| BackgroundLiver fibrosis is the progress of the extracellular matrix proteins excessive accumulation including collagen in most types of chronic liver diseases,which can develop to cirrhosis or cancer.The Liver stromal cells related to hepatic fibrosis include hepatic stellate cells(HSCs),Kupper cells and Hepatic portal fibroblasts,hepatic stellate cells(HSCs)are the main source of collagen accumulation.Hepatic stellate cells are located in the space of Disse between the sinusoidal endothelial cells and hepatic epithelial cells.In a normal liver,HSCs are quiescent and contain numerous vitamin A lipid droplets.When the liver is injured due to chronic diseases,HSCs receive signals secreted by damaged hepatocytes,Kupper cells,endothelial cells and inflammatory cells,causing them to transdifferentiate into activated myofibroblast-like cells.It is well known that HSCs play a central role in the hepatic fibrosis.These activated cells strongly express a-smooth muscle actin(?-SMA),a myofibroblast marker,and synthesize the major composition of extracellular matrix(ECM)fibrillar collagens and collagen-degrading enzymes,such as the degradation factor matrix metalloproteinase(MMP).The persistence process will result an attenuated deposition of collagen and liver injury,which eventually leads to liver fibrosis.Transforming growth factor ?(TGF-p)is a family of structurally homologous dimeric proteins(there mammalian isoforms:TGF-?1 TGF-?2,and TGF-?3).The highest content of biological activity in liver is TGF-?1.TGF-?1 regulates multiple biological processes in the body,and is closely associated with various tissue and organ fibrosis.TGF-?1 is the profibrogenic master cytokine,not only initiates the transdifferentiation of HSC to myofibroblast(MFB),but also enhances matrix gene expression,decreases their degradation by down-regulation of matrix metalloproteases(MMPs)and up-regulation of their specific inhibitiors such as tissue inhibitor of metalloproteases(TIMPs).Early studies showed that TGF-?1 could promote parathyroid hormone-related protein(PTHrP)in granulosa cells and human breast cancer cells,and rencent studies showed that TGF-?1 could also induce expression of PTHrP in human hepatocellular carcinoma cells.PTHrP is a cytokine,which was originally identified as a causative factor of malignant humoral hypercalcemia.PTHrP has a similar structure with Parathyroid hormone(PTH)of a N-terminal amino acid sequence homology.Studies of PTHrP have mainly focused on bones and tumors in recent years.Experiments have demonstrated that PTHrP promotes bone formation and is an excellent osteogenic accelerator that achieved rapid effects and has few adverse reaction,even after years of clinical treatments for osteoporosis(OP).Previous studies demonstrated that PTHrP was markedly induced in hepatocytes during endotoxemia and caused hepatic acute damage.Recent studies had shown that PTHrP could induce the obstructed mouse kidney fibrogenesis with the cooperating of TGF-?1,EGF(endothelial growth factor),and VEGF(vascular endothelial growth factor);PTHrP was elevated in acute pancreatitis induced by alcohol or cerulean,while PTHrP increased expression of procollagen I,fibronectin and inflammatory mediators interleukin-6(IL-6)and played a role in pancreatic fibrosis.Our previous work demonstrated PTHrP could activate HSCs,increase TGF-?1,collagen I and MMP-2 srcretion,and promote the fibrosis process in HSCs.Liver fibrosis is a complex process including multiple cell signaling pathways in which TGF-?