| BackgroundPancreatic stellate cell(PaSC)is a type of pluripotent cell located between pancreatic lobules and the surrounding area of acinars.When activated,PaSC can be transformed into myofibroblast-like cell.A number of evidences suggest that activated PaSC is the main source of the accumulation of extracellular matrix(ECM)protein under the pathological conditions,which lead to pancreatic fibrosis in chronic pancreatitis and pancreatic cancer.Activation of PaSC play a key role in pancreatic fibrosis.Damage to the pancreatic tissue triggers activation of PaSC,in response to inflammatory mediators,alcohol metabolites or growth factors such as the platelet-derived growth factor(PDGF)or transforming growth factors TGF-α and TGF-β.These activating factors are present in the inflamed pancreas and are secreted by infiltrating cells,platelets,endothelial cells or pancreatic acinar cells.Importantly,PaSC themselves may be able to secrete certain growth factors(e.g.PDGF)or cytokines and thus facilitate their activation in auto-or paracrine manner.Many studies search for signaling pathways and factors related to fibrosis in order to reverse the process of pancreatic fibrosis.Bromodomain extral terminus(BET)is a kind of bromodomain protein family which consists of four BET proteins:Brd2,Brd3,Brd4,and bromodomain testis-specific protein(BRDT),the latter of which is expressed in germ cells.These proteins contain two N-terminal bromodomains which bind to acetylated lysine residues on histone tails.Brd4 in particular inhibits tumor progress by downregulation of MYC expression.Kumar et al found that BET inhibitor JQ1 reduces production of collagen type I in PaSC,and inhibits pancreatic fibrosis in PD AC mice.We aim to explore the effects of BET inhibitors on PaSC and the implying mechanism to reverse pancreatic fibrosis.The Hippo/YAP pathway has attracted intense interest as a key regulator of organ size and tissue growth,and more recently of cell differentiation,growth and tumorigenesis in several cancer types including PD AC.The co-transcriptional regulator activity of YAP and its homolog TAZ modulate the expression of genes involved in various aspects of cellular functions,such as proliferation and cell motility.Previous studies demonstrated YAP overexpression in pancreatic disorders,and proposed YAP inhibition as a potential strategy to block immune responses and tumor cell proliferation in PD AC.YAP was reported to take part in the activation of hepatic stellate cell.However,the role(s)of YAP and TAZ in PaSC activation,remained unknown until recently.We aim to explore the role of YAP in PaSC in our research to reverse pancreatic fibrosis.Part One:The effects of BET inhibitors on pancreatic stellate cell activation,proliferation and ECM productionObjective:To evaluate the effects of BET inhibitors on PaSC activation,proliferation and the production of extracellular matrix,as well as to observe the impact of BET inhibitors JQ1 on the level of pancreatic fibrosis in mice with chronic pancreatitis(CP).Methods:(1)Western-Blot was used to measure the effects of iBET151 with different concentrations on protein levels of PaSC marker α-SMA,collagen I,fibronectin(FN),cadherin(CDH11)and MAPK/ERK signaling pathway in both immortalized pancreatic stellate cells(imPaSC)and human pancreatic stellate cells(hPaSC).(2)Cell proliferation was determined by MTT assay.(3)ECM production was studied by quantitative real-time polymerase chain reaction(q-PCR).(4)Western-Blot was performed to measure the effects of 1μM iBET151 on PaSC marker α-SMA,collagen I,FN,CDH11,platelet-derived growth factor receptor-β(PDGFR-β)and MAPK/ERK signaling pathway after stimulation of 5ng/ml TGF-β.(5)ECM production was studied by q-PCR in imPaSC with 5ng/ml TGF-β stimulation.