The Effect Of Decorin On The Growth And Phenotype Change Of Rat Mesangial Cell And Its Relative Signal Transduction Pathway

IntroductionGlomerulosclerosis is a final common outcome of glomerular injury in various types of human glomerular disease, which is characterized with the proliferation of glomerular mesangial cell (MsC) and abnormal accumulation of extracellular matrix (ECM) in glomeruli. The resident MsC functions actively, and acquire a characteristic spectrum of biologic futures which was defined as the inflammatory phenotype during glomerular injury of inflammation in glomerulonephritis. These phenotype changes of MsC contribute to glomerular mesangial expansion resulting in glomerulosclerosis.Decorin associated with the docration and regulation of collange fibrillogenesis is a small leucine-rich proteoglycan and a ECM component of stroma tissue. It can also bind to a viarity of other ECM components such as fibronectin (FN), thrombosponding-1 (TSP-1) and inhibit cell proliferation and migration and neutralize the activity of transforming growth factor-β1 (TGFpi). TGFpi is a multiple functional and pleiotrophic cytokine, regulating a diverse range of cellular response, including proliferation, development, differentiation, apoptosis, and immune response. It was well known that TGFβ1 could play an crucial role in glomerular ECM accumulation in the development and progress of glomerulosclerosis. TGFpi functions through a family of transmembrane protein serine/threonine kinases reffered to as TGFβ1 receptors, type I and II receptor, activating Smads and mitogen-activated protein kinases (MAPKs) downstream molecualr of TGFβ1 receptors. DCN, which has similar structure with TGFp receptor III -betaglycan, can bind to TGFpi and interfere with TGFpi binding to its receptor I and II. However, the mechanism of down-regulated effect of DCN on the TGFPRI and TGFβRII expressions is not still clarified so far. Our former work has proved that DCN can inhibit the proliferation of cultured MsC and antagonize the progress of glomerula lesion in rat experimental glomerulonephritis, but their mechanisms are still not clarified.The main purpose of the present study is to observe the effect of DCN on the expression of TGFβRI and TGFβRII and its possible mechanism; to find whether the influence of DCN to MsC growth is associated with MAPKs activation and P21 protein up-regulation and to investigate the interrelation of DCN and TGFβ1 in order to elucidate the mechanisms of inhibitory effect of DCN on the proliferation, matrix synthesis of cultured MsC and the development of glomerulosclerosis.Part I The Construction of MsC Transfectants Overexpressed DCN Gene Objectives to construct rat MsC transfectant overexpressed DCN geneMethods Cultured rat MsC were transfected stably with recombinant plasmids of pcDNA3.1A-DCN by Lipofectamine^? 2000. After the plasmid was purfied and identified, the positive clones were determined by semiquantitative RT-PCR, Western blot analysis and immunofluorescence respectively.Result After treatment with the enzymes of EcoRI and XbaI, the recombinant plasmid of pcDNA3.1A-DCN was cut into two fragment, carrier and aim gene, whose lengths were 5400bp and 1068bp, respectively. Two cell clones overexpressed DCN mRNA and protein markedly were established.Conclusion Successfully transfecting recombinant plasmid containing DCN to MsC by lipofectin method, two positive cell clones overexpressing markedly DCN mRNA and protein were established.Part II The Changes of TGFpRI and TGFpRII Expressions on MsC by DCN Gene Transfection and Its Possible MechanismObjective To study the influence of DCN gene transfection on the expressions of TGFPRI and TGFpRII on MsC and the possible mechanism.Methoad RT-PCR and Western blot analysis were adopted to detect the expressions of mRNA and protein of both receptors on normal and transfected MsC with or without extrogenous TGFβ1 stimulation. Western blot analysis was used to estimatethe difference of TGFβ1 secretion and p-Smad2 