Tumor metastasis is one of the main causes of the high mortality of hepatocellular carcinoma (HCC). E-Twenty Six variant gene 6 (ETV6) is a strong transcriptional repressor, associated with the development and progression of tumors. However, the exact role and underlying mechanism of ETV6 in HCC remain unclear.were used to detect the expression levels of ETV6, CRKL (v-crk sarcoma virus CT10 oncogene homologue (avian)-like) and miR-429 in HCC tissues and cells; Transwell chamber and F-actin cytoskeleton staining assay to examine the effects of ETV6 and CRKL deregulation on the migration, invasion and cytoskeleton of HCC cells; Co-immunoprecipitation assay to determine the interaction between CRKL and ETV6; Chromatin immunoprecipitation assay to investigate the interaction between ETV6 and miR-429.Western blotting, quantitative real-time PCR and immunohistochemistry CRKL-3′-UTR; ETV6 directly binds to CRKL and positively regulates its expression, which in turn CRKL positively regulates ETV6 expression.We established a novel ETV6-miR-429-CRKL regulatory circuitry contributes to HCC metastasis. ETV6 and CRKL were frequently increased, while miR-429 was downregulated in both hepatocarcinoma tissues and hepatocarcinoma cells. Moreover, ETV6 upregulation was positively correlated with CRKL upregulation, and two negative correlations were also established for ETV6 and CRKL upregulation with miR-429 downregulation in both hepatocarcinoma patients’ tumorous tissues and hepatocarcinoma cells. Functional investigations revealed that overexpression and knockdown of ETV6 was remarkably effective in promoting and suppressing HCC cell migration, invasion, cytoskeleton F-actin expression and arrangement, whereas, CRKL overexpression exhibited similar effects to the overexpression of ETV6. Mechanistically, ETV6 negatively regulates miR-429 expression by directly binding to the promoter region of miR-429; miR-429 negatively regulates CRKL expression by selectively targeting Our data demonstrated that ETV6 promotes migration and invasion of HCC cells by directly binding to promoter region of miR-429 via modulating CRKL expression. The newly identified ETV6-miR-429-CRKL regulatory circuitry contributes to the aggressiveness of HCC, which provides new clues for fundamental research on diagnosis and treatment parameters for HCC. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Its morbidity and mortality rate are among the highest in the world , 2. TumoE-Twenty Six (ETS) variant gene 6 (ETV6), also known as translocation ets leukaemia (Tel), is a member of the transcription factor ETS family , 8. The CRKL, a member of the CRK adapter protein family, is ubiquitously expressed and conserved across eukaryotic organisms . It is cCRKL-3′-UTR at the 3728–3735 bp site by post-transcriptionally mediating CRKL’s functionality, and miR-429 suppressed the migration and invasion of HepG2 cells by targeting CRKL via inhibiting Raf/MEK/ERK  pathway and EMT . The supernatant was collected by centrifugation at 12000 rpm at 4 °C for 15 min. Equal amounts of each group of protein samples determined by Bradford assay were boiled for 5 min in loading buffer and separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein bands were transferred onto a nitrocellulose (NC) membrane , blocked with 5% (w/v) skim milk  in TBST  for 3 h at room temperature (RT) and then incubated with primary antibodies at 4 °C overnight. The primary antibodies were CRKL , ETV6 , ACTB  and GAPDH . The NC membrane was then washed with TBST for 3 × 10 min, incubated with the secondary antibody conjugate for 3 h at RT and washed again with TBST 3 × 10 min. Protein bands were visualized by electrochemiluminescence  and analyzed using the Bio-Rad ChemiDoc™ MP system . GAPDH and ACTB were used as internal references.Total protein was extracted from each group cells using radio-immunoprecipitation assay (RIPA) buffer [50 mM pH 8.0 Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS in the presence of 1 mM NaIHC assay was used to determine the expression levels of CRKL  in HCC tissue arrays. Tissue sections were treated with biotin-streptavidin horseradish peroxidase (HRP) detection systems  according to the manufacturer’s protocol. The images were visualized with a DAB (diaminobenzidin) kit  using a BX63 microscope . IHC immunoreaction intensity was rated into four grades, 0 (negative), 1 (weak), 2 (moderate) and 3 (strong) as Score I. Moreover, based on the detected positively staining cells, DAB staining quantity of each sample was graded as 0 (none), 1 (1–10% cells per field), 2 (10–50%), 3 (51–75%) and 4 (> 76%) as Score II. The multiplication of Score I by Score II ranging from 0 to 12 was used as a measure of IHC immunoreactivity degree. The scores of 0–2, 3–5, 6–8, and 9–12 were considered as negative (−), weak (+), moderate (++) and strong (+++). IHC assays were scored separately by two independent experienced pathologists.-△△CT method.Total RNA was extracted from each group of tissues and cells using Trizol™ reagent  and reversely transcribed into an EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit . qRT-PCR was then performed using TransStart Tip Green qPCR SuperMix  with a StepOnePlus™ Real-Time PCR system . snRNA U6 was used as internal reference for miR-429. The relative expression levels of miR-429 in different groups of cells, and in paired tumor and paracancerous non-tumor tissues from hepatocarcinoma patients were compared using the 2ETV6 was first amplified by RT-PCR using forward primer 5′- TAGCTAGCGCCACCATGTCTGAGACTCCTGCTC-3′ and reverse primer 5′- GCCGCGCTTCGAATCAGCATTCATCTTCTTGG-3′, then inserted into the Nhe I and BstB I sites of a PCDH-EF1-MCS-T2A-Puro vector. The recombinant expression vector PCDH-EF1-MCS-T2A-Puro-ETV6 was used for overexpressing ETV6 in cells, the empty vector PCDH-EF1-MCS-T2A-Puro was used as control.Previously, we constructed the recombinant PCDH-EF1-MCS-T2A-Puro-CRKL expression vector to overexpress CRKL in HCC cell lines . To over5 HepG2, HCCLM3 and HuH7 cells were infected with lentivirus in 6-well plates containing 8 μg/ml polybrene  for 48 h in a humidified incubator at 37 °C with 5% CO2. The cells stably transfected with PCDH-EF1-MCS-T2A-Puro-CRKL, PCDH-EF1-MCS-T2A-Puro-ETV6 and PCDH-EF1-MCS-T2A-Puro were screened against 0.5 μg/ml puromycin for 21 d at 37 °C with 5% CO2.Lentivirus packaging was performed according to the manufacturer′s instructions, the recombinant expression vectors of PCDH-EF1-MCS-T2A-Puro-CRKL, PCDH-EF1-MCS-T2A-Puro-ETV6, PCDH-EF1-MCS-T2A-Puro were separately mixed with the packaging plasmids psPAX2 and pMD2G, and transfected into 293 T cells using Lipofectamine™ 2000  for 48 h. The virus particle supernatants were harvested by centrifugation at 4500 rpm for 10 min and filtered with a 0.22 μm microporous membrane. Then, 1 × 10CRKL sequence  and ETV6 sequence  using Invitrogen, siDirect and Whitehead software. Meanwhile, one siRNA with non-targeting sequence 5′-TTCTCCGAACGTGTCACGT-3′ was designed as a negative control (NC). One day before transfection, 3 × 105 cells/well in 2 ml DMEM supplemented with 15% FBS were seeded into a 6-well plate, and the siRNA mixtures (2 μl siETV6–1 + 2 μl siETV6–2 + 2 μl siETV6–3) were transfected into HepG2, HCCLM3 and HuH7 cells using 5 μl Lipofectamine™ 2000  according to the manufacturer’s instructions for 48 h at 37 °C with 5% CO2, respectively.For CRKL and ETV6 knockdown, targeting siRNAs  were designed according to 4 cells in 200 μl serum-free DMEM were seeded onto the upper compartment of transwell with 8 μm pore size polycarbonate filters . The chambers were then placed into 24-well plates containing 600 μl DMEM with 20% FBS and incubated for 24 h at 37 °C with 5% CO2. For invasion assay, the inserts were first coated with 50 μl 2.5% ECM gel  in DMEM, and incubated at 37 °C for 1 h. 1 × 104 cells in 200 μl serum-free DMEM were seeded onto the upper compartment of the transwell. The chambers were then placed into 24-well plates containing 600 μl DMEM with 20% FBS and incubated for 24 h at 37 °C with 5% CO2. The non-migrated and non-invaded cells on the upper surface of the insert were removed by swabbing, the migrated and invaded cells onto the lower surface were fixed with methanol  for 30 min, stained with 0.1% crystal violet for 40 min, washed with phosphate buffered solution (PBS), counted using an upright light microscope  with 100× magnification. Five random field views were counted and averaged.The effect of CRKL and ETV6 deregulations on the migration and invasion abilities of HepG2, HCCLM3 and HuH7 cells were examined using the Boyden transwell chamber assay. Briefly, 1 × 105 cells from each group were seeded into the cover glasses and cultured for 24 h at 37 °C in humidified incubator with 5% CO2. The cover glasses were then twice washed with 1 ml PBS, fixed in 4% paraformaldehyde for 10 min at RT, washed 3 times with PBS, permeabilized with acetone, and continuously incubated in 100 μl TRITC-Phalloidin (400 nM) containing 1% bovine serum albumin (BSA) for 30 min in the dark at RT. The cover glasses were washed with PBS for 3 times and counterstained in 100 μl 50 nM Hoechst 33342 staining solution  for cell nucleus. Images were immediately taken using a fluorescence microscope  at five randomly selected visual fields of 1000× within 1 h.The TRITC (tetramethyl rhodamin isothiocyanate)-Phalloidin staining assay was performed to investigate the influence of CRKL and ETV6 on the cytoskeleton structure of HepG2, HCCLM3 and HuH7 cells. The 0.17 mm thick round cover glasses of 25 mm diameter were placed into 6-well plates, then 1 × 106 HCCLM3-PCDH-CRKL or HCCLM3-PCDH-NC cells using RIPA buffer, then 500 μg protein was incubated with 2 μl anti-CRKL , 2 μl anti-ETV6 , or 1 μl nonspecific anti-rabbit IgG  for 1 h at 4 °C. The immune complexes were pulled down by 20 μl protein A/G agarose beads  at 4 °C overnight. Then the beads were washed 3 times with 500 μl RIPA buffer at RT, and after centrifugation with 12,000 rpm for 15 min at 4 °C, the beads were mixed with 4 μl 5 × SDS-PAGE loading buffer and 16 μl RIPA and boiled for 10 min. Then the immunoprecipitated proteins were separated by 10% SDS-PAGE and analyzed by WB.Co-immunoprecipitation (Co-IP) assay was performed to determine the interaction between CRKL and ETV6. Total protein was extracted from 5 × 107 HCCLM3 cells were cross-linked with 550 μl 37% formaldehyde for 10 min at RT and treated with 2 ml 10 × glycine for 5 min at RT, then the cells were washed with 2 ml PBS containing 10 μl protease inhibitor cocktail II, scraped and lysed with 1 ml SDS lysis buffer containing 5 μl protease inhibitor cocktail II. Then the lysates were sonicated