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Nephrol Dial Transplant (2010)1of 8

doi:10.1093/ndt/gfq619

Original

Article

Role of Sema4C in TGF-β1-induced mitogen-activated protein kinase activation and epithelial –mesenchymal transition in renal tubular epithelial cells

Rui Zeng 1,*

,Min Han 1,*,Yun Luo 1,Caixia Li 1

,Guangchang Pei 1,Wenhui Liao 2,Shoujun Bai 1,

Shuwang Ge 1,XiaoCheng Liu 1and Gang Xu 11Division of Nephrology,Department of Internal Medicine and 2Department of Geriatrics,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan,Hubei,People ’s Republic of China

Correspondence and offprint requests to :Gang Xu;E-mail:xugang@https://www.wendangku.net/doc/761608015.html, *These authors contributed equally to this work.

Abstract Background.The p38mitogen-activated protein kinase

(p38MAPK)is an important intracellular signal trans-

duction pathway involved in TGF-β1-induced epithelial –mesenchymal transition (EMT).Sema4C,a member of the semaphorin family,was found to be essential for the ac-tivation of p38MAPK.However,the role of Sema4C in promoting TGF-β1-induced EMT is unclear.Methods.Renal fibrosis was induced by 5/6subtotal neph-rectomy rat model.In vitro ,Sema4C was induced in human proximal tubular epithelial cells (HKC)by treatment with TGF-β1,or was inhibited by siRNA or was over-expressed by Sema4C transfection.The selective p38MAPK inhibi-tor,SB203580,was administered to inhibit the p38path-way.The expression of Sema4C,the markers of EMT,p38phosphorylation and fibronectin secretion were mea-sured by western blotting,immunohistochemistry,immuno-cytochemistry or enzyme-linked immunosorbent assay.Results.The expression of Sema4C increased in HKC cells that were treated with TGF-β1.Knockdown of Se-ma4C potently inhibited phosphorylation of p38MAPK and reversed TGF-β1-induced EMT.Over-expression of Sema4C via Sema4C transfection elicited p38MAPK phosphorylation and promoted EMT.The effects of Se-ma4C during EMT were blocked by a p38-specific in-hibitor.In vivo ,the expression of Sema4C increased in the tubular epithelia of 5/6-nephrectomized rats and hu-man fibrotic renal tissue,and similar localization of phosphorylated p38and Sema4C was demonstrated by immunohistochemistry on serial sections.Conclusions.Our findings suggest that Sema4C plays an important role in TGF-β1-induced EMT through activa-tion of p38MAPK in proximal tubular epithelial cells.Keywords:epithelial to mesenchymal transition;p38MAPK;Sema4C;TGF-β1Introduction

The progression of chronic kidney disease leads to wide-spread tissue fibrosis and irreversible loss of renal func-tion.Epithelial –mesenchymal transition (EMT)of tubular epithelial cells contributes significantly to the onset and pathogenesis of renal fibrosis [1–4].An extracellular stimulus usually initiates this process,which leads to the loss of junctional contacts,expression of mesenchymal markers,development of cell motility and production of extracellular matrix (ECM)[2,5].Of the many factors that trigger EMT,transforming growth factor-β1(TGF-β1)is the most important and well studied [6].TGF-β1mediates the EMT process via numerous intracellular signal trans-duction pathways,including the canonical Smad pathway,mitogen-activated protein kinases (MAPK),PI3K/Akt and small GTPases that control the activity or expression of factors related to EMT [7,8].

In recent years,significant evidence suggests that p38MAPK pathway is an important intracellular signal trans-duction pathway involved in TGF-β1-induced EMT in renal tubular epithelial cells [9,10].The activated p38MAPK could directly regulate the protein synthesis of α-smooth muscle cell actin (α-SMA)[10],indirectly activate Smad pathway [9],lead to excessive matrix deposition and finally induce the fibrotic process.However,the molecular details of how TGF-β1could potentially induce p38MAPK in renal tubular cells have not been elucidated yet.Sema4C,a member of the semaphorin family,has been shown to be essential for the activation of p38MAPK [11].The semaphorins are a large family of secreted or mem-brane-bound proteins that all have a conserved Sema do-

main,which is known to regulate tumor progression [12],

angiogenesis [13],nervous system development [14]and immune cell interactions [15].Our previous microarray

analysis of metastatic human cervical cancer tissue indi-

the Creative Commons Attribution License (https://www.wendangku.net/doc/761608015.html,/licenses?by-nc/2.0/uk),which permits unrestricted non-commercial use distribution,and reproduction in any medium,provided the original work is properly cited.

