL) Dulbecco’s modified Eagle medium  supplemented with 10% foetal bovine serum , 0.29 mg/mL L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM Hepes, 1 mM sodium pyruvate  and maintained at 37 °C with 5% CO2. Medium was replaced twice a week. When confluent, the cells were detached with 0.05% trypsin/0.053 mM EDTA  and plated at 5 × 103 cells/cm2 until they reached passage 3.To isolate cells, NP, AF, and EP were minced in small pieces and digested under constant agitation with type II collagenase 37 °C, 22 h) , at the concentration of 224 U/mL for NP, 560 U/mL for AF  and 336  °C, 22 hFF while the remaining half carried the Ff genotypes.FokI VDR genotypes of disc cells were determined after genomic DNA extraction with Pure LinkTM Genomic DNA Mini kit  and spectrophotometric quantification  by using TaqMan SNP Genotyping Assay  for rs2228570 polymorphism and StepOne Plus instrument  as previously reported . Half of4 cells/cm2 in standard culture medium for 24 h, thereafter the standard medium was replaced with low serum (5% FBS) medium in order to limit the binding between vitamin D and serum protein. After 24 h from the medium shift, 10−8 M 1,25(OH)2D3 or vehicle (0.1% DMSO)  with or without 1 ng/mL of IL-1β were added to the cultures. The cells were collected after 48 h of treatment, counted, and pooled per treatment and genotype (2 × 105 cells for each cell type for each genotype), resulting in eight final samples that were maintained at −80 °C until RNA extraction.NP, AF, and EP cells at passage 3 were detached and seeded at 10RNA was extracted from lysates of pooled cells by RNeasy Plus Mini Kit . For residual genomic DNA digestion, RNase-Free DNase Set (Qiagen) was used and the isolated RNA was quantified spectrophotometrically . To evaluate the RNA integrity number (RIN), Agilent RNA ScreenTape System  was used, values ranged from 10 (intact) to 1  . All RNAhttps://earray.chem.agilent.com/earray/, date of access 12 February 2018) to analyze a huge panel of genes of interest  [A custom gene expression microarray was designed through a suitable algorithm of the Agilent Technologies (ch gene) ,39.Each RNA sample was added with a spike mix (prepared with the One Color RNA Spike-In kit) in order to assess the correct annealing of 10 optimized positive control transcripts to the complementary probes of the array and to evaluate the auto- and cross-hybridization. 100 ng of RNA was labeled and amplified by Low Input Quick Amp Labeling Kit one-color to obtain cRNA that was purified using RNeasy Plus Mini Kit . The samples were then hybridized on the microarray slide with the Gene Expression Hybridization Kit. The slide was than washed and scanned using SureScan Microarray Scanner to obtain a high-resolution image of the fluorescence values for each probe.Unless otherwise specified, all the reagents, instruments and software were purchased by Agilent Technologies .http://www.pantherdb.org/, 12 February 2018).Data were extracted by Feature Extraction v.12.0 software (Agilent). To evaluate the reproducibility and reliability of microarray experiments, quality control report was generated for each sample. The data analysis was performed by Genespring GX software 14.9 and fold change Fc > 2 or <0.5 in the cells exposed to different treatments were described and considered of interest. Pathway analysis was carried out by Panther database  , detection sensitivity up to pg/mL of protein, according to manufacturer’s instruction.The levels of inflammatory mediators in culture media obtained from disc cells, stimulated or not with IL-1β and vitamin D, were evaluated with ELISA-based protein arrays AAH-CYT-3  and pooled according to genotype and treatment.The conditioned media were obtained for NP, AF, and EP cells, from four donors . The results were generated by quantifying the mean spot pixel density using Image Lab Software 2.4 (Biorad). The signal intensities were normalized to the background and positive control and signal intensity results was obtained by the average pixel density of two spots per each inflammatory mediator. The relative concentration of the antigen in the treated sample was proportional to the signal intensity for each control spot. Fold change Fc > 2 or <0.5 in the cells exposed to different treatments were described and considered of interest.Ff genotype. A panel-relevant proteins identified by gene and protein arrays of pooled samples has been assayed and validated in each single population of NP, AF, and EP cells of seven donors bearing the most responsive A custom panel  for the multiple detection of BMP-2, BMP-7, IL-1Ra, IL-2, IL-3, IL-6, IL-17A, MMP-1, MMP-3, and MMP-13 was created. Detection range and sensitivity for these analytes are reported in The assay was performed on a MagPix™ Luminex System  following the manufacturer instructions.t-test was performed in case of gaussian distribution whereas Wilcoxon test was performed if data were not normally distributed to establish significative differences between cells treated with vitamin D in comparison with controls. A p value ≤ 0.05 was considered significative, 0.09 ≤ p > 0.05 was considered as a tendency.Data are showed as mean with standard error mean (SEM). Kolmogorov–Sminorv test was perfomed to assess the normal distribution of the data. Student’s The strength of the present study was to expand the knowledge of the pathways modulated by vitamin D in IVD cells in relation to the presence of functional variants in VDR by using omics methods. This allowed to overcome the restricted evaluation of panels of small number of molecules, by providing a more complete identification of specific areas of the hormone’s action. These findings pose robust basis for a clinical investigation aimed to assess the potential of the administration of vitamin D in patients affected by disc degeneration. In fact, only this approach would permit to evaluate both the response to the treatment according to the VDR genotype of the patients and the effect of the hormone on matrix remodeling and on hypertrophy/calcification of the tissues involved.