on ice for 6 × 15 s to shear the DNA and the debris was removed by centrifugation at 12000 rpm for 10 min at 4 °C. A small part of the lysate was used as DNA input control, the remaining part was diluted 10-fold with dilution buffer and incubated with 4 μl anti-ETV6  or 1 μl anti-RNA polymerase II, 1 μl normal mouse IgG, 4 μl anti-rabbit IgG at 4 °C overnight. Immunoprecipitated complexes were collected using 60 μl protein A/G agarose beads at 4 °C for 1 h. After centrifugation at 5000 rpm for 1 min at 4 °C, the precipitates were washed with 1 ml washing buffer at 4 °C for 3 × 5 min, and eluted with elution buffer (20% SDS and 1 M NaHCO3) for 15 min at RT. Cross-linking of protein-DNA complexes was reversed with 8 μl 5 M NaCl at 65 °C overnight, followed by treatment with 1 μl RNase A at 37 °C for 30 min, and 4 μl 0.5 M EDTA, 8 μl 1 M Tris-HCl, 1 μl Proteinase K at 45 °C for 2 h. Then, DNA was extracted using a spin column according to the manufacturer’s instructions. The DNA was subjected to PCR to amplify the 262 bp promoter region of miR-429 using specifically the designed primers, F: 5′-ACCTCGCCACCGCCTCCCATTGTCCCGTCG-3′ and R: 5′-TGCCAGGCCCGGGTGGGTGTGAACCGGCTTC-3′.ChIP assay was performed to investigate the interaction between ETV6 and miR-429 using an EZ-ChIP™ Chromatin Immunoprecipitation Kit . Briefly, 2 × 10P ≤ 0.05 are statistically significant.Statistical analyses were performed using GraphPad Prism 5.0 software. The data were presented as mean ± SD of at least three independent experiments. The statistical differences between groups were evaluated by Student’s t-test analysis. As most of the distributions deviated from the normal Gaussian distribution, non-parametric tests were used for further analyses. The correlations between the expression of ETV6, CRKL and miR-429 were analyzed using the Spearman’s rank correlation coefficient. Differences with P = 0.0005, Fig. P = 0.0499, Fig. P = 0.0284, Fig. P = 0.0166, Fig. To investigate the potential role of ETV6, CRKL and miR-429 in hepatocarcinoma, we detected the expression levels of ETV6, CRKL and miR-429 in HCC tissues and cells. WB results showed that compared with non-tumor liver tissues, ETV6 expression in tumorous tissues of hepatocarcinoma patients was increased by 127.6% (P = 0.0041) in the tumorous tissues from hepatocarcinoma patients. Meanwhile, CRKL expression levels were increased by 47.2% (P = 0.0221), 101.7% (P = 0.0068) and 85.5% (P = 0.0134) in HepG2, HCCLM3 and HuH7 cells compared with LO2 cell  in the tumorous tissues from hepatocarcinoma patients. Furthermore, miR-429 was expressed in all the tested cell lines and a comparatively high expression was shown in normal liver LO2 cells compared to hepatocarcinoma cells. miR-429 expression levels were decreased by 83.0% (P = 0.0002), 93.3% (P < 0.0001) and 95.1% (P < 0.0001) in HepG2, HCCLM3 and HuH7 cells compared with LO2 cell  and 61.7% (P = 0.003) in HCCLM3-PCDH-ETV6 and HuH7-PCDH-ETV6 cells, respectively  and 45.3% (P = 0.0044)  and 125.6% (P = 0.0001) than those of HCCLM3-PCDH (57.0 ± 0.7) and HuH7-PCDH cells (38.8 ± 2.3). The numbers of invaded HCCLM3-PCDH-ETV6 (104.6 ± 3.4) and HuH7-PCDH-ETV6 (116.2 ± 2.9) cells increased by 110.0% (P = 0.0001) and 96.6% (P = 0.0003) than those of HCCLM3-PCDH (50.4 ± 1.2) and HuH7-PCDH cells (58.8 ± 4.4). Consistently, ETV6 downregulation inhibited the migration and invasion abilities of HCCLM3 and HuH7 cells  and 57.3% (P = 0.0003) than HCCLM3-siNC (59.8 ± 1.7) and HuH7-siNC cells (61.0 ± 4.3). The numbers of invaded HCCLM3-siETV6 (39.2 ± 0.6) and HuH7-siETV6 (38.4 ± 1.2) cells decreased by 32.8% (P = 0.0001) and 45.9% (P = 0.0003) than HCCLM3-siNC (57.8 ± 0.9) and HuH7-siNC cells (88.8 ± 1.2). Clearly, ETV6 significantly affected the in vitro migration and invasion abilities of HCCLM3 and HuH7 cells. Our results indicated that ETV6 acts as a tumor promoter in hepatocarcinoma by promoting the aggressiveness of tumor cells.ETV6 dysexpression apparently affects the in vitro migration and invasion capacities of HCCLM3 and HuH7 cells. ETV6 expression level was positively correlated with the migration and invasion abilities of HCCLM3 and HuH7 cells. ETV6 overexpression enhanced the migration and invasion abilities of HCCLM3 and HuH7 Fig. c. The nuIt is well established that cytoskeleton elements are closely related to cell movement. The changes of F-actin microfilament organization in HCCLM3 and HuH7 cells after ETV6 deregulation was investigated by fluorescence microscopy using FITC-labeled phalloidin. ETV6 deregulation significantly affected F-actin cytoskeleton protein expression of HCCLM3 and HuH7 cells. As shown in Fig. P = 0.0106), 162.0% (P = 0.0001) and 82.1% (P = 0.0004) in HepG2-PCDH-CRKL, HCCLM3-PCDH-CRKL and HuH7-PCDH-CRKL cells, respectively, , 77.4% (P = 0.0004), 148.5% (P < 0.0001) compared with HepG2-PCDH (69.2 ± 4.8), HCCLM3-PCDH (56.6 ± 4.8) and HuH7-PCDH (47.8 ± 3.2) cells, respectively. The numbers of invaded HepG2-PCDH-CRKL (204.4 ± 7.2), HCCLM3-PCDH-CRKL (100.4 ± 5.8) and HuH7-PCDH-CRKL (137.8 ± 6.9) cells increased by 108.6% (P < 0.0001), 41.2% (P = 0.0004), 88.3% (P < 0.0001) compared with HepG2-PCDH (98.0 ± 5.0), HCCLM3-PCDH (69.4 ± 5.3) and HuH7-PCDH (73.2 ± 3.7) cells, respectively , 45.2% (P = 0.0075), 51.5% (P = 0.0338) in HepG2-PCDH-CRKL, HCCLM3-PCDH-CRKL and HuH7-PCDH-CRKL cells. CRKL knockdown consistently resulted in decreased ETV6 expression by 37.0% (P = 0.0253), 33.9% (P = 0.0082), 26.7% (P = 0.0295) in HepG2, HCCLM3 and HuH7 cells , 33.7% (P = 0.0224), 19.1% (P = 0.0181) in HepG2-PCDH-CRKL-siETV6, HCCLM3-PCDH-CRKL-siETV6 and HuH7-PCDH-CRKL-siETV6 cells. The results demonstrated ETV6 expression was positively correlated with CRKL expression in hepatocarcinoma cells.After, we observed a significant positive correlation between the expression levels of ETV6 and CRKL in hepatocarcinoma tumor tissues and hepatocarcinoma cell lines, then we investigated the mutual influence between the dysexpression of ETV6 and CRKL. CRKL overexpression increased endogenous ETV6 expression levels Fig. a. In comTel-ABL fusion protein may form complexes with CRKL , and thuP = 0.0091), 8.4-fold (P = 0.0050) and 4.2-fold (P = 0.0031) in HepG2-PCDH-CRKL-siETV6, HCCLM3-PCDH-CRKL-siETV6 and HuH7-PCDH-CRKL-siETV6 cells. Our results further demonstrated that ETV6 expression was negatively correlated with miR-429 expression.We observed a significant negative correlation between the expression level of ETV6 and miR-429 in hepatocarcinoma tumor tissues and hepatocarcinoma cell lines. To further confirm the negative correlation between ETV6 and miR-429 expression levels, we detected the effect of ETV6 knockdown on the expression level of miR-429 by transfecting si-ETV6 in CRKL-overexpression HepG2, HCCLM3 and HuH7 cells. ETV6 downregulation resulted in increased expression levels of miR-429 Fig. a. In comhttp://jaspar2016.genereg.net/cgi-bin/jaspar_db.pl?rm=browse&db=core&tax_group=vertebrates) indicated that ETV6 protein potentially binds to the DNA promoter region at the 696–705 site of miR-429 , 41.5% (P = 0.0311), 73.1% (P = 0.0030) in HepG2-PCDH-CRKL, HCCLM3-PCDH-CRKL and HuH7-PCDH-CRKL cells. Consistently, CRKL knockdown resulted in increased endogenous miR-429 expression levels by 94.5% (P = 0.0100), 90.1% (P = 0.0462), 89.7% (P = 0.0218) in HepG2, HCCLM3 and HuH7 cells  patients’ tumorous tissues and matched paracancerous nontumoral renal tissues, and bone marrow (BM) of chronic myeloid leukemia (CML) patients and peripheral blood (PB) of normal samples, the expression patterns and the correlations of ETV6, CRKL and miR-429 in ccRCC and CML consistent with those in liver cancer (unpublished). Previously, we have detected the effect of ETV6 on proliferation ability of HCC cells, we found re-expression or silencing of ETV6 slightly affected the proliferation and colony formation abilities of HCCLM3 and HuH7 cells. Meanwhile, re-expression or silencing of miR-429 cannot affect HCC cells proliferation and colony formation abilities, and CRKL only deregulated to a certain degree, which can affect HCC cells proliferation ability . Our resETV6 is a leukemia-associated gene located on 12p13, which has been shown to play a wide role in hematopoiesis and hematological malignancies , 37, 38.P = 0.0041) in the tumor from 16 additional hepatocarcinoma patients  in the tumor from 16 additional hepatocarcinoma patients  fusion gene was common in cancers such as secretory breast carcinoma, mesoblastic nephroma and congenital fibrosarcoma [The osarcoma –55, ETV6osarcoma , committosarcoma . ETV6-CRThe current work also establishes the association of ETV6 with miR-429 in hepatocarcinoma. First, we found that ETV6 upregulation was negatively correlated with miR-429 deficiency in tumorous tissues from hepatocarcinoma patients Fig. a. ETV6 aPreviously, we reported that miR-429 negatively regulates CRKL expression in HepG2 cells by selectively targeting its 3′-UTR at the 3728–3735 bp site and we established the primary association of miR-429 with CRKL in hepatocarcinoma . Herein,CRKL-3′-UTR at the 3728–3735 bp site. Meanwhile, ETV6 forms complexes with CRKL and promotes CRKL expression, which in turn promotes ETV6 expression. Furthermore, CRKL overexpression potentially promotes hepatocarcinoma malignancy by activating ERK pathway.As illustrated in Fig. Hepatocarcinogenesis is a very complex biological process associated with environmental risk factors and hereditary factors, including abnormal activation of Wnt/β-catenin, MAPK, AKT, and ERK signaling pathways, and the balance between activation and inactivation of proto-oncogenes and anti-oncogenes, and the differentiation of liver cancer stem cells . Ras/RafOur results showed that an ETV6-miR-429-CRKL regulatory circuitry contributes to aggressiveness of HCC by activating Ras/Raf/ERK pathway. ETV6 is known to forms fusion protein with PDGFR, CRKL can function in cellular signaling cascades by directly forms complex with downstream receptor protein to regulate cellular tyrosine kinase activity, meanwhile, some miRNAs have been evaluated for the prediction of response to sorafenib treatment in HCC . While, CRKL, ETV6 acts as tumor promotors and miR-429 acts as a tumor suppressor in HCC, their dysexpressions are involved in the carcinogenesis and progression of hepatocarcinoma. We have uncovered a novel ETV6-miR-429-CRKL regulatory circuitry that contributes to hepatocarcinoma tumorigenesis. The newly identified ETV6-miR-429-CRKL regulatory circuitry from the current work provides new insights into the underlying mechanism of pathogenesis in HCC and represents a potential therapeutic target for diagnosis and treatment of HCC.Additional file 1.