NDT Advance Access published October 19, 2010 at Guangdong Medical College on January 20, 2011

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cated a significant up-regulation of Sema4C during cancer invasion and metastasis (Table 1;this data had not been published elsewhere),a process that is analogous to that observed during tubular EMT [6,16].However,it is not known whether Sema4C is involved in EMT.We therefore examined whether TGF-β1-induced EMT is mediated by Sema4C –MAPK pathway.For this purpose,we measured Sema4C in the tubular epithelia of fibrotic renal tissue and in renal tubular cells treated with TGF-β1,examined the effect of Sema4C siRNA on TGF-β1-induced MAPK ac-tivation,EMT and fibronectin secretion,and measured the p38phosphorylation and EMT in Sema4C over-expressed cells.Materials and methods Animal model

Male Sprague –Dawley rats (150–200g)were obtained from the Tongji

Laboratory Animal Center (Wuhan,China).All rats were sacrificed 20weeks after nephrectomy,and serum was collected for determination of creatinine and urea nitrogen.Kidneys were immediately excised;some were fixed with 4%paraformaldehyde and others were frozen in liquid ni-trogen for later use.For histological examination,renal tissues were stained with periodic acid –Schiff and Masson's trichrome.All procedures were per-formed in accordance with our university ’s guidelines for animal care.Cell cultures

HKC cells were cultured in DMEM/F12(Invitrogen,Inc.,Carlsbad,CA,USA)supplemented with 10%fetal bovine serum (Gibco,USA).HKC

cells were cultured in serum-free medium for 24h before being used for experiments.To induce renal tubular EMT,cells were then treated with recombinant TGF-β1(10ng/mL;B&D Systems Inc.,Minneapolis,MN,USA)for 72h.SiRNA for Sema4C For siRNA experiments,RNA primers complementary to human Sema4C were designed and synthesized by the Shanghai Invitrogen Biotechnology Company.HKC cells were transfected with the annealed RNA primer pair by using Lipofectamine 2000(Invitrogen Inc.),according to the manufac-turer's instructions.Five hours after transfection,cells were incubated with 10ng/mL of TGF-β1for 72h to observe the effect of Sema4C silencing.Production of stable HKC clones over-expressing human Sema4C

The plasmid pcDNA 4.0–Sema4C was transfected into HKC cells with

Lipofectamine 2000according to the manufacturer's instructions.Cells were plated at ~70–90%confluence in six-well plates 1day before transfection.The cells were washed and seeded in growth medium con-taining zeocin.This selection procedure lasted for 2weeks,and then Zeocin-resistant clones were harvested and analysed.To block the p38MAPK pathway,Sema4C-transfected HKC cells were treated with 20mM SB203580for 48h.

Western blotting Kidney tissues and cells were extracted with lysis buffer containing 1%Triton X-100,0.5%Nonidet P-40,20mM Tris –HCl,15mM NaCl,1mmol/L EDTA,1mmol/L egtazic acid,1mM Na 3VO 4·10H 2O,2mM NaF ,2mM Na 2P 2O 4·10H 2O,10mM β-glycerophosphate disodium salt (pH 8.0)and four protease inhibitors (5mM phenylmethanesulfonyl fluor-ide,5μg/mL leupeptin,5μg/mL pepstatin and 5μg/mL aprotinin)for 30min at ?20°C.Then,the extract was centrifuged at 12000g for

20min at 4°C to remove cell debris.Protein concentration was deter-mined by the Bradford protein assay,80μg of protein was subjected to

SDS –PAGE and proteins were transferred to nitrocellulose membranes.After transfer,the membranes were blocked with 5%non-fat dry milk dissolved in TBS containing 0.1%Tween-20for 1h at 37°C and blotted routinely with Sema4C (BD Biosciences,San Jose,CA,USA),E-cadherin