(6)In this study,24 male C57BL/6 male mice(Envigo,Placentia,CA)were subjected to repeated episodes of acute cerulein pancreatitis starting at 6-7 weeks of age.CP group(n=6)was induced by repeated cerulein AP episodes(twice a week for 4 weeks;total 8 episodes),and mice were sacrificed 4 days after the last AP episode.JQ1 group(n=6)was established by 50 mg/kg JQ1 in DMSO/(2-hydroxypropyl)-β-cyclodextrin(5 days per week).The control group(n=6)was peritoneal injection with the same amount of DMSO/(2-hydroxypropyl)-β-cyclodextrin.Body weight and were measured daily.HE staining and Mason staining were used to evaluate histopathological changes and collagen deposition.Protein expressions related to pancreatic fibrosis were detected by Western blot.Activation of pancreatic stellate cells(PaSC)in pancreatic exocrine of CP mice were evaluated by immunofluorescence staining.Results:(1)After 6h,24h and 48h intervention,BET inhibitors induced stellate cell quiescence,as suggested by marked decreases in protein levels of the activation markers alpha-SMA,collagen I,FN,CDH11,PDGFR-β,and sustained expression of the quiescent marker GFAP in a dose-dependent manner.(2)Compared with control group,iBET151 reduced proliferation of imPaSC obviously by MTT assay.(3)iBET151 decreased RNA levels of the activation markers α-SMA,collagen I,and increased the quiescent marker of PaSC,Glial fibrillary acidic protein(GFAP).In addition,the gene expressions of some inflammatory cytokines including interleukin 6(IL-6),monocyte chemotactic protein-1(MCP-1),fibroblast activation protein(FAP),and hepatocyte growth factor(HGF)were reduced by iBET151.(4)5ng/ml TGF-β was found to increase protein levels of α-SMA,fibronectin,CDH11 in imPaSC,and 1μM iBET151 helped reduce the activation of PaSC by TGF-β.(5)q-PCR indicated that 5ng/ml TGF-β elevated RNA levels of α-SMA,fibronectin,CDH11,IL-6 and MCP-1 in imPaSC,and 1μM iBET151 helped reverse the increased RNA levels stimulated by TGF-β.(6)JQ1 inhibited the weight gain of mice obviously;H&E staining showed obvious pancreatic acinar atrophy,fibrosis and inflammatory cell infiltration in both CP group and JQ1+CP group,and JQ1 didn’t reverse the destruction level.Trichrome staining showed a large amount of collagen deposition in pancreas in both CP and CP+JQ1 group,and JQ1 didn’t decrease the collagen deposition.IF staining showed some α-SMA(+)PaSC in both CP and CP+JQ1 group,but no significant difference was found between CP and CP+JQ1 group.Western blot showed increased protein levels of α-SMA、FN、ColIal、CDH11、PDGFR-β in both CP and CP+JQ1 group,but no significant difference was found between CP and CP+JQ1 group.Conclusion:BET inhibitors reduced PaSC activation,proliferation and ECM production,as well as the secretion of some inflammatory cytokines and growth factors.In addition,BET inhbitors decreased the activation of PaSC by TGF-β.Part Two:The role of BRD2 and BRD4 in pancreatic stellate cell activation,proliferation and ECM productionObjective:In part one,we found inhibitory effects of BET inhibitors on PaSC activation,proliferation and the production of extracellular matrix.siRNA againt BRD2 and BRD4 was used to further illuminate the role of BRDs in PaSC activation and regulating pro-fibrosing responses.Methods:(1)siRNA against BRD2 and BRD4(siBRD2,siBRD4)and nonspecific control siRNAs(siControl)were synthesized by Dharmacon.In vitro transcription steps were according to the RNAi manual of Dharmacon.qPCR was used to evaluate the effects of RNAi.(2)Western-Blot was used to measure the effects of siBRD2 and siBRD4 on protein levels of PaSC marker α-SMA,collagen Ial,FN,CDH11,p-ERK and PDGFR-β.(3)Cell proliferation was determined by MTT assay in PaSC with BRD2 siRNA or BRD4 siRNA.(4)RNA levels related to ECM production including α-SMA,collagen Ial,FN,CDH11,MCP-1,IL-6,HGF were studied in PaSC with BRD2 siRNA or BRD4 siRNA by q-PCR.(5)IF staining was performed to evaluate α-SMA and FN expression in PaSC with BRD2 or BRD4 gene silence.