Ucp1 and Pgc-1α. The mechanisms underlying ATF2 target activation are unknown. Here we demonstrate that p62 (Sqstm1) binds to ATF2 to orchestrate activation of the Ucp1 enhancer and Pgc-1α promoter. P62Δ69-251 mice show reduced expression of Ucp1 and Pgc-1α with impaired ATF2 genomic binding. Modulation of Ucp1 and Pgc-1α expression through p62 regulation of ATF2 signaling is demonstrated in vitro and in vivo in p62Δ69-251 mice, global p62−/− and Ucp1-Cre p62flx/flx mice. BAT dysfunction resulting from p62 deficiency is manifest after birth and obesity subsequently develops despite normal food intake, intestinal nutrient absorption and locomotor activity. In summary, our data identify p62 as a master regulator of BAT function in that it controls the Ucp1 pathway through regulation of ATF2 genomic binding.During β-adrenergic stimulation of brown adipose tissue (BAT), p38 phosphorylates the activating transcription factor 2 (ATF2) which then translocates to the nucleus to activate the expression of  Ucp1 and Pgc-1α. Here, the authors show that the scaffold protein p62 regulates brown adipose tissue function through modifying ATF2 genomic binding and subsequent Ucp1 and Pgc-1α induction.Beta-adrenergic stimulation of brown adipose tissue leads to thermogenesis via the activating transcription factor 2 (ATF2) mediated expression of the thermogenic genes  In adult humans, the mass and activity of BAT inversely correlates with body mass index (BMI) and percent body fat6, and sympathetic nervous system (SNS) activation by cold exposure9, or by β3-adrenergic receptor agonism10 enhances BAT thermogenesis and energy expenditure. The mitogen-activated protein kinase (MAPK) p38α, the key regulator of adaptive thermogenesis, modulates uncoupling protein 1 (Ucp1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α) expression via phosphorylation, and action of the activating transcription factor 2 (ATF2)12. Upon activation by p38α, p-ATF2 translocates to the nucleus and initiates expression of Ucp1 and Ppargc1a (Pgc-1α) via binding to cAMP responsive elements (CRE) in the Ucp1 enhancer and Pgc-1α promoter11. Highlighting the key role of this pathway in UCP1 action, cold-induced β-adrenergic receptor stimulation fails to promote Ucp1 expression in mouse BAT primary cells pretreated with the p38α/β MAPK inhibitor SB20219012. While nuclear entry and action of ATF2 is crucial for BAT adaptive thermogenesis12, the mechanism underlying ATF2 target activation is unknown13.Having long been considered dispensable, brown adipose tissue (BAT) in adult humans is now recognized today as an organ of physiological relevance for energy metabolism14. Mice with global15 or adipose-specific16 deletion of p62 have a severe obese phenotype with normal food intake but decreased energy expenditure and impaired BAT function. Global p62−/− mice also have enhanced adipogenesis with hyperphosphorylation of the extracellular signal-regulated kinase (ERK1/2) in the white adipose tissue (WAT)15. Loss of adipogenic capacity by ERK1/2 deletion can prevent obesity in global p62−/− mice17, emphasizing uncertainty as to whether the dysregulated energy metabolism in p62-deficient mice originates from enhanced adipogenesis and/or impaired energy expenditure16. The aim of this paper was to dissect the molecular foundations underlying energy metabolism control by p62 and to identify the molecular mechanisms of how p62 regulates BAT thermogenesis. Our data demonstrate that mice that lack the amino acids (aa) 69–251 of the p62 protein (p62Δ69-251 mice) have normal protein levels of p-ERK1/2 in WAT and show no changes in adipogenesis or adipocyte differentiation. However, these mice develop a severe obese phenotype that is accompanied by impaired energy expenditure and dysfunctional BAT. In a series of in vitro and in vivo experiments using p62Δ69-251 mice, global p62-deficient mice (p62−/−) and Ucp1-Cre p62flx/flx mice, we demonstrate that p62 is a key signaling node of the UCP1 pathway. p62 directly binds to ATF2 to orchestrate its genomic binding to and activation of the Ucp1 enhancer and Pgc-1α promoter. As demonstrated in p62Δ69-251 mice, global p62−/− mice and Ucp1-Cre p62flx/flx mice, lack of p62 action leads to failure of ATF2 to activate its nuclear targets Ucp1 and Pgc1α and results in impaired BAT function and increased body weight. The cell autonomous effect of p62 to modulate ATF2 nuclear target activation is confirmed in BAT primary cells from p62Δ69-251 mice and is verified in cultured BAT cells of global p62−/− mice and Ucp1-Cre p62flx/flx mice. Our data establish p62 as a key regulator of adaptive thermogenesis in that it regulates the UCP1 pathway via modulation of ATF2 genomic target activation.Scaffold proteins are key mediators of selective and efficient cell signal transduction, which they achieve through direct and specific interaction with their target proteins. The scaffold protein p62  is a multimodular adaptor protein involved in key metabolic processes like tissue inflammation, cell differentiation, cell growth, and tumorigenesisΔ69-251 mice). Mice were bred on the C57BL/6J background and were designed to yield a truncated p62 protein of ∼37 kDa that lacks the zinc finger (zz) domain, the TB1 domain and one of the two p38 interacting motifs, but to otherwise maintain regular p62 function  . This is further supported by unaltered Oil Red O staining during differentiation of BAT and iWAT primary cells   following administration of 18F-FDG, is reduced in p62Δ69-251 mice  and thus functional mitochondrial complex IV , and cell death-inducing DNA fragmentation factor alpha subunit-like effector A (Cidea) in BAT  increased in BAT of obese p62Δ69-251 mice, with unchanged levels of p38 and p-p38  Fig. . These dice Fig.  despite  IV Fig. . The impBAT Fig. . Expressups Fig. . NotablyBAT Fig. , we see p38 Fig. . Since pΔ69-251 pups relative to wt controls, as assessed by infrared thermography , Dio2, and Cidea are decreased in BAT  are confirmed also in BAT and iWAT primary cells harvested from young non-obese p62Δ69-251 mice . In BAT of these mice, expression of p62 is decreased by ~75% relative to wt controls  are decreased in in vitro cultured brown adipocytes of the Ucp1-Cre p62flx/flx mice  and promoter (CRE4), and the Pgc-1α promoter11. Importantly, ATF2 binding to target loci was severely impaired in BAT from p62Δ69-251 mice  using a targeting vector that replaces exons two to four of p62 with a neomycin-resistance cassette. Ucp1-Cre p62flx/flx mice were generated by pairing p62flx/flx mice with mice expressing Cre recombinase under control of the Ucp1 promoter.Mice were kept at a constant environment with ambient temperature set to 22 ± 2 °C with constant humidity (45–65%) and a 12 h/12 h light/dark cycle. Mice had free access to water and were fed ad libitum with either a standard chow  or HFD . Animal experiments were performed in accordance with European guidelines under permission of the local animal ethics committee of the state of Bavaria or the Regional Council Tübingen. Whole-body composition (fat and lean tissue mass) was measured using nuclear magnetic resonance technology . For glucose and insulin tolerance test, mice were fasted for 6 h and then challenged with 1.75 g glucose per kg body weight or 0.75 U insulin  per kg body weight. Generation of full-length p62 knock-out mouse lines p622 and CO2 were measured every 10 min for 4–5 days. Data for energy expenditure were analyzed using ANCOVA with body weight as covariate as previously suggested18. For ANCOVA analysis, energy expenditure raw data were blotted against the individual body weight. Every individual data point was then slighted up or down the group’s own regression line until all data line up at a predefined body weight of 40 g. The result of this ANCOVA correction by body weight was then plotted as a bar graph as previously suggested24.Energy expenditure and home-cage activity were assessed using a climate-controlled combined indirect calorimetric system . After acclimatization for 24 h, levels of O−1) or saline control. Afterward, the temperature of the climate chamber was gradually decreased from 30 to 23 °C (2 days), to 10 °C (1 day), to 5 °C (1 day). Tissues were collected at the end of the study.Body weight and body composition was measured at the beginning and end of the study. Mice were acclimatized to thermoneutrality (30 °C) for 1 day, followed by subsequent measurement of energy expenditure starting at 30 °C for another day. On Day 3, a non-shivering thermogenesis (NST) test was performed at 30 °C. For the NST test, mice were i.p. injected with a single bolus of Norepinephrine  were performed using a dedicated micro-PET scanner . The spatial resolution was 1.4 mm (center of field of view), the axial field of view was 12.7 cm. All PET scans were acquired with an energy window of 350–650 keV and a coincidence timing window of 3.432 ns. All major corrections were applied to the PET scans , but no attenuation correction was used as only mice on thin plastic beds were scanned. PET-images were reconstructed using the three-dimensional ordered-subset-expectation-maximization (OSEM3D) algorithm with a 128 × 128 matrix and a final pixel size of 0.79 × 0.79 ×0.80 mm³.Animals were injected i.p. with 11.7 ± 0.3 MBq 2-weighted anatomical MR-images were acquired using the following 3D-spoiled turbo spin echo sequence: matrix size: 256 × 256, FOV: 35 × 57 mm2, repetition time = 800 ms, echo time = 30.8 ms, slice thickness = 0.25 mm, flip angle 90°, echo train length 16.Animals were transferred on-bed to a 7-Tesla small animal MRI scanner  to ensure identical mouse positioning on-bed for PET and MRI acquisitions. TInveon Research Workplace  was used for data analysis. PET and MRI data were co-registered for exact, MRI-guided delineation of volumes of interest (VOI), drawn manually for BAT based on the MR image. PET data were expressed as standardized uptake value (SUV), calculated as measured activity in BAT divided by injected activity and body weight.For surface body temperature measurement, 7-d-old pups were temporarily placed in a 6-well dish for temperature recording via a thermal imaging camera Optris, OPTPI400 .Eout) from the energy intake (Ein). Chow control diet was combusted to calculate energy content of the absorbed food. Bomb calorimetric analysis was performed using a 6300 Oxygen Bomb Calorimeter .For assessment of assimilated energy, food intake and feces production were assessed over a 5-d period. Feces and food were dessicated in a drying oven (50 °C) for bomb calorimetric combustion. The assimilated energy was then calculated by subtracting the energy lost with feces  or saline control and BAT was collected after 20 min in fractionation buffer  with proteinase and phosphatase inhibitors (Thermo Fisher). After homogenization at 16 Hz for 2 min, samples were centrifuged at 750 × g for 5 min at 4 °C. The supernatant was collected and centrifuged at 10,000 rpm, 10 min (cytoplasmic fraction). This process was repeated four times to remove nuclear contamination. The pellet was washed with fractionation buffer and centrifuged at 750 × g, 5 min to remove cytoplasmic contamination at least three times. The nuclear/membrane fraction was dissolved in RIPA buffer.For the protein fractionation, p62n-dodecyl-β-D-maltoside) and subjected to a temperature-controlled reaction chamber containing 130 μM cytochrome c from horse heart  and 18 mM ascorbate in measuring buffer .Cox activity of interscapular BAT was assessed polarographically using a Clark type electrode  and a Powerlab for data processing . Tissue was weighed and homogenized mechanically in tissue buffer  using QIAGEN TissueLyser . Homogenates were treated with detergent  neutrally buffered formalin, embedded in paraffin, cut into 3-µm slices and a hematoxylin and eosin (H&E) staining was performed. The stained tissue sections were scanned with an AxioScan.Z1 digital slide scanner  equipped with a ×20 magnification objective. Quantification of adipocytes was performed using ImageJ software.