(BD Biosciences),vimentin (Abcam,Cambridge,MA,USA),GAPDH

(Proteintech Group,Inc.),phosphorylated p38MAPK and p38primary antibodies (Cell Signaling Technology,Danvers,MA,USA)at 37°C for 1h.The bound antibody complexes were visualized by enhanced chemi-

luminescence (SuperSignal West Femto Kit;Pierce,Rockford,IL,USA),

and X-ray films were scanned with a ChemiImager 5500image analysis

system (Alpha Innotech,San Leandro,CA,USA).Quantity One software

(Bio-Rad)was used to quantify band density.

Renal biopsy specimens

Renal biopsy specimens were from patients with sclerosing glomerulo-nephritis or obstructive nephropathy,diagnosed between 2008and 2010in the Division of Nephrology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and https://www.wendangku.net/doc/761608015.html,rmed consent was obtained from each patient when the renal biopsy was performed.The research was in compliance with the Declaration of Helsinki.In all of

the renal biopsy specimens,the tubulointerstitial fibrosis was dominant.

Immunohistochemistry and immunocytochemistry

For immunohistochemical analysis,paraffin sections or serial sections were incubated with either primary anti-Sema4C antibody or phosphory-lated p38MAPK antibody at 4°C overnight.The sections were then in-cubated with biotinylated goat anti-mouse Ig antibody as the secondary

antibody,and the antibody reactions were visualized by using diamino

benzidine (DAKO,Tokyo,Japan).The serial sections were then analysed

to clarify the colocalization of Sema4C and phosphorylated p38MAPK in these kidneys.

For immunocytochemical analysis,HKC cells were cultured on sterile glass coverslips in six-well plates.The slides were incubated overnight at 4°C with anti-E-cadherin,anti-vimentin or anti-phosphorylated p38anti-body,followed by incubation with FITC-conjugated secondary antibody at room temperature for 1h.Finally,slides were counterstained with propi-

dium iodide for E-cadherin,DAPI for vimentin and visualized by confocal

laser scanning microscopy.

ELISA assay

Fibronectin (FN)secretion was determined by a competitive ELLSA assay kit (Boster Biological Technology,Wuhan,China)according to the manufacturer's instructions.The OD value was detected by an ELISA Reader in 450-nm wavelength and calculated in the linear part of the curve.

Statistical analyses

All data were analysed by Student ’s t -test or a one-way ANOV A using SPSS (version 12.0).Data are expressed as means ±standard errors of the mean (SEM).Significance was assessed at P <0.05.

Table 1.Genes up-regulated in metastatic human cervical cancer tissue

Probe set ID Gene symbol Description P-value 46665_at Sema4c Nervous system and cell differentiation development 0.000219217022_s_at IGHA1Immune response,protein –chromophore linkage 0.000052221748_s_at TNS1Intracellular signalling cascade 0.000147229152_at C4orf7Chromosome 4open reading frame 70.001953234379_at FLT -4Protein –tyrosine kinase activity and receptor activity 0.014212R.Zeng et al. at Guangdong Medical College on January 20, 2011

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Results

Sema4C is involved in renal fibrosis in vivo

First,we examined the distribution of Sema4C in the fi-brotic kidney by use of the5/6subtotal nephrectomy rat model.As shown in Table2,serum urea nitrogen and cre-atinine were significantly elevated in5/6-nephrectomized rats compared with sham-operated rats.Light microscopy indicated glomerular sclerosis and interstitial fibrosis in the5/6-nephrectomized rats,but not in the sham-operated rats(Figure1A).

Immunohistochemical staining showed that Sema4C was mainly expressed in renal tubular cells of5/6-nephrecto-mized rats,with very little staining in the renal tubules of sham-operated rats(Figure1B).Western blotting also in-dicated that Sema4C protein expression was significantly elevated in the kidney of5/6-nephrectomized rats com-pared with sham-operated rats(Figure1C).These results suggest that Sema4C is involved in renal fibrosis in this animal model.