(6)Western-Blot was performed to measure the effects of siBRD2 or siBRD4 on PaSC markerα-SMA,collagen I,FN,CDH11,platelet-derived growth factor receptor-β(PDGFR-β)and MAPK/ERK signaling pathway after stimulation of 5ng/ml TGF-β.In addition,ECM production was studied by q-PCR in TGF-β stimulated imPaSC with siBRD2 or siBRD4.Results:(1)BRD4 siRNA treatment resulted in 85%reduction in BRD4 mRNA levels at at 48h post-transfection.BRD2 siRNA treatment resulted in 60%reduction in BRD2 mRNA levels at at 32h post-transfection.(2)Western blot showed that 48h BRD2 siRNA treatment reduced protein levels of α-SMA,CDH11 and PDGFR-β in PaSC,but no reductions in FN,collagen Ial and p-ERK.In addition,48h BRD4 siRNA treatment decreased protein levels of FN,collagen Ial,CDH11 and PDGFR-β in PaSC,but no reductions in α-SMA and p-ERK.(3)MTT assay indicated an obvious decrease of PaSC proliferation with BRD4 siRNA treatment but no changes with BRD2 siRNA treatment.(4)q-PCR showed that 32h BRD2 siRNA treatment reduced mRNA levels of α-SMA,CDH11 and IL-6 in PaSC,but no reductions in FN,collagen Ial,MCP-1,HGF or FAP.In addition,48h BRD4 siRNA treatment decreased mRNA levels of FN,collagen Ial,CDH11 and HGF in PaSC,but no reductions in α-SMA,FAP,MCP-1 or IL-6.(5)IF staining indicated that 48h BRD2 siRNA treatment reduced expression of α-SMA in PaSC,but no changes in FN.In contrast,48h BRD4 siRNA treatment reduced expression of FN,but no changes in α-SMA.Conclusions:BRD2 participated in modulating expression of PaSC marker α-SMA.BRD4 plays a role in cell proliferation and ECM production in PaSC,relating to pancreatic fibrosis.Part Three:The effects of BET inhibitors on YAP in pancreatic stellate cell and the the underlying molecular mechanismObjective:To evaluate the effects of BET inhibitors on YAP expression in PaSC.In addition,siRNA againt BRD2 and BRD4 was used to further illuminate the role of BRDs in YAP regulation in PaSC.Methods:(1)Western-Blot was used to measure the effects of iBET151 with different concentrations on protein levels of YAP and p-YAP(Ser127)in both imPaSC and hPaSC.(2)q-PCR was performed to measure the effects of iBET151 on mRNA levels of YAP in both imPaSC.(3)Protein levels of YAP and p-YAP(Ser127)were measured in imPaSC with both BRD2 siRNA and BRD4 siRNA treatments.(4)mRNA level of YAP was measured in imPaSC with both BRD2 siRNA and BRD4 siRNA treatments.Results:(1)After 6h,24h and 48h intervention,BET inhibitors decreased protein level of YAP and p-YAP(Ser127)in both imPaSC and hPaSC in a dose-dependent manner.(2)iBET151 decreased RNA levels of the YAP in imPaSC and hPaSC.(3)Western blot showed that 48h BRD4 siRNA treatment reduced protein levels of YAP and p-YAP(Ser127).In addition,48h BRD2 siRNA treatment had no effects on YAP and p-YAP protein levels.(4)q-PCR showed that 48h BRD4 siRNA treatment reduced mRNAlevels of YAP in imPaSC,however,32h BRD2 siRNA treatment had no effects on YAP mRNA.Conclusions:BET inhibitors reduced YAP expression in both imPaSC and hPaSC,and BRD4 participated in regulating expression of YAP in PaSC.Part Four:The role of YAP in pancreatic stellate cell activation,proliferation and ECM productionObjective:To evaluate the YAP expression in mouse pancreas tissues with pancreatitis and pancreatic cancer.PaSC.In addition,siRNA againt YAP was used to further illuminate the role of YAP in regulating PaSC activation,proliferation and ECM production.Methods:(1)C57BL/6 male mice were subjected to repeated episodes of acute cerulein pancreatitis starting at 6-7 weeks of age.Each acute pancreatitis(AP)episode consist of 7 hourly intraperitoneal injections of saline or 50 μg/kg cerulein Recurrent AP(RAP)was characterized in mice subjected to two episodes of cerulein AP,the first one at day 1(dl)and the second at day 3(d3).In this RAP model,mice were sacrificed during the acute phase of pancreatitis(1 h after the last cerulein injection,at dl and d3)and during the recovery phase(at d5).Chronic pancreatitis(CP)was induced by repeated cerulein AP episodes(twice a week for 4 weeks;total 8 episodes),and mice were sacrificed 4 days after the last AP episode.At sacrificed,pancreatic tissues were collected and snap-frozen for subsequent Western blotting analysis(YAP,CDH11 and PDGFR-β)or formalin-fixed for IF analysis of YAP and PaSC activation markers αSMA.