−1 Collagenase IV ; 3 U/ml Dispase II ; 0,01 mM CaCl2 in PBS). Cell suspension was filtered through a 100 µm cell strainer, centrifuged (500 × g for 10 min) and resuspended in growth medium DMEM/F12 (1:1) plus Glutamax , 1% Pen/Strep and 10% FBS after each step. Homogenate was filtered through a 70 µm cell strainer. Primary white adipocytes were plated onto collagen-coated dishes  and grown to confluence . At confluence, cells were exposed to an adipogenic cocktail containing dexamethasone (1 µM), isobutylmethylxanthine (0.5 mM), rosiglitazone (1 µM) and insulin (5 µg ml−1) in growth medium. After 48-h induction, cells were maintained in culture medium containing insulin (5 µg ml−1) and rosiglitazone (1 µM). On Day 4 of differentiation, cells were cultured in growth medium containing insulin (5 µg ml−1). To stimulate thermogenesis, cells were treated with 0.5 µM Isoproterenol , dissolved in serum-free growth medium, for 6 h on Day 6 of adipocyte differentiation.Inguinal white adipose tissue was obtained from 6- to 8-wk-old male C57Bl/6J  mice. Fat pads were minced and digested for 40 min at 37 °C (1 mg ml−1) in growth medium. Two days post-induction of differentiation, cells were maintained in culture medium containing rosiglitazone (1 µM), T3 (1 nM), and insulin (0.5 µg ml−1). On Day 4 of differentiation, cells were cultured in growth medium containing T3 (1 nM) and insulin (0.5 µg ml−1).Primary brown adipocytes were isolated via the same procedure with the exception that 1 mg/ml Collagenase II  was used for digestion. For differentiation of primary brown adipocytes, the induction cocktail contained dexamethasone (5 µM), isobutylmethylxanthine (0.5 mM), rosiglitazone (1 µM), indomethacin (125 µM), T3 (1 nM), and insulin  during different days of differentiation. Adipocytes were stained with Oil Red O  for 10 min and immediately washed with HN = 3 per group) was profiled with quantitative PCR-based (qPCR) techniques using SYBR green or TaqMan Single Probes . The relative expression of the selected genes was measured using the 7900HT Fast Real-Time PCR System . The relative expression levels of each gene were normalized to the housekeeping gene peptidylprolyl isomerase B (Ppib) or Hypoxanthine Phosphoribosyltransferase (Hprt). TaqMan Low Density Array  was performed according to instructions. The following primers were used: Ucp1 forward 5ʹ-GGCCTCTACGACTCAGTCCA-3ʹ, Ucp1 reverse 5ʹ-TAAGCCGGCTGAGATCTTGT-3ʹ, Pgc-1α forward 5ʹ-AGCCGTGACCACTGACAACGAG-3ʹ, Pgc-1α reverse 5ʹ-GCTGCATGGTTCTGAGTGCTAAG-3ʹ, Prdm16 forward 5ʹ-CCGCTGTGATGAGTGTGATG-3ʹ, Prdm16 reverse 5ʹ-GGACGATCATGTGTTGCTCC-3ʹ, Bmp8b forward 5ʹ-TCCACCAACCACGCCACTAT-3ʹ, Bmp8b reverse 5ʹ-CAGTAGGCACACAGCACACCT-3ʹ, p62/Sqstm1 wild type forward 5ʹ-CCAGTGATGAGGAGCTGACA-3ʹ, p62/Sqstm1 wild type reverse 5ʹ-CCGTTGCAACCATCACAGAT-3ʹ, p62/Sqstm1 mutant delta 69–251 forward 5ʹ-CACTACCGCGGCATTGAG-3ʹ, p62/Sqstm1 mutant delta 69–251 reverse 5ʹ-AGGTTTGCTGACTTCCGAAG-3ʹ. Cidea forward 5ʹ-AATGGACACCGGGTAGTAAGT-3ʹ, Cidea reverse 5ʹ-CAGCCTGTATAGGTCGAAGGT-3ʹ, Dio2 forward 5ʹ-TGCCACCTTCTTGACTTTGC-3ʹ, Dio2 reverse 5ʹ-GGTTCCGGTGCTTCTTAACC-3ʹ, Adiponectin forward 5ʹ-GGTCCTAAGGGTGAGACAGG-3ʹ, Adiponectin reverse 5ʹ-AGTCCCGGAATGTTGCAGTA-3ʹ, Fasn forward 5ʹ-AGAGATCCCGAGACGCTTCT-3ʹ, Fasn reverse 5ʹ-GCTTGGTCCTTTGAAGTCGAAGA-3ʹ, Fabp4 forward 5ʹ-CAGCGTAAATGGGGATTTGG-3ʹ, Fabp4 reverse 5ʹ-CCGCCATCTAGGGTTATGAT-3ʹ, Pparg forward 5ʹ-TCGCTGATGCACTGCCTATG-3ʹ, Pparg reverse 5ʹ-GAGAGGTCCACAGAGCTGATT-3ʹ. The following taqman probes were used: p62 Mm01070495_m1, Ucp1 Mm01244861_m1, Pgc-1α Mm01208835_m1, Dio2 Mm00515664_m1, Cidea Mm00432554_m1, Bmp8b Mm00432115_g1. Hprt Mm01545399_m1, Jun Mm00495062_s, Fos Mm00487425_m1, JunB Mm04243546_s1, Atf3 Mm00476033_m1, Atm Mm01177457_m1, Xpa Mm00457111_m1, Rad23b Mm00772280_m1, Msh6 Mm00487761_m1, Nos2 Mm00440502_m1, Hspa5 Mm00517691_m1, Ccna1 Mm01292554_m1, Ccnd1 Mm00432359_m1, Tnf Mm00443258_m1, Il8 Mm00441263_m1, Il1b Mm00434228_m1, Il6 Mm00446190_m1, Th Mm00447557_m1, Col1a1 Mm00801666_g1, Mmp2 Mm00439498_m1, Sele Mm00441278_m1, Selp Mm01295931_m1, Vcam1 Mm01320970_m1, Ppargc1 Mm01208835_m1, Ucp1 Mm01244861_m1, Zfp516 Mm00813584_m1, Mapk14 Mm01301009_m1, Creb1 Mm00501607_m1, Brca1 Mm00515386_m1, Bcl2L1 Mm00437783_m1, Ddit3 Mm01135937_g1, Adrb2 Mm02524224_s1, Map2k6 Mm00803694_m1, Ep300 Mm00625535_m1, Bcl6 Mm00477633_m1, Nfil3 Mm00600292_s1, Pdgfra Mm00440701_m1, Atf6 Mm01295319_m1, Atf4 Mm00515325_g1, Ulk1 Mm00437238_m1, Atg7 Mm00512209_m1, Atf13 Mm00521135_m1, Map1lc3a Mm00458724_m1.Total RNA was prepared using RNeasy Kit  according to manufacturer’s instructions. cDNA synthesis was performed with QuantiTect Reverse Transcription Kit  according to manufacturer’s instructions. Gene expression of cell samples , isoproterenol , 2,4-Dinitrophenol (DNP) , rotenone (5 µM)/antimycin (2 µM)/2-Deoxyglucose (100 mM) (15 min). For normalization, the cell plate was subsequently co-stained with Dapi and Nile red and the fluorescence signal was detected to correct for cell number and differentiation. Proton leak respiration was calculated by subtraction of Rot/AA/2DG-OCR from Oligomycin-OCR.Primary BAT cells were isolated and differentiated for 5 d on a collagen-coated XF24 well plate. On Day 5, cells were washed with DMEM XF Assay medium , supplemented with 25 mM glucose, no pyruvate and no fatty-acid free BSA. All port compounds were dissolved in pure DMEM XF Assay medium without supplements and 10-fold higher concentrated compounds were loaded into the ports of a XF Assay Cartridge. Oxygen consumption rate (OCR) was measured using an extracellular flux analyzer . Basal OCR was recorded for 15 min followed by measurement of OCR after injection of oligomycin  or HEK293T cells  were cultured in 6-well plate and transfected with 1.5 μg Flag-p62 and 1.5 μg HA-ATF2 plasmid per well by polyethylenimine  for 24 h. Isoproterenol (10 μM) was added half an hour before the cells were collected. The protein lysate of 1500 μg was incubated with anti-Flag-agarose  overnight for immunoprecipitation, together with 50 μg lysate as input to be loaded for western blotting detection of HA  or Flag tag (anti-Flag). Antibodies were obtained from the following sources: Anti-HA (C29F4) , anti-FLAG-M2 , anti-beta-actin , anti-rabbit , anti-mouse .2, tissues were rapidly frozen on dry ice and stored at −80 °C until further analysis. Fat tissues were lysed in ice-cold RIPA buffer  containing protease/phosphatase inhibitor , 1 mM PMSF , and 10 nM Calyculin A  using a polytron. Proteins were separated on Criterion gel  and transferred onto nitrocellulose membranes. Membranes were incubated with primary antibody  overnight at 4 °C, and HRP-coupled secondary antibody  was utilized to detect protein signal via the LI-COR imaging system or the BioRad Chemidoc system. The following antibodies were used:, ATF2 , ATF2 , ATF2 ; dilution 1:70), GAPDH , GAPDH , H3 , p-ATF2 , p-ATF2 , p-p38 , p-p38 , p38 , p38alpha , Ucp1 , Ucp1 , p-PKC , ERK1/2 , LC3A/B , ATG7 , alpha Tubulin , p-p44/42 MAPK (ERK1/2) . Images of uncropped blots are provided in the Source data file.For protein quantification, mice were euthanized by CO25, using two different ATF2 antibodies ATF2  or rabbit IgG  and two different p62 antibodies  and p62 . Immunoprecipitated DNA was decrosslinked, purified and used for quantitative real-time PCR. Primer sequences used are the following: Foxl2 forward 5ʹ-GCTGGCAGAATAGCATCCG-3ʹ, Foxl2 reverse 5ʹ-TGATGAAGCACTCGTTGAGGC-3ʹ, Jun forward 5ʹ-CGCAGCGGAGCATTACCTC-3ʹ, Jun reverse 5ʹ-GCCTGAGCTCAACACTTATCTG-3ʹ, Fos forward 5ʹ-GCTGAGGCGCCTACTACTCC-3ʹ, Fos reverse 5ʹ-TCATGGTCGAAGTTTGGGGAAA-3ʹ, Atf3 forward 5ʹ-TTAGCCGATTGGCTCCACTG-3ʹ, Atf3 reverse 5ʹ-CGTCAGCCTGGGATTGGTAA-3ʹ, Ucp1 (CRE2) forward 5ʹ-AGTGAAGCTTGCTGTCACTC-3ʹ, Ucp1 (CRE2) reverse 5ʹ-GTCTGAGGAAAGGGTTGACC-3ʹ, Ucp1 (CRE4) forward 5ʹ-GAGTGACGCGCGGCTGGG-3ʹ, Ucp1 (CRE4) reverse 5ʹ-GGGCTAGGTAGTGCCAGT-3ʹ, Pgc-1α forward 5ʹ-ACTATAGGGCACGCGTGGTCGACG-3ʹ, Pgc-1α reverse 5ʹ-ACACAGAGCACACACTCATGCAGG-3ʹ.ChIP-qPCR of mouse brown adipose tissue was performed with sonicated nuclear extract prepared from formaldehyde-cross-linked tissue, according to Mir et al.26, pcDNA3.1-C-DYK-p62wt, pcDNA3.1-C-DYK-p62∆69-251, pcDNA3.1. The ATF2 reporter (pCRE-Luc) was a kind gift from Takayuki Akimoto.ATF2 transcriptional activity was monitored in vitro using a reporter construct consisting of four tandem copies of the CRE-binding sequence TGACGTCA linked to luciferase. HEK293T cells  were transiently transfected with the following plasmids using Lipofectamine 2000 according to manufacturer’s : pCRE-Luc, pRL-TK, pcDNA3-Flag MKK6(K82A) Transfection was performed in collagen A-coated 96-well plates using 0.25 µg plasmid DNA and 0.875 µl Lipofectamine 2000 reagent in a volume of 25 µl Optimem media. To warrant equal DNA uptake of the cells, the plasmid DNA concentration was adjusted with an empty vector (pcDNA3.1). CRE-binding activity reporter assay was performed using the Dual-Glo luciferase assay , which allows normalization to transfection efficiency by simultaneous measurement of firefly and renilla (pRL-TK) luminescence. Therefore, the pRL-TK construct was routineously co-transfected to all samples. Luciferase assay was performed according to protocol and luminescence signal was measured using a PHERAstar plate reader.−/− mice were transiently transfected with the following plasmids using Lipfectamine P3000 according to manufacturer’s protocol : pcDNA3.1 and pcDNA3.1-C-DYK-p62wt. Transfection was performed on Day 4 of differentiation in 6-well plates using 4 µg plasmid DNA and 12 µl Lipofectamine reagent in 250 µl OptiMem medium. Cells were harvested in RIPA buffer for western blot analysis on Day 6 of differentiation.Primary brown adipocytes isolated from p62t-test, one-way ANOVA, or two-way ANOVA with time and genotype as co-variants followed by Bonferroni’s post hoc multiple comparison testing for individual time points. All results are given as mean ± SEM. P < 0.05 was considered statistically significant. Differences in energy expenditure were calculated using ANCOVA with body weight as covariate using SPSS (version 24). For animal studies, sample sizes were calculated based on a power analysis assuming that a ≥5 g difference in body weight between genotypes can be assessed with a power of ≥75% when using a two-sided statistical test under the assumption of a standard deviation of 3.5 and an alpha level of 0.05.Statistical analyses were performed using statistical tools in GraphPad Prism8 (version 8.3.0). Differences between groups were assessed by Student’s two-sided two-tailed Further information on research design is available in the Supplementary InformationReporting Summary