TGF-β1increases the expression of Sema4C in HKC cells and Sema4C depletion inhibits TGF-β1-induced EMT Tubular epithelial cells are the natural targets of TGF-β1in vivo,which plays a critical role in renal fibrosis in the5/6 subtotal nephrectomy rat model[17].Therefore,we exam-ined the expression of Sema4C in proximal epithelial cells (HKC cells)that were treated with TGF-β1in vitro.The result indicates that Sema4C was significantly elevated in HKC cells after incubation for72h with10ng/mL TGF-β1(Figure2A).

We also investigated whether Sema4C could affect TGF-β1-dependent EMT by use of siRNA experiments. As shown in Figure2A,cells developed EMT after72h of incubation with TGF-β1.This was manifested as down-regulation of E-cadherin,an epithelial marker,the decrease of which is a hallmark of EMT.In contrast,the protein vimentin,a mesenchymal marker,was up-regulated in TGF-β1-treated cells as compared with control cells (Figure2A).

Confocal laser microscopy showed that E-cadherin had a continuous distribution near the perimeter of control cells[Figure2B(I)],but had a discontinuous distribution near the perimeter of TGF-β1-treated cells[Figure2B (II)].Vimentin was present exclusively in the cytosol of TGF-β1-treated cells[Figure2B(V)],and there was little endogenous expression in control cells[Figure2B(IV)].

HKC cells transfected with Sema4C-specific siRNA were resistant to TGF-β1-induced EMT.As shown in Figure2A,Sema4C after silencing almost touched the ba-sal level,which is about62%lower than that of TGF-β1-treated cells.The EMT resistance manifested as elevated levels of E-cadherin protein(Figure2A),relocalization of E-cadherin protein to the cell perimeter[Figure2B

Table2.Renal function in sham-operated rats and5/6-nephrectomized rats

Groups n Bun(mmol/L)Scr(μmol/L) Sham-operated8 6.25±1.0227.13±6.13 5/6-nephrectomized836.79±15.93*125.29±58.35* *P<0.01compared with sham-operated rats.

Bun,serum urea nitrogen;Scr,serum

creatinine.Fig.1.Expression of Sema4C is increased in the kidney tissue of5/6 nephrectomized rats.(A)Representative histology of kidney sections from sham-operated and5/6-nephrectomized(5/6NX)rats stained with periodic acid–Schiff(upper)or Masson's trichrome stain(lower). Interstitial fibrosis and glomerular sclerosis are extensive in5/6NX rats.The tissue of sham rats was normal.Magnification×200.(B) Representative immunohistochemical detection of Sema4C in the kidney tissue of sham-operated and5/6-nephrectomized rats.Sema4C immunostaining is strong in the epithelial tubular cells in5/6NX rats and much weaker in sham rats.Magnification×200.(C)Representative western blot of Sema4C in the kidney tissue of sham-operated and5/6-nephrectomized rats.The expression of the Sema4C protein was elevated in the kidney tissue of5/6-nephrectomized rats.The histogram shows the average volume density corrected for the loading control,GAPDH (n=6).*P<0.05compared with sham-operated rats.

Sema4C mediates TGF-β1stimulation of EMT via p38MAPK3

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(III)]and reduced vimentin expression compared with TGF-β1-treated cells [Figure 2A and 2B (VI)].All of these results suggest that Sema4C depletion inhibits TGF-β1-induced EMT.As accumulated interstitial matrix components were a consequence of EMT,fibronectin secretion by HKC cells was measured in culture supernatants.Treatment of HKC cells for 72h with TGF-β1resulted in a significant mean 3.8-fold increase (N =4)in fibronectin protein compared with control cells (Figure 2C).HKC cells transfected with Sema4C-specific siRNA were resistant to TGF-β1-induced fibronectin secretion (Figure 2C).This result was consist-ent with the finding that Sema4C depletion prevented TGF-β1-induced EMT.Sema4C is an activator of p38MAPK in TGF-β1-induced EMT Sema4C has been reported to be a key inducer of p38MAPK pathway signalling [11].To investigate whether Sema4C is involved in the TGF-β1-induced EMT through the activation of p38MAPK,we examined the phosphor-ylation of p38MAPK in TGF-β1-treated cells.Western blotting showed that TGF-β1significantly increased phos-phorylation of p38MAPK in HKC cells after 72h of treat-ment (Figure 3).The phosphorylated p38was about 2-fold higher than that of control,and it could be strikingly inhib-ited,almost to basal level,by Sema4C-specific siRNA (Figure 3).

