Role Of Cross Talk Between The Smad2 And MAPK Pathways In TGF-β1-induced Collagen Ⅳ Expression In Mesangial Cells

Glomerulosclerosis is very common in the end stage of various glomerular nephritis and other renal diseases, which is the result of structural remodeling and excessive deposition of extracellular matrix (ECM) in the glomeruli and eventually leads to the renal failure. Mesangial cells (MC) have been identified as the main producer of glomerular ECM and critically contribute to the structural and functional integrity of the glomerulus. CollagenⅣis a component of ECM in the glomerulus. Expression of collagenⅣwill be greatly enhanced in the mesangial region or capillary membrane of glomeruli by MC during the glomerulonephritis or glomerulosclerosis. So it is a significant topic of investigating the expression of collagen IV and its related mechanisms in MC.MC excrete various cytokines, including transforming growth factorβ1 (TGF-β1). TGF-β1 is a member of TGF-βsuperfamily. Promoting the expression of ECM genes in parenchymal cells such as MC is one of its functions. Therefore, TGF-β1 plays an important role in the development of glomerulosclerosis. TGF-β1 mediates the transcription of target genes through its downstream signal pathways. Smads family are delineated as the classic signal transducers of the downstream of TGF-β1. Besides that, other signal pathways such as MAPK or AKT/PI3K pathway have also been reported to play important roles in transducing downstream of TGF-β1 signals in recent years. MAP kinase pathway is a more elucidated downstream of TGF-β1 pathway besides the classic Smad pathway. Many reports show that there exist cross talk between MAPK and Smad pathways,but much is known about the relationship between ERK1/2 and Smad pathways, relatively little is known about the relationship between JNK, p38 and Smad or the reverse reactions.Decorin (DCN) is one of the small leucine-rich proteoglycans and excreted by MC in the glomerulus. It is a component of ECM mainly existing in connective tissues. Present studies showed that DCN took part in regulating the proliferation and transformation of cells, synthesis and deposition of ECM of cells, functions of cytokines, and so on. DCN can antagonize functions of TGF-β1 at several levels, so it is an important inhibitor of TGF-β1. We previously reported that DCN could repress the proliferation of MC and deposition of ECM in the glomerulus. In addition, DCN could antaganize the development of the glomerular lesions in the rat anti-Thy-1.1 nephritis model. However, the functions of DCN in the glomerular diseases are not fully elucidated.MC are the main target cells of TGF-β1 in glomerular nephritis. Studies have already shown that both the Smad and MAPK pathways are activated by TGF-β1 in the glomerular diseases [21]. But researches on the cross talk between MAPK and Smad pathways in MC are a few. Hayashida once reported that ERK-dependent Smad3 linker region phosphorylation enhanced collagen I synthesis in human mesangial cells. However, the complex mechanisms for the interaction are still needed to be further investigated. In the present study, we continue to investigate the cross talk between MAPK and Smad pathways, especially the crosstalk between JNK, p38 and Smad2 pathways besides ERK pathway. Moreover, we also investigated that whether Smad2 could reversely influence the phosphorylation of MAPK. We try to determine the role of the crosstalk between MAPK and Smad2 in the process of TGF-β1 induced collagenⅣexpression. Meanwhile, we initially attempted to study the effects of DCN on the expression of collagen IV and phosphorylation of MAPK and Smad2 induced by TGF-β1. PartⅠEffects of MAPK on Smad pathway in the process of collagen IV synthesis in cultured rat mesangial cells induced by TGF-β1Objective To explore the effects of exogenous TGF-β1 on the expression of collagenⅣand the phosphorylation of MAPKs and Smad2, and observe the influence of MAPK on the Smad pathway in rat MC in vitro.Methods Cultured MC were treated with exogenous TGF-β1 in different concentrations or in a single concentration for different time. Next the cultured MC were pretreated with inhibitors of ERK1/2, JNK and p38 (U0126, SP600125 and SB203580), respectively, and then the different groups of cells were treated with TGF-β1 (2.5ng/ml) for indicated time. The expression of collagenⅣ, MAPKs and P-MAPKs, Smad2 and P-Smad2 were examined by Western Blot analysis, respectively. The mRNA level of collagenⅣwas examined by Real time RT-PCR.Results The cultured MC treated with different concentrations of TGF-β1 showed increased expression of collagenⅣfrom lng/ml to lOng/ml, and reached a peak at 2.5ng/ml(P<0.05). Then the cells were treated with TGF-β1 (2.5ng/ml) for indicated time showed the expression of collagenⅣand phosphorylation of MAPKs increased, and the phosphorylation of Smad2 both at the C terminal (P-Smad2C) and the linker sites (P-Smad2L) also increased. The enhanced expression of collagenⅣreached a peak at 6h after the stimulation (P<0.05). The