E)-3-(2-aryl-2-oxoethylidene)indolin-2-ones, also in excellent yield, and again characterized spectroscopically. The structures of four of the former and five of the latter are reported, and all show different patterns of supramolecular assembly.Ten novel 3-(2-aryl-2-oxoethyl)-3-hydroxyindolin-2-ones have been synthesized, mostly in excellent yield, and characterized spectroscopically, and nine of these have been dehydrated to the corresponding ( N-substituted 3-(2-aryl-2-oxoethyl)-3-hydroxyindolin-2-ones by a piperidine-catalysed aldol reaction between aryl methyl ketones and N-alkylisatins. These aldol products were used successfully as strategic intermediates for the preparation of N-substituted (E)-3-(2-hetaryl-2-oxoethylidene)indolin-2-ones by a stereoselective dehydration reaction under acidic conditions. The products have all been fully characterized by 1H and 13C NMR spectroscopy, by mass spectrometry and, for a representative selection, by crystal structure analysis. In each of (RS)-1-benzyl-3-hydroxy-3-[2-(4-methoxyphenyl)-2-oxoethyl]indolin-2-one, C24H21NO4, (Ic), and (RS)-1-benzyl-3-{2-[4-(dimethylamino)phenyl]-2-oxoethyl}-3-hydroxyindolin-2-one, C25H24N2O3, (Id), inversion-related pairs of molecules are linked by O—H⋯O hydrogen bonds to form R22(10) rings, which are further linked into chains of rings by a combination of C—H⋯O and C—H⋯π(arene) hydrogen bonds in (Ic) and by C—H⋯π(arene) hydrogen bonds in (Id). The molecules of (RS)-1-benzyl-3-hydroxy-3-[2-oxo-2-(pyridin-4-yl)ethyl]indolin-2-one, C22H18N2O3, (Ie), are linked into a three-dimensional framework structure by a combination of O—H⋯N, C—H⋯O and C—H⋯π(arene) hydrogen bonds. (RS)-3-[2--2-oxoethyl]-1-benzyl-3-hydroxyindolin-2-one, C24H19NO5, (If), crystallizes with Z′ = 2 in the space group PE)-1-benzyl-3-[2-(4-fluorophenyl)-2-oxoethylidene]indolin-2-one, C23H16FNO2, (IIa), and (E)-1-benzyl-3-[2-oxo-2-(thiophen-2-yl)ethylidene]indolin-2-one, C21H15NO2S, (IIg), the molecules are linked into simple chains by a single C—H⋯O hydrogen bond, while those of (E)-1-benzyl-3-[2-oxo-2-(pyridin-4-yl)ethylidene]indolin-2-one, C22H16N2O2, (IIe), are linked by three C—H⋯O hydrogen bonds to form sheets which are further linked into a three-dimensional structure by C—H⋯π(arene) hydrogen bonds. There are no hydrogen bonds in the structures of either (E)-1-benzyl-3-[2-(4-methoxyphenyl)-2-oxoethylidene]indolin-2-one, C24H19NO3, (IIc), or (E)-1-benzyl-5-chloro-3-[2-(4-chlorophenyl)-2-oxoethylidene]indolin-2-one, C23H15Cl2NO2, (IIh), but the molecules of (IIh) are linked into chains of π-stacked dimers by a combination of C—Cl⋯π(arene) and aromatic π–π stacking interactions.An operationally simple and time-efficient approach has been developed for the synthesis of racemic  For the synthesis of the 3-hydroxyindolin-2-ones, (I)  (1.0 mmol), the appropriate aryl methyl ketone (1.0 mmol) and piperidine (0.2 mmol) in ethanol (10 ml) was stirred at 298 K for 6 h [24 h in the case of compound (Id)], after which time the starting materials were no longer detectable using thin-layer chromatography (TLC). The resulting solid products were collected by filtration, washed with cold ethanol (2 ml) and dried in air to give the products of type (I). Analytical data for compound (Ia): yield 93%, m.p. 437–438 K : yield 91%, m.p. 430–431 K : yield 85%, m.p. 430 K : yield 36%, m.p. 450 K; compound (Ie): yield 92%, m.p. 479–481 K; compound (If): yield 87%, m.p. 452–453 K; compound (Ig): yield 82%, m.p. 421–422 K : yield 89%, m.p. 423 K; compound (Ii): yield 83%, m.p. 398 K. Colourless crystals of compounds (Ic), (Id), (Ie) and (If) suitable for single-crystal X-ray diffraction analysis were grown by slow evaporation, at ambient temperature and in the presence of air, of solutions in ethanol–dimethylformamide (6:1 v/v).Isatins (a): yield 92%, m.p. 425–426 K; compound (IIb): yield 85%, m.p. 417–418 K; compound (IIc): yield 93%, m.p. 393 K; compound (IIe): yield 97%, m.p. 398 K; compound (IIf): yield 88%, m.p. 388 K; compound (IIg): yield 91%, m.p. 388–389 K; compound (IIh): yield 89%, m.p. 401–403 K; compound (IIi): yield 83%, m.p. 376–378 K. Crystals of compounds (IIa) (orange), and (IIb), (IIc), (IIe) and (IIg)  suitable for single-crystal X-ray diffraction analysis were grown by slow evaporation, at ambient temperature and in the presence of air, of solutions in ethanol–dimethylformamide .For the conversion of the 3-hydroxy compounds (I) into the ethylidene products (II), a solution of the appropriate 3-hydroxy compound (I) (0.50 mmol) in glacial acetic acid (2 ml) was stirred at ambient temperature for 10 min, during which time concentrated hydrochloric acid (0.1 ml) was added slowly. The resulting mixtures were then stirred at 333 K for a further 2 h. For each mixture, the pH was then brought to 7.0 by the addition of a concentrated aqueous solution of ammonia, and the resulting solid products were collected by filtration, washed with cold water and dried in air to give the products of type (II). Analytical data for compound (IIa), (Ib), (Ic) and (Ig) have been reported recently in the literature  for the other compounds reported here are provided in the supporting information.Spectroscopic data for compounds (Ia) was handled as a non-merohedral twin with the twin matrix (e) was also handled as a non-merohedral twin, with the twin matrix (g), the thiophene unit is disordered over two sets of atomic sites having unequal occupancies for the minor-disorder component, and the bonded and -nonbonded distances were restrained to be the same as the corresponding distances in the major-disorder component, subject to s.u. values of 0.01 and 0.02 Å, respectively; in addition, the anisotropic displacement parameters of pairs of partial-occupancy atoms occupying essentially the same physical space were constrained to be equal. All H atoms, apart from those in the minor-disorder component of compound (IIg), were located in difference maps. H atoms bonded to C atoms were then treated as riding atoms in geometrically idealized positions, with C—H = 0.95 , 0.98 (CH3) or 0.99 Å (CH2) and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms; the H atoms in the minor-disorder component of compound (IIg) were included on the same basis, giving refined disorder occupancies of 0.9387 (19) and 0.0613 (19). For the H atoms bonded to O atoms, the atomic coordinates were refined with Uiso(H) = 1.5Ueq(O), giving the O—H distances shown in Table 2Crystal data, data collection and structure refinement details are summarized in Table 1A),  consistently in excess of 90%. Focusing primarily on the N-benzyl intermediate of type (B), the subsequent reactions with aryl methyl ketones in the presence of piperidine did indeed provide generally much higher yields of the products of type (I), usually well above 80%, than had previously been achieved using isatin carrying no substituent at the N atom, which is consistent with the idea of partial proton transfer from the N-unsubstituted isatin to piperidine. The yields in both steps appear to be much the same regardless of whether the substituent at the N atom is benzyl or 1-hexyl, or whether the substituent at C5 is H or Cl. The only exception was found for compound (Id), where the yield was quite low, 36%, even after a much longer reaction time than that required for all the other type (I) products; this may be associated with the strongly electron-donating nature of the dimethylamino group. Acid-catalysed dehydration of nine of products (I) gave the N-substituted (E)-3-(2-aryl-2-oxoethylidene)indolin-2-ones (II), again with yields well above 80%, although, because of the slow formation and poor yields of (Id) in the first step, the dehydration of this intermediate was not pursued.The title compounds were synthesized starting from the readily available isatins (c), (Id) and (Ie), and at atoms C13 and C23 in the two independent molecules in (If). In every case, the reference molecule was selected as one having the R configuration at this atom, but the space groups  and (II)  (Table 2a) and (IIe), but not in any of (IIc), (IIg) and (IIh). On the other hand, the conformations of the rest of the molecule relative to the indolinone unit is broadly similar in both type (I) and type (II) compounds, aside from the pyridyl derivative (Ie). In each of 4-methoxy derivatives (Ic) and (IIc), the two exocyclic C—C—O angles at atom C324  and (IIc), respectively.In each of the aldol compounds of type (I) Figs. 1 ▸ ▸, tc) are linked into a chain of rings by a combination of O—H⋯O, C—H⋯O and C—H⋯π(arene) hydrogen bonds  chain motif  is very simple, taking the form of a chain of rings running parallel to the  direction  is likewise very simple, with a single C—H⋯O hydrogen bond linking molecules which are related by the a-glide plane at z = C(7) chain running parallel to the [100] direction  means that the hydrogen bonding in these structures is simpler than that found in compounds of type (I). Thus, for each of compounds  into a sheet lying parallel to (010) and lying in the domain y < 1.0 , the molecules are nonetheless linked into a chain by the combination of a C—Cl⋯π(arene) interaction and a π–π stacking interaction. In the first of these interactions, atom Cl34 in the molecule at  forms a short contact with the C3A/C4–C7/C7A ring in the molecule at , with geometric parameters Cl⋯Cg = 3.6055 (8) Å and C—Cl⋯Cg = 88.71 (5)°, where Cg represents the centroid of the aryl ring. The Cl⋯Cg distance here may be compared with the average value of 2.6° deduced from a database analysis of such contacts  and the C321–C326 ring at  make a dihedral angle of only 7.58 (7)°. The ring-centroid separation is 3.7374 (9) Å and the shortest perpendicular distance from the centroid of one ring to the plane of the other is 3.3592 (6) Å, corresponding to a ring-centroid offset of ca 1.64 Å. The combination of these two interactions thus generates a chain of π-stacked dimers lying parallel to the [100] direction , (Id) and (If), paired O—H⋯O hydrogen bonds generate c) and (Id), although the ring in (If) exhibits no crystallographic symmetry. By contrast, the structure of pyridyl derivative (Ie) contains