Next,we examined the phosphorylation of p38and mesenchymal phenotype in Sema4C-transfected HKC cells.The results confirmed high expression of Sema4C

and p38phosphorylation and typical mesenchymal pheno-type in Sema4C-transfected cells (Figure 4).As shown in Figure 4B,Sema4C transfection significantly increased the phosphorylation of p38MAPK.Treatment with SB203580(a p38MAPK inhibitor)decreased phosphorylation of p38MAPK by 31%(Figure 4B).Confocal laser microscopy

showed that E-cadherin was linearly localized at cell bor-

ders of control cells [Figure 4C (I)],but formed a zipper-like pattern near the perimeter of Sema4C-transfected cells

[Figure 4C (II)].Vimentin was present exclusively in the

cytosol of Sema4C-transfected cells [Figure 4C (V)],and there was little endogenous expression in control cells [Figure 4C (IV)].Treatment with SB203580inhibited Sema4C-mediated EMT [Figures 4A and 4C (III and VI)].Fibronectin secretion of HKC cells was also regulated by Sema4C.When HKC cells were transfected with

Sema4C,Fig.2.Expression of Sema4C is increased in TGF-β1-treated cells,and depletion of Sema4C represses TGF-β1-induced EMT.(A )Representative western blot of Sema4C,E-cadherin and vimentin in control cells,TGF-β1-treated cells and TGF-β1-treated Sema4C-siRNA cells.The histogram shows the average volume density corrected for the loading control,GAPDH (n =4).*P <0.05compared with control cells.#P <0.05compared with TGF-β1-treated cells.(B )Representative confocal microscopy of E-cadherin and vimentin in monolayers of control cells,TGF-β1-treated cells and TGF-β1-treated Sema4C-siRNA cells.Cell nuclei were enhanced by staining of cell nuclei with propidium iodide for E-cadherin and DAPI for Vimentin.Magnification ×400.(C )Representative fibronectin secretion in culture media of control cells,TGF-β1-treated cells and TGF-β1-treated Sema4C-siRNA cells (n =4).*P <0.05compared with control cells.#P <0.05compared with TGF-β1-treated cells.

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there was a significant increase in fibronectin accumulation in culture supernatants (Figure 4D).However,treatment with SB203580inhibited the secretion of fibronectin pro-tein compared with Sema4C-transfected cells (Figure 4D).Those results indicate that Sema4C can phosphorylate p38MAPK in cultured human tubular epithelial cells and induce EMT.To further confirm the relationship between Sema4C and activation of p38MAPK on tubules in vivo ,we eval-uated sequential kidney sections from 5/6-nephrectomized rats and patients with renal fibrosis.Sema4C expression in tubules was highly correlated with p-p38MAPK expres-sion in these kidneys (Figure 5).In fibrotic kidney from either rats or human beings,along with an increase in Sema4C,phosphorylated p38MAPK was over-expressed in the renal epithelia,and the distribution patterns of phos-phorylated p38MAPK and Sema4C were highly congru-ent in the renal tubules (Figure 5C –H).In contrast,tubules in normal rats barely expressed either Sema4C or p-p38MAPK (Figure 5A –B).These results suggest that Sema4C is involved in TGF-β1-induced renal fibrosis via the p38MAPK pathway.Taken together,our results indicate that Sema4C directly activates p38kinase in TGF-β1-treated cells,and that Sema4C-MAPK signalling pathway is important in TGF-β1-induced EMT.Discussion The epithelial –mesenchymal transition (EMT)of tubular epithelial cells is a critical process of renal tubulointersti-tial fibrosis [2,18].TGF-β1signalling pathway plays a central role in regulating tubular EMT [5,18].TGF-β1transduces cellular signals via heterotetrameric complexes of type II (T βR-II)and type I (T βR-I)receptors [19].The binding of TGF-β1to T βR-II leads to phosphorylation of T βR-I and then activates receptor-activated (R)Smads (Smad2and Smad3)and triggers EMT [20].However,the activation of EMT is thought to occur through the sig-nals arising not only from Smad2/3but also those activated in response to p38MAPK [21–23].Recent studies have established a direct link between the TGF-βreceptors and p38MAPK [10,21,22].TGF-β1enables src to phos-phorylate T βR-II on Tyr284.Phosphorylated T βR-II func-tions as an Src homology 2(SH2)domain-binding site for growth factor receptor-bound protein 2(Grb2),leading to substantial phosphorylation of p38MAPK [21,22].This,in turn,triggers intracellular signalling pathways related to EMT.Despite recent advances in understanding this pathway,the molecular mechanisms that enable TGF-β1to activate p38MAPK remain to be fully elucidated.In the current study,Sema4C,a transmembrane protein and member of the semaphorin family,was reported to be essential for the activation of p38MAPK [11].The cyto-plasmic domain of Sema4C contains a proline-rich region that may interact with Src homology 3(SH3)domains and mediate intracellular signalling via interaction with a SH3domain-containing protein.The adapter protein Grb2is composed almost exclusively of SH3domains [24].Thus,it is possible that Sema4C may recruit Grb2to the cellular membrane,promote association of T βR-II and Grb2,and ultimately facilitate the activation of p38MAPK.There-fore,we postulated that Sema4C promote p38MAPK sig-nalling in the TGF-β1-induced renal tubular EMT.The findings of our study support the hypothesis that Sema4C plays an important role in mediating renal tubular