-Smad signaling pathway,Mitogen activated protein kinase(MAPK)signaling pathway and Rho-ROCK signaling pathway are well studied,these signaling pathways play a role in the different links,and jointly promote development of liver fibrosis.The mitogen-activated protein kinases(MAPKs),are mainly composed of three serine/threonine related kinases:extracellular signal-related kinases(ERKs),c-Jun NH2-terminal kinases(JNKs)and p38 MAPKs.TGF-?1 can activate both Smad and MAPKs signaling to produce excess ECM,leading to liver fibrosis.TGF-?1 is a recognized factor promoting liver fibrosis,and PTHrP is a good prospectly promoter of bone formation can promote liver fibrosis.According to the domestic and foreign rearch as well as our previous results,we propose the following hypothesis:TGF-?1 and PTHrP established a positive feedback loop on HSCs,activating Smad and MAPK signaling pathways to promote liver fibrosis.The assumption can not only clarify the relationship of PTHrP and TGF-?1,deepen the understanding of liver fibrosis,also contribute to the pathological mechanisms of liver fibrosis induced by PTHrP and TGF-?1,and provide a new way for prevention and treatment for liver fibrosis.ObjectivesThe activation of hepatic stellate cells has been a popular issue in liver fibrosis.Interaction of TGF-?1 and PTHrP on HSCs provides us a new starting point to the treatment of liver fibrosis,and also reveals the pathological mechanism of fibrosis caused by TGF-?1 and PTHrP.Materials and methods1.We bought one commercial cell lines the human hepatic stellate cell-LX-2.We examined what effects TGF-?1 act in LX-2,treated with different concentration(1 ng/ml,5 ng/ml,10 ng/ml,30 ng/ml,50 ng/ml)and different time period(1 h,3 h,6 h,12 h,24 h,48 h,72 h,96h)of TGF-?1.Then the total protein and total RNA were collected.2.We used reverse transcriptase polymerase chain reaction(PCR)and relative quantitative reverse transcriptase polymerase chain reaction(RT-qPCR)detected the following factor that related to activated:PTHrP,collagen I,MMP-2,and Western-blot detect the following factor:PTHrP,collagen I,MMP-2,Smad2/p-Smad2,Smad3/p-Smad3,Smadl/p-Smadl,Smad5/p-Smad5,JNK/p-JNK,p44/42/p-p44/42,p38/p-p38.3.Immunofluorescence was performed to identify the expression of PTHrP after treated with only TGF-?1(10 ng/ml)for 48h,or LY2157299 +TGF-?1(10 ng/ml),or SB203580+TGF-?1(10 ng/ml).collagen I and MMP-2 were also detected after treated with PTHrP(7-34)+TGF-?1(10 ng/ml).4.The data were shown as mean ? standard error of the mean(S.E.M.)based on experiments repeated in triplicate.Multiple comparisons were analyzed using one-way analysis of variance(ANOVA)with Statistical Package for the Social Sciences(SPSS)13.0 software(Chicago,IL).Probability(p)-value less than 0.05 were considered statistically significant.The results of Ctrl group were set to 1 in Western-blot and qPCR.Results1.After treated with TGF-?1 in LX-2,PTHrP was detected by mRNA(q-PCR)and protein(western-blot),found that PTHrP was increased in the concentration of lng/ml for 48h with 3.2-to 3.3-fold(P<0.05).In response to TGF-?1 at 5-50ng/ml for 48h,PTHrP was significant increased,as assessed by qPCR mRNA levels with 13.2-14.6-fold while protein levels with 5.0-to 7.4-fold campared with Ctrls(P<0.05).Treated with TGF-?1(lOng/ml)for 1-96h,the gene expression of PTHrP was higher than that of control group(P<0.01),and the effect of 3h group was the most obvious.Treated with TGF-?1(lOng/ml)for 3-96h,PTHrP protein increased 1.3-to 2.3-fold at 6-24h(P<0.01)compared with Ctrl groups in the ris,and 3.1-to 3.4-fold at 48-96h(P<0.01),but there was no significant difference with 3h group and Ctrl group.2.Smad,JNK,ERK,and p38 signaling pathways were detected after treated with TGF-?1(10 ng/ml)for different