no O—H⋯O hydrogen bonds -2-oxocyclohexane-1,3-diyl]bis[-3-hydroxyindolin-2-one] which was crystallized as a dehydrate  global, Ic, Id, Ie, If, IIa, IIc, IIe, IIg, IIh. DOI: Click here for additional data file.10.1107/S2053229620004143/sk3748Icsup11.cmlSupporting information file. DOI: 10.1107/S2053229620004143/sk3748Icsup2.hklStructure factors: contains datablock(s) Ic. DOI: Click here for additional data file.10.1107/S2053229620004143/sk3748Idsup12.cmlSupporting information file. DOI: 10.1107/S2053229620004143/sk3748Idsup3.hklStructure factors: contains datablock(s) Id. DOI: Click here for additional data file.10.1107/S2053229620004143/sk3748Iesup13.cmlSupporting information file. DOI: 10.1107/S2053229620004143/sk3748Iesup4.hklStructure factors: contains datablock(s) Ie. DOI: Click here for additional data file.10.1107/S2053229620004143/sk3748Ifsup14.cmlSupporting information file. DOI: 10.1107/S2053229620004143/sk3748Ifsup5.hklStructure factors: contains datablock(s) If. DOI: Click here for additional data file.10.1107/S2053229620004143/sk3748IIasup15.cmlSupporting information file. DOI: 10.1107/S2053229620004143/sk3748IIasup6.hklStructure factors: contains datablock(s) IIa. DOI: Click here for additional data file.10.1107/S2053229620004143/sk3748IIcsup16.cmlSupporting information file. DOI: 10.1107/S2053229620004143/sk3748IIcsup7.hklStructure factors: contains datablock(s) IIc. DOI: Click here for additional data file.10.1107/S2053229620004143/sk3748IIesup17.cmlSupporting information file. DOI: 10.1107/S2053229620004143/sk3748IIesup8.hklStructure factors: contains datablock(s) IIe. DOI: Click here for additional data file.10.1107/S2053229620004143/sk3748IIgsup18.cmlSupporting information file. DOI: 10.1107/S2053229620004143/sk3748IIgsup9.hklStructure factors: contains datablock(s) IIg. DOI: 10.1107/S2053229620004143/sk3748IIhsup10.hklStructure factors: contains datablock(s) IIh. DOI: Click here for additional data file.10.1107/S2053229620004143/sk3748IIhsup19.cmlSupporting information file. DOI: 10.1107/S2053229620004143/sk3748sup20.txtSpectroscopic data for compounds (Id)-(If), (Ih), (Ii), (IIa)-(IIc) and (IIe)-(IIi). DOI: 1992762, 1992761, 1992760, 1992759, 1992758, 1992757, 1992756, 1992755, 1992754CCDC references:


MO1–MO9) containing the morpholine moiety were assessed for their inhibitory activities against monoamine oxidases (MAOs) and acetylcholinesterase (AChE). Most of the compounds potently inhibited MAO-B; MO1 most potently inhibited with an IC50 value of 0.030 µM, followed by MO7 (0.25 µM). MO5 most potently inhibited AChE (IC50 = 6.1 µM), followed by MO9 (IC50 = 12.01 µM) and MO7 most potently inhibited MAO-A (IC50 = 7.1 µM). MO1 was a reversible mixed-type inhibitor of MAO-B (iK = 0.018 µM); MO5 reversibly competitively inhibited AChE (iK = 2.52 µM); and MO9 reversibly noncompetitively inhibited AChE (iK = 7.04 µM). MO1, MO5 and MO9 crossed the blood–brain barrier, and were non-toxic to normal VERO cells. These results show that MO1 is a selective inhibitor of MAO-B and that MO5 is a dual-acting inhibitor of AChE and MAO-B, and that both should be considered candidates for the treatment of Alzheimer’s disease.Nine compounds ( The isoenzymes of MAO-A and MAO-B are considered major therapeutic targets in various neuropsychiatric illnesses and neurodegenerative disorders like Alzheimer’s disease (AD) and Parkinson’s disease (PD)Morpholine is a tetrahydro-1,4-oxazine with a saturated heterocyclic ring and provides a promising developmental starting point due to its biological profile with metabolic stability. The presence of a heteroatom like oxygen or nitrogen facilitates hydrogen bonding, and thus, interactions with enzymes, and the presence of electron-deficient atoms may also impart hydrophobic interactions with morpholine. From the synthetic perspective, various molecular scaffolds have been added to morpholine by replacing its secondary nitrogenA and B rings of chalcones can impart electrophilic character to the Michael acceptor present. The introductions of electron-donating/lipophilic halogens onto the phenyl B ring of chalcones have resulted in the syntheses of highly selective MAO-B inhibitorsA ring provides AChE inhibitory activityA ring favoured hMAO-B inhibitory activity,B ring was substituted using a fluoro or trifluoromethyl groupThe chalcones framework is considered an excellent starting point for the design for MAO-B and AChE enzyme inhibitors2.2.1.para substituted aldehydes and 4-morpholine acetophenone in the presence of an alcoholic basic medium1 NMR showed that the ring protons N-CH2 and O-CH2 of morpholine ring resonated at 3.32–3.31 and 3.75–3.88 ppm as triplets, respectively. The large coupling constant (J = 15 Hz) value of Hα and Hβ protons confirmed the trans configuration of the morpholine chalcones. The deshielded values between 186.88–186.07 in C13-NMR clearly evidenced the formation of sp2 carbonyl functional group of α,β-unsaturated ketones. The identities of the compounds were determined by 1H-NMR, 13 C-NMR and Mass spectrometry .Morpholine-containing α,β-unsaturated ketones were prepared by condensation between various aromatic/2.2.2.2.1.MO1–MO9) were analysed with respect to their abilities to inhibit MAO-A, MAO-B and AChE. Most of the compounds inhibited MAO-B by ∼ 50% at 1.0 µM, except MO3 and MO5 (MO1 most potently inhibited MAO-B (IC50 = 0.030 µM), followed by MO7, MO8, MO4, MO9, MO6, MO2, MO3 and MO5 . In addition, MO5 most potently inhibited AChE (IC50 = 6.1 µM), followed by MO9 and MO8 . MO7 most potently inhibited MAO-A (IC50 = 7.1 µM), followed by MO6 (IC50 = 8.7 µM). MO5 most potently inhibited BChE (IC50 = 18.09 µM), with weaker inhibitory activity compared to AChE, followed by MO7 (IC50 = 24.83 µM)  .para position of the phenyl B ring of the basic chalcone scaffold afforded different derivatives. Surprisingly, unsubstituted MO1 inhibited MAO-B more potently than the other eight derivatives. All nine derivatives exhibited MAO-B selectivity. In particular, MO1 (IC50 = 0.030 µM) was ∼2 times more potent than the reversible MAO-B inhibitor lazabemide (IC50 = 0.063 µM), and had a high selectivity index (SI = >1333.3), which meant that it was the best inhibitor in the series. Regarding MAO-B inhibition, MO1 was slightly less potent than the reference irreversible MAO-B inhibitor pargyline (IC50 = 0.028 µM). The presence of dimethylamino, chloro and bromo substituents at the para position of the chalcone B ring conferred moderate AChE inhibition. Recently, it was reported that shifting of morpholine ring to the phenyl B ring of chalcones showed moderate AChE inhibitionMorpholine-containing compounds in this study shared a 1–(4-morpholinophenyl) prop-2-en-1-one structure. Introduction of various electron-donating and withdrawing groups onto the 2.2.2.MO1 showed that MO1 is a mixed inhibitor of MAO-B (iK = 0.018 ± 0.002)  . These r2.2.3.MO5 and MO9 competitively and non-competitively, respectively, inhibited AChE  to 77.1% (AD), which was similar to that shown by lazabemide (from 38.8 to 90.0%), a reversible MAO-B inhibitor  to 79.2% (AD) and from 33.8 to 80.7%, respectively, which were similar to that observed for tacrine (from 32.8 to 91.7%), a reversible AChE inhibitor  > 4.00, CNS- (low): Pe (10−6 cm s−1) <2.00 and CNS± (uncertain): Pe (10−6 cm s−1) from 4.00 to 2.00. Our results suggest that all nine synthesised derivatives could cross the BBB, and that MO1, MO5 and MO9 would be the most effective.The synthesised derivatives were screened for their abilities to cross the BBB using the parallel artificial membrane permeability assay (PAMPA) because this ability is a critical developmental requirement for any drug targeting neurodegenerative disorders or depression. The PAMPA used was validated by comparing the experimentally determined permeabilities of eight commercial drugs with reported values . Accordi2.4.MO1, MO5 and MO9 were investigated using an MTT -2,5-diphenyltetrazolium bromide) assay and normal VERO cellsMO1, MO5 and MO9 were non-toxic to normal VERO cells with IC50 values of 195.14, 185.44 and 188.34 µg/mL, respectively, indicating that the therapeutic potentials of these compounds would be suitable.The biocompatibilities of 2.5.MO1, MO5 and MO9 were subjected to ROS assays using Hela cells. ROS levels significantly increased in H2O2-treated Hela cells as determined by fluorescent image analyses  as entries 2Z5X, 2VZ5 and 4EY7MO1 and MO5 and MAO-A, MAO-B and AChE. The docking scores resulting from in silico simulations are reported in MO1 and MO5 hydrogen-bonded with F295 and formed π − π interactions with the side chain of W286 at the peripheral anionic subsite (PAS) of AChE . MO1 and MO5 formed π − π interactions with the selective MAO-B residue Y326  inhibited MAO-B inhibition in the low nanomolar range and was more potent than lazabemide (the reference compound). The low cytotoxicity and the ability of MO1 to transit the BBB support our drug design strategy. In addition, the ROS scavenging efficacy of MO1 suggests improved neuroprotective effects.We describe the synthesis of morpholine-containing α,β-unsaturated ketones, and the results of an investigation of their MAOs and AChE inhibition profiles. Most of the nine compounds synthesised exhibited potent MAO-B inhibition with moderate AChE inhibition. Interestingly, 4.4.1.,S-butyrylthiocholine iodide (BTCI), respectively, as substrates for 10 min at 412 nmMAO-A activity was measured continuously for 20 min at 316 nm using 0.06 mM kynuramine as substrate, as described previously4.2.50 values were determined by measuring the residual enzyme activities. Toloxatone, lazabemide and tacrine, were used as reference reversible inhibitors of MAO-A, MAO-B and AChE, respectively, and clorgyline and pargyline as reference irreversible inhibitors of MAO-A and MAO-B, respectively. iK values and inhibitor types were determined by kinetic testing, as previously described50, IC50, and 2 × IC50 values. Lineweaver–Burk plots and their secondary plots were used to determine iK values and inhibitor types.MAO-A, MAO-B, AChE and BChE activities were measured after exposure to inhibitors at a concentration of 10 µM. Inhibitions of MAO-B at 10 µM of these compounds tested were too excessive, and thus, a concentration of 1.0 µM was used. IC4.3.51,52, but by using 0.06 mM kynuramine and 0.3 mM benzylamine. For the experiment of AChE, 0.5 mM ATCI was used. Dialysis experiments were performed by preincubating enzymes and inhibitors (or reference inhibitors) at ∼2 × IC50 in 0.1 M sodium phosphate buffer (pH 7.2) for 30 min, and then dialysing solutions with stirring for 6 h with a buffer change at 3 h. Residual activities were calculated using the measured activities of undialyzed (AU) and dialysed (AD) solutions and the activities of untreated controls (i.e. without inhibitor).Inhibition types (reversible or irreversible) were determined by dialysis, as previously describedClick here for additional data file.