maximum activation of ERK1/2, p38 and Smad2 was observed at 1h after the stimulation (P<0.05), and that of JNK was observed at 2h after the stimulation (P<0.05).Pretreatment of cells with U0126, SP600125 or SB203580 significantly inhibited the phosphorylation ERK, JNK or p38, respectively (P<0.05). Further more, U0126 for ERK block and SB203580 for p38 block obviously suppressed the phosphorylation of Smad2 both at C-terminal and linker sites induced by TGF-β1(P<0.05), while the SP600125 for JNK block also inhibited Smad2 phosphorylation at C-terminal sites but had little effect at linker sites. The pretreatment of the three specific inhibitors did not affect the total amount of Smad2. All the three MAPK inhibitors decreased the enhanced expression of collagenⅣinduced by TGF-β1 at both protein and mRNA levels (P<0.05). Summary TGF-β1 can up-regulate the expression of collagenⅣin dose and time-dependent manners in cultured rat MC. Activation of MAPKs and Smad induced by TGF-β1 takes part in the regulation of collagenⅣexpression. Moreover, there exists crosstalk between MAPK (ERK1/2, p38 and JNK) and Smad pathways. However, there is also some difference in the crosstalk among ERK1/2, p38, JNK and Smad pathways.PartⅡEffects of Smad on MAPK pathway in the process of collagen IV synthesis in cultured rat mesangial cells induced by TGF-β1Objective To investigate the role of Smad2 in the process of TGF-β1-induced expression of collagenⅣand to determine whether Smad2 could reversely influence MAPKs.Methods Cultured MC were transfected with Dominant Negative Smad2 (DN-Smad2) for 48 h, then treated with 2.5 ng/ml TGF-β1 for indicated time. The expression of collagen IV and P-MAPKs, P-Smad2 were examined by Western Blot analysis, respectively. The mRNA level of collagenⅣwas examined by Real time RT-PCR.Results Transfection of DN-Smad2 reduced the phosphorylation of Smad2 and expression of collagenⅣinduced by TGF-β1(P<0.05). Meanwhile, DN-Smad2 also downregulated the phosphorylation of ERK1/2 and JNK, but had little effects on that of p38 (P<0.05).Summary These results indicate that activation of Smad pathway directly mediate the expression of collagenⅣinduced by TGF-β1. Furthermore, Smad2 may also indirectly regulate the expression of collagenⅣby interacting with MAPKs pathways.PartⅢEffects of DCN on the expression of collagenⅣand activation of MAPK and Smad pathways induced by TGF-β1 Objective To preliminary explore the effects of DCN on the expression of collagenⅣand activation of MAPK and Smad pathways.Methods Cultured MC were transfected with pcDNA3.1-DCN or DCN siRNA for 48h, and then treated with or without TGF-β1 for indicated time. The expression of collagenⅣand MAPKs, Smad2 were examined by Western Blot analysis, respectively. The mRN A level of collagenⅣwas examined by Real time RT-PCR.Results Both protein and mRNA levels of DCN were increased after transfection with DCN plasmid, comparing with the control cells (P<0.05). Otherwise, both levels decreased after albation of DCN by siRNA interference (P<0.05).In addition, collagenⅣexpression was reduced or heightened both at protein and mRNA levels in MC after transfected with a DCN plasmid or DCN siRNA, respectively (P<0.05).Furthermore, transfection of DCN reduced phosphorylation of ERK1/2, JNK, p38 and phosphorylation of Smad2 induced by TGF-β1(P<0.05). However, there were not significant changes in the protein level of P-ERK1/2, P-JNK, P-p383和P-Smad2 stimulated by TGF-β1.Summary pcDNA3.1-DCN和DCN siRNA were successfully transfected into the rat MC. DCN could inhibite the expression of collagenⅣwhile TGF-β1 promoted expression of collagenⅣ, which suggests an antagonistic effect of DCN on TGF-β1. Moreover, DCN could down regulate the phosphorylation of ERK1/2, JNK, p38 and phosphorylation of Smad2 induced by TGF-β1, which indicates that DCN could interfere TGF-β1-induced activation of ERK, JNK, p38 and Smad2. RNA interference of DCN does not influence the expression of P-ERK1/2, P-JNK, P-p38and P-Smad2, implying that physiological state of endogenous DCN in MC have little effect on the activation of such MAPK and Smad. 1. TGF-β1 can promote the expression of collagenⅣin dose and time-dependent manners, which may indicate the controllability of collagenⅣgene as one of TGF-β1-responsive genes. Meanwhile, TGF-β1 could activate both MAPKs and Smad pathways. Moreover, the block of MAPK or Smad phosphorylation can reduce the collagenⅣexpression, which suggests the activation of two pathways involve in the expression of collagenⅣ.2. The block of MAPK obviously suppressed the phosphorylation of Smad2 both at C-terminal and linker sites induced by TGF-β1, while DN-Smad2 also down regulated the phosphorylation of ERK1/2 and JNK. There is mutual cross-talk between MAPK and Smad pathways which may further affect the expression of collagenⅣ.3. DCN inhibites expression of collagenⅣwhile TGF-β1 promotes expression of collagenⅣ. Otherwise, overexpression of DCN down regulates the phosphorylation of ERK1/2, JNK, p38 and P-Smad2 induced by TGF-β1.