EMT through p38MAPK signalling.Our in vivo experi-

ments indicated that Sema4C increased in the tubular epi-

thelial cells of fibrotic kidneys,and in vitro experiments

indicated that TGF-β1treatment induced over-expression

of Sema4C in human tubular epithelial cells (HKC)accom-

panying characteristic changes of EMT.Loss of E-cadherin (a cell adhesion molecule present in the membranes of most epithelial cells)occurred,and this protein developed a dis-continuous distribution along the cell perimeters.Vimentin,a cytoskeletal protein in many mesenchymal cells,was also induced.Fibronectin secretion,a consequence of EMT,was significantly increased in HKC cell culture supernatants.Over-expression of Sema4C,performed with a Sema4C-transfected cell culture system,also remarkably accelerated the differentiation of epithelial HKC into mesenchymal cells.In addition,Sema4C siRNA knockdown in TGF-β1-treated HKC cells maintained E-cadherin,blocked

vimentin Fig.3.Depletion of Sema4C represses TGF-β1-induced activation of p38MAPK.Representative western blot of phosphorylated p38MAPK in control cells,TGF-β1-treated cells and TGF-β1-treated Sema4C-siRNA cells.The histogram shows the average volume density of phosphorylated p38MAPK corrected for the loading control,total p38MAPK (n =4).*P <0.05compared with control cells.#P <0.05compared with TGF-β1-treated cells.Sema4C mediates TGF-β1stimulation of EMT via p38MAPK 5 at Guangdong Medical College on January 20, 2011

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expression and inhibited fibronectin secretion,suggesting a delay of the EMT process.Taken together,these results suggest that Sema4C contributes to TGF-β1-induced EMT.Haitao Wu et al.[11]have previously demonstrated that p38MAPK is a key element for Sema4C signalling,and Sema4C is an activator for p38MAPK.In this study,we confirmed that p38MAPK requires Sema4C for the regu-lation of EMT.Sema4C initiates p38MAPK phosphoryl-ation in Sema4C-transfected cells,and SB203580(a p38MAPK inhibitor)suppresses the activation of p38MAPK and EMT.Knockdown of Sema4C dramatically impairs the phosphorylation of p38MAPK during TGF-β1treat-ment (Figure 3).Those results indicated that Sema4C mediated TGF-β1-induced EMT through the activation of p38MAPK.Furthermore,we demonstrated in vivo that the distribution pattern of phosphorylated p38MAPK is highly congruent with that of Sema4C in tubules of