Scientific Reports 10.1038/s41598-019-47350-x, published online 26 July 2019Correction to: Figure 2F contains errors. The panel labelled as ‘Lawsone, 5 days, 10 μM’ was inadvertently duplicated from the ‘DMSO, 10 days, 10 μM’. The correct Figure 2 appears below as Figure


Scutellaria nepetoides M. Pop. and its structure has been established by X-ray crystallographic analysis.5-Hydroxy-3,6,7,8-tetramethoxyflavone was isolated from a butanol extract of the herb  H-chromen-4-one), C19H18O7, is a flavone that was isolated from a butanol extract of the herb Scutellaria nepetoides M. Pop. The flavone molecule is almost planar, with a dihedral angle between the planes of the benzopyran-4-one group and the attached phenyl ring of 6.4 (4)°. The 5-hydroxy group forms a strong intramolecular hydrogen bond with the carbonyl group, resulting in a six-membered hydrogen-bonded ring. The crystal structure has triclinic  and H⋯O/O⋯H (20.9%) interactions.The title compound (systematic name: 5-hydroxy-3,6,7,8-tetramethoxy-2-phenyl-4 Scutellaria L. are widespread in Europe, North America, East Asia and are extensively used in traditional Chinese medicine   with butanol (5 l) at 353 K. The butanol filtrates were collected and concentrated under reduced pressure to provide 10.2 g of butanol extract. The butanol extract (1 g) was subjected to silica gel (60–100 mesh) column  as eluent, and five fractions were collected according to TLC analysis. All fractions were concentrated under reduced pressure. A crystallization procedure with different solvents at high temperature was used to obtain the pure compounds. Fraction 5 (0.23 g) was eluted with butanol (100%) at 353 K and with ethanol (95%) at 343 K. The obtained polycrystals were removed from the butanol solution by filtration. Yellow prismatic single crystals were prepared by slow evaporation of butanol solution at room temperature.Air-dried whole plants (1.1 kg) of 3), 0.93 Å (aryl H) and O—H = 0.82 Å and with Uiso(H) = 1.2Ueq(C) (aryl H) and 1.5Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020013596/zn2001sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020013596/zn2001Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020013596/zn2001Isup3.cmlSupporting information file. DOI: 2036551CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Cassia auriculata seeds was found to inhibit melanogenesis in B16 melanoma 4A5 cells under conditions of theophylline stimulation. Two new phlegmacin-type anthracenone dimer glycosides, auriculataosides A and B, were isolated from the active methanol fraction, and their inhibitory effects were observed in the concentration range of 0.03 to 0.3 μM. Inhibition of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 protein expression was observed, suggesting that the inhibition of these factors is part of the mechanism of action underlying melanogenesis inhibition.The methanol extract of The online version of this article (10.1007/s11418-018-01276-2) contains supplementary material, which is available to authorized users. Melanocytes originate from the ganglion in the embryonic phase and are distributed in the epidermal basal layer, hair follicles, uvea of the eye, soft brain membrane, oral mucosa, inner ear, peritoneal membrane, and eye socket. The formation of cutaneous melanin is a normal physiological phenomenon that plays an important role in protecting skin from UV injury , 2. HoweCassia auriculata is a perennial evergreen shrub that is native to India, Sri Lanka, and other parts of Asia. The flower, leaves, stem, root, and unripe fruit of this shrub have been used for the treatment of diabetes, conjunctivitis, and skin diseases in Ayurveda, Indian traditional medicine. We have reported the isolation of various active compounds, including benzocoumarins, flavones, flavonols, and a flavanol dimer, with hepatoprotective effects in vitro from the leaves of C. auriculate , two methylenes , two methoxy groups , five aromatic protons , and a β-d-glucopyranosyl moiety .Auriculataosides A  and B   -anhydrophlegmacin-9,10-quinone 8′-O-methyl ether  having the S configuration at the 7–10′ stereostructure exhibited a positive Cotton effect (Δε + 22.6) at 267 nm and a negative Cotton effect (Δε − 34.4) at 283 nm  to give nine fractions (Fr.) .The seeds of 2O  → MeOH]  to  give 13 fractions . Fr. 5-5 (1991 mg) was separated and purified by HPLC [MeOH:H2O  or CH3CN:MeOH:H2O ] to give rumejaposide E  and rumejaposide F . Fr. 5-6 (588 mg) was also subjected to HPLC [MeOH:H2O ] to give rumejaposide E  and rumejaposide F . Fr. 5-8 (609 mg) was subjected to silica gel column chromatography [CHCl3 → CHCl3:MeOH  → CHCl3:MeOH:H2O ] and HPLC [MeOH:H2O ] to give auriculataoside A . Fr. 5-9 (269 mg) was separated by HPLC [MeOH:H2O ] to furnish auriculataoside A  and auriculataoside B . Fr. 5-10 (400 mg) was purified by HPLC [MeOH:H2O ] to obtain auriculataoside B .Fr. 5 (10.4 g) was further separated by ODS column chromatography D24 – 129.5 ; UV (MeOH) λmax (log ε) 225 (4.58), 273 (4.71), 399 (4.09), 433 (4.02) nm; CD (MeOH) nm (Δε) 217 (+ 49.0), 234 (− 17.2), 242 (−6.7), 270 (− 38.0), 284 (+ 25.5); IR (KBr) vmax 3450, 2948, 1655, 1070 cm−1; 1H-NMR  1.15, 2.24 , 2.61 , 2.74, 2.88 , 3.21 , 3.35 , 3.43 , 3.47 , 3.48 , 3.65, 3.89 , 3.74 , 4.79 , 5.04 , 6.20, 6.88 , 7.22, 7.59 , 7.55 , 11.88 , 12.19 , 19.20 ; 13C-NMR data, see Table m/z 755 [M+Na]+; high-resolution (HR)FAB-MS: m/z 755.1958 .Red powder; [α]D24 – 41.3 ; UV (MeOH) λmax (log ε) 225 (4.62), 273 (4.74), 399 (3.72), 433 (4.05) nm; CD (MeOH) nm (Δε) 217 (– 42.8), 233 (+ 20.5), 268 (+ 23.8), 282 (− 33.9); IR (KBr) vmax 3450, 2948, 1655, 1070 cm−1; 1H-NMR  1.18, 2.24 , 2.57, 2.75 , 2.72, 2.90 , 3.22 , 3.35 , 3.44 , 3.47 , 3.50 , 3.66, 3.85 , 3.75 , 4.78 , 5.02 , 6.20, 6.89 , 7.18, 7.53 , 7.52 , 11.90 , 12.27 , 19.20 ; 13C-NMR data, see Table m/z 755 [M+Na]+; HRFAB-MS: m/z 755.1952 .Red powder;  equipped with an optical rotation detector . d-glucose (from 1 and 2) was confirmed by comparing its retention time (tR) and optical rotation with those of an authentic sample; tR = 11.5 min .Compounds Dulbecco’s modified Eagle’s medium  was purchased from Sigma-Aldrich ; fetal bovine serum (FBS). Penicillin, and streptomycin were purchased from Gibco . The tetrazolium salt MTT was obtained from Dojindo Laboratories ; Soluene-350 was obtained from PerkinElmer, Inc. ; Blocking One was from Nacalai Tesque ; phosphatase inhibitor was purchased from Roche ; proteinase inhibitor, the Protein Assay kit, and other chemicals were purchased from Wako Pure Chemical Industries, Ltd. Six- and 24-well multiwell plates and 96-well microplates were purchased from Greiner Japan .2 and 37 °C. The HDF cells were grown in DMEM (1000 mg/L glucose) medium. Cells were harvested by incubation in phosphate-buffered saline (PBS) containing 1 mM EDTA and 0.25% trypsin for approximately 5 min at 37 °C and were used for subsequent bioassays.Murine B16 melanoma 4A5 cells (RCB0557) were obtained from Riken Cell Bank  and HDF (code no. CA10605n) were purchased from Toyobo Co., Ltd. . The B16 melanoma 4A5 cells were grown in DMEM (4500 mg/L glucose) medium (Sigma-Aldrich) containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin under conditions of 5% CO4 cells/400 μL/well) were seeded in a 24-well multiwell plate and pre-cultured for 24 h , following which the test sample and theophylline  were added. After culturing for 72 h, the cells were detached and recovered by trypsin treatment, washed with PBS, and dissolved in NaOH aq. . The cell lysate was fractionated (100 μL/well) in a 96-well microplate, and the absorbance of melanin produced was measured with a microplate reader  (wavelength 405 nm). The test sample was dissolved in DMSO and added to the medium . Inhibition was measured as:A is the absorbance without test sample (control); B is the absorbance with test sample; C is cell viability (%) with test sample.The screening test for melanogenesis using B16 melanoma 4A5 cells was performed as described previously . B16 mel3 cells/100 μL/well) and HDF (5.0 × 103 cells/100 μL/well) were seeded in a 96-well microplate and pre-cultured for 24 h , following which the test sample and theophylline  were added. After culturing for 71 h , 10 μL of MTT (0.5%) was added. After incubation for 1 h , the formazan present, which was produced as a catalytic product of MTT, was dissolved in 100 µL of 2-propanol containing 0.04 M HCl, and the absorbance was measured with a microplate reader . The test sample was dissolved in DMSO and added to the medium . Viability of the cells was measured as:A is the absorbance without test sample (control); B is the absorbance with test sample.B16 melanoma 4A5 cells (5.0 × 10A is the absorbance without test sample (control); B is the absorbance with test sample.Inhibitory effects on mushroom tyrosinase activity were examined as reported elsewhere , 31. A pg for 10 min. The supernatant was used as the crude enzyme solution, and the amount of enzyme was quantified using an enzymatic reaction, as follows. Phosphate buffer solution (pH 6.5) containing 2.5 mM L-DOPA (80 μL/well) was dispensed into a 96-well microplate, and 10 μL of test substance (DMSO solution) was added. Next, crude tyrosinase solution was diluted with phosphate buffer solution (pH 6.5) to 10 µg/mL protein. The diluted enzyme solution was add to each well (10 μL/well), and the enzymatic reaction (37 °C) was carried out for 24 h. A microplate reader  was used to measure the absorbance of melanin (measurement wavelength 405 nm). Inhibition was measured as:A is the absorbance without test sample (control); B is the absorbance with test sample.Inhibitory effects on melanoma tyrosinase activity were examined using methods described elsewhere, with slight modifications –33. B16 g for 15 min. After the residue was washed with ethanol, it was dissolved in 1 mL of Soluene-350 (60 °C), and the absorbance of the melanin produced was measured using a microplate reader  (wavelength 405 nm). Inhibition was measured as:A is the absorbance without test sample (control); B is the absorbance with test sample.The autoxidation of dopachrome was performed according to previously reported methods, with slight modifications , 37. A p5 cells/2 mL/well) were seeded in a 6-well multiwell plate and pre-incubated for 24 h, following which the test sample and theophylline  were added and the cell solution incubated for a further 72 h. Then, 140 μL of lysis buffer was added after washing with PBS. Cells detached with a cell scraper were disrupted with an ultrasonic cell disruptor under cooling with ice to extract the protein. The supernatant was centrifuged at 11,000 g for 10 min. The amount of protein was determined by the Lowry method and denatured at 100 °C for 5 min.B16 melanoma 4A5 cells  was electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels for 1 h, washed with Tris-buffered saline (T-TBS), and transferred to a membrane (PVDF membrane) using a transfer device. After washing with T-TBS, blocking was carried out with Blocking One  for 1 h, and incubation was commenced with primary antibody against each protein in 5% Blocking One/T-TBS solution (0.2 μg/mL) for 1 h. After washing with T-TBS, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody in 5% Blocking One/T-TBS solution (0.3 μg/mL) for 1 h. Immunoreactive proteins were detected using a chemiluminescence kit  according to the manufacturer’s instructions. Each target band was measured by a lumino image analyzer ; Multi Gauge V3.0 software was used to analyze the data (Fujifilm).The primary antibody was diluted 1000-fold. Anti-tyrosinase (goat), anti-TRP-1 (rabbit), and anti-TRP-2 (rabbit) were purchased from Santa Cruz Biotechnology . Anti-MITF (rabbit) and anti-β-actin (rabbit) were from Cell Signaling Technology . The secondary antibody was diluted 4000-fold. Anti-rabbit antibody was from Cell Signaling Technology and rabbit anti-goat antibody was from Thermo Fisher Scientific .P values of < 0.05 were regarded as significant.Values were expressed as the mean ± standard error of the mean. A one-way analysis of variance followed by Dunnett’s test was used to determine the statistical significance of the differences between the control group and test sample-treated groups. Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 533 kb)