peroids.P-Smad2 in 10min to 24h was higher than control group,reached the peak at 30min with 46.6-fold(P<0.01),and decreased after 1h.P-Smad3 was also higher than control group in 10min to 24h(P<0.01),the most significant time was 10-30min for 24.1-to 25.3-fold at 1-24h(P<0.01)and decreased after 1h.P-Smadl increased 3.8-to 13.4-fold at 10-30min(P<0.01),21.1-fold at 1h reaching the peak(P<0.01)and 2.2-to 2.4-fold at 3-6h(P<0.01),while there was no significant difference with 12-24h group and Ctrl group.The tendency of p-Smad5 was same as p-Smad1,p-Smad5increased 3.4-to 7.4-fold at 10-30min(P<0.01),12.8-fold at 1h(P<0.01)and 1.8-fold at 3-6h(P<0.01).While there were no significant differences with 12-24h group and Ctrl group in p-Smadl and p-Smad5.P-jnk increased 1.8-fold at 12h(P<0.01),2.9-to 3.1-fold at 24-72h(P<0.01),and 1.9-fold at 96h(P<0.01),while there were no significant differences with 3-6h group and Ctrl group.P-p44/42 increased 3.6-to 10.0-fold at 6-96h(P<0.01)and there was no significant differences with 3h group and Ctrl group.P-p38 showed 3.9-5.7-fold increase at 3-96h(P<0.01).3.Pre-incubation with inhibitor of TGF-? receptors:SD 208(2?M,10?M)the inhibitor of T?R I,LY2157299(4?M,10?M)the inhibitor of T?R ?/?,PTHrP mRNA and protein increased 2.3-to 3.2-fold and 1.5-1.8-fold respectively in SD 208(2?M,10?M)+TGF-?1 group,which had statistical significance in both TGF-?1 group and Ctrl group(P<0.01),while there was no significant differences in LY2157299(4?M,10?M)+TGF-?1 group and Ctrl group but had statistical significance with TGF-?1 group(P<0.01).Pre-incubation with inhibitor of p38 signaling pathway,PTHrP mRNA and protein increased 4.9-fold and 3.0-fold respectively in SB203580(20?M)+TGF-?1 group,which had statistical significance in both TGF-?1 group and Ctrl group(P<0.05),while SB203580(50?M)+TGF-?1 group had statistical significance in TGF-?1 group(P<0.01),no statistical significance in Ctrl group.Pre-incubation with inhibitors of JNK and ERK signaling pathway,both of SP600125(10?M,20?M)+TGF-?1 group and PD98059(10?M,25?M)+TGF-?1 group had statistical significance in Ctrl group(P<0.01)about the expression of PTHrP mRNA and protein,no statistical significance with TGF-?1 group.4.Pre-incubation with inhibitor of PTHrP,collagen I mRNA and protein increased 7.0-fold and 2.3-fold respectively in PTHrP(7-34)(1?M)+TGF-?1 group which had statistical significance in both TGF-?1 group and Ctrl group(P<0.05),and MMP-2 mRNA and protein increased 3.3-fold and 1.4-fold respectively in PTHrP(7-34)(1?M)+TGF-?1 group which also had statistical significance in both TGF-?1 group and Ctrl group(P<0.05).5.LX-2 stained strongly for PTHrP compared with Ctrl group after incubation with TGF-?1 at lOng/ml for 48h when immunofluorescence was performed.Pre-incubation with LY2157299(10?M)or SB203580(50?M),then treated with TGF-?1(10 ng/ml)for 48 h,PTHrP expression decreased in LY2157299(10?M)+TGF-?1 group and SB203580(50?M)+TGF-?1 group,and had no statistical significance in Ctrl group.Pre-incubation with PTHrP(7-34)at 1?M,then treated with TGF-?1(10 ng/ml)for 48 h,expression of collagen I and MMP-2 decreased but still higher than Ctrl group.ConclusionTGF-?1 increased PTHrP expression depended on dose and time.TGF-?treatment leaded to phosphorylation of smad,JNK,p38 MAPK and ERK in LX2 cells,but TGF-?-induced PTHrP production in LX2 cells was T?R and p38 dependent.PTHrP(7-34)inhibited TGF-?1-induced collagen I and MMP2 protein and mRNA levels.In summary,TGF-?1 and PTHrP may establish a positive feedback loop on LX-2 and work together to promote liver fibrosis. |
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