activity in both normal control and trandfected cell clones. Immunoprecipation was used to detect the combination of DCN and TGFpi in cultured supernatatent.Result The expressions of TGF-pRI and TGF-pR II mRNA and their proteins on normal MsC stimulated by exogenous TGF-pl inceased in time-dependent fashion and got to peak at 24h. Compared with normal and untransfected MsC (1P-1), the mRNA and protein expressions of TGF-pRI and TGF-pRII on MsC (3D-5, 7D-1) transfected by DCN gene decreased significantly. The expression of TGF-|3RI mRNA reduced to 20% and 32%, that of TGF-PRII mRNA to 64% and 59%, that of TGF-pRI protein to 34% and 25% and that of TGF-pRII protein to 49% and 57%. DCN gene transfection can suppress the increase of mRNA and protein expression of both receptors caused by exogenous TGF-β1. DCN could bind to TGFpi and the secretion of TGFpi autocrine in supernatant decreased notably in transfected cell clone. The activity of p-Smad2 reduced in cell clone 3D-5 and 7D-1 after stimulated with exogenous TGF-β1.Conclusions TGFβ1 can up-regulate protein expressions of both TGFpi receptors on MsC in time-dependent manner, whereas DCN gene transfection can down-regulate their protein level obviously. Their down-regulation may be associated with the binding of DCN to TGFβ1, and the decrease of phosphorylation of Smad2 and autocrine TGF-β1 secretion.Part III Study on the Inhibitory Effect of Decorin on Rat MsC Growth and Its Signal Transduction PathwayObjective To explore the inhibitory effect of decorin on MsC growth and MAPKs and P21 protein expressions.Methods We collected DCN-containing supernatant and added it into the culture medium of normal MsC , then used flow cytometer to detect the cell cycle. Western blot analysis was used to detect the changes of MAPK molecule and P21 protein. Inmmunofluorescence was adopted to observe the expression of P21 on cultured MsC. Inmmunoprecipation was used to check the binding of P21 and CDK2.Results Compared with normal MsC, the number of G2-M cells affected by DCN-containing supernatant decreased to 35% (p<0.01) , while the expression of p-ERKl/2 and p-SAPK/JNK on MsC increased obviously, reached to 2.2,1.4 times and 1.7, 1.8 times respectively (compared with normal MsC , PO.05). When giving antibody against DCN to MsC, the increase of p-ERKl/2 and p-SAPK/JNK was suppressed in dose-dependence. Western blot analysis and inmunofluorescence revealed that expression of both total and nuclear P21 proteins on MsC affected by DCN-containing supernatant increased and was inhibited dose-dependently when giving DCN antibody to MsC. Inhibitors U0126 and circumin of ERK and SAPK/JNK pathway could inhibit total and nuclear P21 up-regulation caused by DCN-containing supernatant, and the expression of total P21 protein reduced to 64% and 68% respectively (PO.05), while the inhibitor SB203580 of P38 pathway had no this effect on MsC. The change of P21 located in nucleare. P21 could bind to CDK2 in dose-dependently.Conclusions DCN can suppress the cultured MsC growth, possibly associated with the increase of signaling molecule ERK 1/2 and SAPK/JNK and the decrease of P21 protein expression.Part IV The Study of the Interaction between DCN and TGF-plObjective to expore the interaction between DCN and TGF-pi on MsC proliferation and their related signal pathwaysMethod Flow cytometer was adopted to estimate the cell cycle and Western blot analysis was used to detect the protein expressions of p-ERKl/2, p-SAPK/JNK, p-Smad2 and total P21 on MsC stimulated with DCN or/and TGF-pi.Result DCN or TGF-pi (2ng/ml) alone could suppress MsC proliferation, but their growth cycle did not differ from the control group when stimulated by both DCN and TGF-pl. After stimulated by DCN or TGF-pl, the p-ERKl/2 and p-SAPK/JNK expressions were increased, while the combinative use of both DCN and TGF-pi could not change their expessions compared with control group. The protein level of p-Smad2 was increased with stimulation of TGF-pi and P21 expression was...