Ceramide, a bioactive lipid, plays an essential role in the development of several pulmonary inflammatory diseases. Matrix metallopeptidase 9 (MMP-9) regulates the synthesis and degradation of extracellular matrix, and is associated with airway remodeling and tissue injury. This study was conducted to investigate the effects and underlying mechanisms of ceramide on MMP-9 expression in airway epithelium.signal transducer and activator of transcription 3 (STAT3) inhibitor. The cells were then stimulated with C6-ceramide. The levels of MMP-9 were determined by ELISA and real-time quantitative PCR (RT-qPCR). JAK2, phosphorylated JAK2 (p-JAK2), STAT3, and phosphorylated STAT3 (p-STAT3) expression was examined by Western blotting. BALB/c mice were pretreated with AG490 or Stattic before intratracheally instillated with C6-ceramide. Pathological changes in lung tissues were examined by Hematoxylin and Eosin staining, Periodic-acid Schiff staining, and Masson’s trichrome staining. MMP-9, JAK2, p-JAK2, STAT3, and p-STAT3 expression in the lung tissues was examined by Western blotting.BEAS-2B cells, normal human bronchial epithelium cell lines, were pretreated with AG490, a selective janus tyrosine kinase 2 (JAK2) inhibitor, or Stattic, a selective The expression of MMP-9, p-JAK2 and p-STAT3 in BEAS-2B cells was significantly increased after the treatment of C6-ceramide. Furthermore, the increased expression of MMP-9 induced by C6-ceramide was inhibited by AG490 and Stattic. Similar results were obtained in the lung tissues of C6-ceramide-exposed mice which were treated with AG490 or Stattic.Ceramide could up-regulate MMP-9 expression through the activation of the JAK2/STAT3 pathway in airway epithelium. Targeted modulation of the ceramide signaling pathway may offer a potential therapeutic approach for inhibiting MMP-9 expression. This study points to a potentially novel approach to alleviating airway remodeling in inflammatory airway diseases. Airway epithelium is a pivotal structure with the greatest surface exposed to a large number of external stimulus, including respiratory viruses, air pollutants, and allergens. The airway epithelium also plays a significant role in the first line of immunological defense by supporting the activation, recruitment, and mobilization of immune cells . HoweverCeramide, a bioactive lipid, has been implicated in a variety of physiological and pathological processes, such as cell proliferation, differentiation, apoptosis, and inflammation , 11. VarMatrix metalloproteinase 9 (MMP-9), an important enzyme regulating the synthesis and degradation of ECM, is associated with airway remodeling and lung injury in asthma and COPD. MMP-9 directly degrades ECM proteins and is central in facilitating the trafficking of inflammatory cells from the parenchyma to the airways during inflammation . CeramidThe objective of this study was therefore to investigate whether ceramide could stimulate MMP-9 expression in airway epithelium. We also aimed to determine the mechanisms of ceramide on MMP-9 expression.BEAS-2B cell lines were acquired from the American Type Culture Collection . C6-ceramide, ammonium pyrrolidinedithiocarbamate (PDTC), tyrphostin B42 (AG490), and 6-Nitrobenzo[b]thiophene-1, 1-dioxide (Stattic) were acquired from Sigma-Aldrich . Dulbecco’s Modified Eagle’s Medium (DMEM) and Fetal bovine serum (FBS) were obtained from HyClone . Anti-JAK2, anti-phosphorylated JAK2 (p-JAK2), anti-STAT3, anti-phosphorylated STAT3 (p-STAT3), anti-CD68, and anti-β-actin antibodies were acquired from Cell Signaling Technology . IRDye 680RD Goat anti-Rabbit and Goat anti-Mouse IgG (H + L) was acquired from LI-COR Biosciences . TRIzol was acquired from Invitrogen Corporation . TB Green Mixture was acquired from TaKaRa .4 cells/well in 96-well plates and cultured overnight. BEAS-2B cells were then treated with AG490 at 20, 10, 5, or 2.5 μM or Stattic at 2, 1, 0.5, or 0.25 μM for 24 h. We added 0.1% DMSO to control wells. The media was then changed to fresh media, and 10 μL of CCK8 solution was added per well. The optical densities (OD) at 450 nm were measured 4 h later using a microplate reader . Cell viabilities were expressed as percentage of controls cultured with 0.1% DMSO.Cell viability was determined by the Cell Counting Kit-8 (CCK8) assay. BEAS-2B cells were seeded at a density of 1 × 105 cells/well in 6-well plates and cultured overnight. BEAS-2B cells were treated with C6-ceramide  for 24 h in serum-free media. DMSO (0.1%) was added to control wells. In the experiments involving AG490 and Stattic, AG490  and Stattic  were added to the cultures 2 h prior to the addition of C6-ceramide. First-strand cDNAs were synthesized using Prime Script RT Reagent kit . Real-time quantitative PCR (RT-qPCR) was performed using TB Green Mixture . The primer sequences were as follows: human MMP-9, forward: 5′-GATCATTCCTCAGTGCCGGA-3′, reverse: 5′-TTCAGGGCGAGGACCATAGA-3′; human GAPDH, forward: 5′-CCACATCGCTCAGACACCAT − 3′, reverse: 5′- TTGACGGTGCCATGGAATTT-3′. The relative mRNA levels of MMP-9 were determined with GAPDH as control and were represented as 2-△△Ct. MMP-9 protein levels in cell culture supernatant were determined by ELISA .BEAS-2B cells were seeded at a density of 3 × 105 cells/well in 6-well plates and incubated overnight. Then the cells were treated with C6-ceramide  for 24 h in serum-free media. DMSO (0.1%) was added to control wells. JAK2, p-JAK2, STAT3, and p-STAT3 levels were examined by Western blotting. Band density was quantified by QuantiScan Version 11 .BEAS-2B cells were seeded at a density of 3 × 10n = 6): the naive normal control group was exposed to vehicle control; the C6-ceramide group was exposed to C6-ceramide and pretreated with 5% DMSO; the AG490 group was exposed to C6-ceramide and was pretreated with AG490 ; and the Stattic group was exposed to C6-ceramide and was pretreated with Stattic . These animals were exposed to C6-ceramide at 5 mg / kg of body weight or vehicle control by intratracheal instillation for 3 days. One hour before C6-ceramide exposure on each day, the animals were intraperitoneally injected with AG490, Stattic, or 5% DMSO as indicated above. The lung tissues were then fixed with 4% buffered formalin solution. Pathological changes in the lung tissues were examined using Hematoxylin and Eosin (H&E) staining, Periodic Acid-Schiff (PAS) staining, and Masson’s trichrome staining. CD68 staining was conducted by immunohistochemistry. MMP-9, JAK2, p-JAK2, STAT3, and p-STAT3 levels in lung tissues were examined by Western blotting.Specifc pathogen free male BALB/c mice  were randomly divided into 4 groups , 4.68 ± 0.32 (P < 0.01), and 3.82 ± 0.15 (P < 0.01), respectively , 3361.10 ± 139.19 pg/mL (P < 0.01), and 1536.25 ± 53.05 pg/mL (P < 0.01), respectively  and 76.10% (range 76.19–82.40%) of control (P = 0.01)  Fig. , respectP < 0.01), 2.21 ± 0.12 (P < 0.01), and 3.17 ± 0.67 (P < 0.01), respectively , 2553.77 ± 111.76 pg/mL (P < 0.01), and 3674.43 ± 448.99 pg/mL (P < 0.01), respectively  and 1997.43 ± 38.07 pg/mL (P < 0.01), respectively, similar to the effect of 10 μM AG490. Meanwhile, when the cells were pretreated with 1 μM, 0.5 μM, and 0.25 μM of Stattic for 2 h, the relative MMP-9 mRNA levels decreased from 4.75 ± 0.05 to 2.00 ± 0.02 (P < 0.01), 2.94 ± 0.08 (P < 0.01), and 3.59 ± 0.06 (P < 0.01), respectively , 2879.90 ± 303.29 pg/mL (P < 0.01), and 3194.83 ± 318.90 pg/mL (P < 0.01), respectively  and 1997.43 ± 38.07 pg/mL (P < 0.01), respectively, similar to the effect of 1 μM Stattic. Meanwhile, AG490 and Stattic did not affect the expression of MMP-9 in the absence C6-ceramide . Treatment with PDTC, the NF-κB inhibitor, had no significant effect on MMP-9 expression (data not shown). These results suggested that the JAK2/STAT3 signaling pathway may be involved in ceramide-induced MMP-9 expression.BEAS-2B cells were pretreated with signal pathway inhibitors, such as PDTC (NF-κB inhibitor), AG490 (JAK2 inhibitor), and Stattic (STAT3 inhibitor), for 2 h before being incubated with 10 μM of C6-ceramide for 24 h. MMP-9 levels were determined by RT-qPCR and ELISA. Compared to the cells incubated with C6-ceramide only, pretreatment with 10 μM, 5 μM, and 2.5 μM of AG490 decreased the relative MMP-9 mRNA levels from 4.52 ± 0.59 to 1.69 ± 0.05 , 0.79 ± 0.05 (P = 0.04), and 0.75 ± 0.08 (P = 0.05), respectively , 0.68 ± 0.04 (P < 0.01), and 0.64 ± 0.02 (P < 0.01), respectively , while the pretreatment with AG490 and Stattic decreased this level to 3.65 ± 0.28 (P = 0.03) and 4.82 ± 0.26 (P = 0.02), respectively , while the pretreatment with AG490 and Stattic decreased this level to 0.60 ± 0.02 (P = 0.03) and 0.59 ± 0.07 (P = 0.02), respectively     and 0.56 ± 0.03 (P = 0.04), respectively  Fig. , and inc01) Fig. . Treatme04) Fig. . In a siely Fig. .Fig. 5DIncreased ceramide level is associated with chronic lung diseases, including asthma, COPD, and emphysema , 13, 19.MMPs play an essential role in tissue remodeling and they also contribute to excessive breakdown of connective tissue in inflammatory disorders, such as asthma, COPD, and rheumatoid arthritis. MMP-7 and MMP-9 are the predominant forms of MMPs expressed by epithelial cells in lung tissues . MMP-9 dCeramide could induce the activation of various inflammatory signaling pathways, including NLRP3/Caspase1/IL-1β, JAK/STAT and NF-κB –26, 36. The strengths of this study were listed as following: First, the study reported the effect of ceramide on MMP-9 production in airway epithelium which has not been reported. Second, this study clarify the pathway involved in ceramide-induced MMP-9 expression in airway epithelium. Some limitations of the study deserve to be mentioned. Firstly, JAK2 knock-out mice and STAT3 knock-out mice are needed to confirm these results. Secondly, MMP-9 expression was only partly inhibited by AG490 and Stattic. In airway epithelium, other signaling pathway may be involved in ceramide-induced MMP-9 expresison. Thus, further studies are needed to investigate the involvement of other signaling pathway in ceramide-induced MMP-9 expression in airway epithelium.In conclusion, the findings of the present study suggest that ceramide may induce MMP-9 expression in airway epithelium via the JAK2/STAT3 pathway. Therefore, targeted modulation of the ceramide signaling pathway may offer a potential therapeutic approach for inhibiting MMP-9 expression in airway epithelium. Further understanding of the mechanisms responsible for pathological remodeling may highlight novel therapeutic strategies in chronic inflammatory airway disease, such as asthma and COPD.Additional file 1: Figure S1.