(2)Ptfl-Cre;LSL-KrasG12D/+(KC)mice,a GEMM model used to study pancreatic ductal adenocarcinoma(PDAC)or wild-type(WT)mice were sacrificed at 3 months and pancreas tissues harvested for histological and Western blotting protein analysis of YAP,CDH11,CK19 and PDGFR-β.(3)Primary mouse PaSC(mPaSC)were obtained from wild-type and KC pancreas tissues,and hPaSC were obtained from pancreatic cadaveric tissues from organ donors or pancreatic ductal adenocarcinoma(PDAC)surgical resections.q-PCR and IF staning were used to measure the expression of YAP and α-SMA in mPaSC and hPaSC with different activation states.(4)siRNA against YAP(siYAP)and nonspecific control siRNAs(siControl)were synthesized by ThermoFisher Scientific.In vitro transcription steps were according to the RNAi manual of company.qPCR was used to evaluate the effects of RNAi.Cell proliferation was determined by MTT assay in PaSC with YAP siRNA.RNA levels related to ECM production including α-SMA,collagen Ial,FN were studied in PaSC with YAP siRNA by q-PCR.Results:(1)We found negligible levels of YAP protein expression in pancreas of untreated,control mice or mice subjected to one episode of AP(day 1).YAP levels increased moderately after the second episode of AP(day 3)but markedly 2 days after RAP induction(day 5)and remained elevated during CP.YAP upregulation was concomitant with upregulation of the PaSC markers CDH11 and PDGFR-β.Moreover,aSMA positive,activated PaSC in RAP and CP pancreas exhibited marked YAP nuclear staining,suggesting YAP transcriptional activity in these cells.(2)We found that YAP expression is linked to the activated state of PaSC.Quiescent,freshly isolated hPaSC and mPaSC did not express YAP,but YAP levels markedly increased during cell activation in culture and remained elevated during passages.In KC mice,at three months of age,pancreas of KC mice displayed significant upregulation of protein levels of YAP,the PaSC markers PDGFR-β and CDH11,and the ductal marker cytokeratin 19(CK19).(3)To further explore the role of YAP in PaSC biology,we measured levels and cellular location of YAP in quiescent and activated PaSC obtained from human(hPaSC)and mouse(mPaSC)tissues.Moreover,activated PaSC isolated from resected human PDAC tumors or KC mice displayed robust YAP nuclear staining in culture conditions,consistent with YAP transcriptional activity.(4)YAP siRNA treatment resulted in 82%reduction in YAP mRNA levels at 24 h post-transfection and 60%reduction at 48h post-transfection.At 48h,we found a 50%reduction in cell proliferation in siRNA treated imPaSC.YAP siRNA knockdown reduced a subset of genes including the previously identified YAP gene targets Connective tissue growth factor(Ctgf)and Cysteine rich angiogenic inducer 61(Cyr61)that are involved in cell adhesion and ECM remodeling;the PaSC markers αSMA,Collal and FN(one of the most abundant collagen chains in fibrotic tissues)that participate in PaSC morphology,matrix tension and collagen deposition and TGFβ,that stimulates fibroinflammatory responses in these cells.Interestingly,YAP siRNA treatment markedly increased levels of metalloproteinase 3(MMP3),a protease involved in degradation of collagens and other ECM proteins during tissue remodeling.Conclusions:YAP plays a role in PaSC activation and fibroimflammatory responses,inhibition of YAP helps to reduce PaSC activation and ECM production. |
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