fibrot-Fig.4.Over-expression of human Sema4C promotes EMT via activation of p38MAPK.(A )Representative western blots of Sema4C,E-cadherin and vimentin in control cells,Sema4C-transfected cells and SB203580-treated Sema4C-transfected cells.The histogram shows the average volume density corrected for the loading control,GAPDH (n =4).*P <0.05compared with control cells.#P <0.05compared with Sema4C-transfected cells.(B )Representative western blot of phosphorylated p38MAPK in control cells,Sema4C-transfected cells and SB203580-treated Sema4C-transfected cells.The histogram shows the average volume density of phosphorylated p38MAPK corrected for the loading control,total p38(n =4).*P <0.05compared with control cells.#P <0.05compared with Sema4C-transfected cells.(C )Representative confocal microscopy of E-cadherin and vimentin in monolayers of control cells,Sema4C-transfected cells and SB203580-treated Sema4C-transfected cells.Cell nuclei were enhanced by staining of cell nuclei with propidium iodide for E-cadherin and DAPI for vimentin.Magnification,×400.(D )Representative fibronectin secretion in culture media of control cells,Sema4C-transfected cells and SB203580-treated Sema4C-transfected cells (n =4).*P <0.05compared with control cells.#P <0.05compared with Sema4C-transfected cells.

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ic kidney (Figure 5).As tubular epithelial cells are the nat-ural targets of TGF-β1in vivo [17],this result further sup-ported that TGF-β1exerts its fibrogenic effect through Sema4C-mediated activation of p38MAPK.Our study provides the first evidence for this hypothesis and shows that the TGF-β1stimulation of tubular EMT is intimately linked to the Sema4C and the associated phosphorylation of p38MAPK.From these findings,we propose to identify the formation and distribution of Sema4C –Grb2complex and indicate its necessity for the activation of p38MAPK during TGF-β1treatment in fu-ture studies.Future studies are also needed to determine

whether therapeutic targeting of Sema4C may function in

alleviating the development of the TGF-β1-induced EMT.Furthermore,the knockdown of Sema4C (Figure 2A –B)or inhibition of p38MAPK (Figure 4A,C)did not substantial-ly preserve EMT,suggesting that other activated pathways are involved.The crosstalk between Sema4C/p38MAPK and other intracellular signal transduction pathways and the therapeutic strategy targeting multiple kinases may need to be studied in the future.

In summary,the results presented here demonstrate that TGF-β1-induced activation of p38MAPK and EMT is regulated by Sema4C in renal proximal tubular epithelial cells.Depletion of Sema4C inhibited activation of p38MAPK and blocked TGF-β1-induced EMT.Our results in-dicate the importance of Sema4C-MAPK signalling path-way in the development and progression of renal fibrosis,and thus suggest that this is a potential therapeutic target for the treatment of renal fibrosis.

Acknowledgements.This work was supported by the National Natural

Sciences Foundation of China (30800525and 30971372),Doctoral Fund for Y outh Scholars of Ministry of Education of China (200804871042)

and under the auspices of the New Century Excellent Talents Grant by the Ministry of Education of China (NCET004-0712).We greatly thank Prof.Ming Fan (Chinese Academy of Medical Sciences)for the generous gift of the pcDNA 4.0–Sema4C plasmid.We also thank Prof.Changlin

Mei (The Kidney Center of PLA)for the generous gift of HKC

cells.Fig.5.Immunohistochemistry of Sema4C and phosphorylated p38MAPK on serial sections of rats or patients with renal fibrosis.Sema4C and p38MAPK were co-expressed in tubules from rats or patients with tubulointerstitial fibrosis.These were representative photomicrographs from kidneys of sham rats immunostained with Sema4C (A )or p-p38MAPK (B ),5/6-nephrectomized rats immunostained with Sema4C (C )or p-p38MAPK (D ),patients with sclerosing glomerulonephritis immunostained with Sema4C (E )or p-p38MAPK (F )and patients with obstructive nephropathy immunostained with Sema4C (G )or p-p38MAPK (H ).Magnification,×200.

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Conflict of interest statement.None declared.

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Received for publication:6.1.10;Accepted in revised form:16.9.10

8R.Zeng et al.

at Guangdong Medical College on January 20, https://www.wendangku.net/doc/761608015.html,

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