The TGFβ1/Smad Signaling Pathway Is Functional In Cultured Rat Mesangial Cells

IntroductionProgressive glomerulosclerosis, characterized by extracellular matrix (ECM) accumulation and glomerular collapse, represents a common pathway for the loss of functioning glomeruli associated with primary diseases as disparate as glomerulonephritis, IgA nephropathy, diabetic nephropathy, and lupus nephritis. There is increasing evidence that the imbalance of ECM synthesis and degradation may be an important mechanism of glomerulosclerosis. Recent research points are focused on the regulation ECM metabolism, and its correlation with the development of glomerulosclerosis.Transforming growth factor-β1 (TGFβ1) is a multiple functional and pleiotrophic cytokine, regulating a diverse range of cellular response, including proliferation, development, differentiation, apoptosis, and immune response. A significant body of literature suggests that TGFβ1 could play an crucial role in glomerular ECM accumulation in the process of glomerulosclerosis, but the mechanism, especially the intracellular signals resulting in increased accumulation of ECM in mesangial cells is unclear. The proteins activated by the TGFβ superfamily are called Smad proteins. It is proved that TGFβ1/Smad signaling pathway is very important for transmiting signals from the cytoplasm into the nucleus. The Smad family can be subdivided into three classes based on their function in the signaling cascade: the receptor-regulated Smads (R-Smads), including Smad1, 2, 3, and Smad8 which propagate the specificity of the signal in response to ligands; the common Smads (Smad4 in vertebrates), which are thought to function as mediators for signaling by all TGFβ family members; and the inhibitory Smads, including Smad6 and Smad7 that function to regulate the ligand response by blocking signaling transduction.The main purpose of the present research is to elucidate the effect of the TGFβ1/Smad signaling pathway on the expression of ECM components, such as fibronectin (FN), type Ⅰcollagen (ColⅠ), type Ⅳ collagen (Col Ⅳ), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in cultured rat MsC through the methods of gene transfection; to explore the crosstalk between Smad and MAPK signaling pathways in the transcription of Smad7 gene; and/ to try to elucidate the TGFβ1/Smad signaling pathway on the pathogenesis of glomerulosclerosis, providing the therotical and experimental bases for therapeutic intervention in animal models with chronic progressive glomerulonephritis.PartⅠ The effect of R-Smads, Smad4 genes on extracellular matrix metabolism in cultured rat MsCObjective To explore the effect of TGFβ1/Smad signaling pathway on the expression and or enzymatic activity of ColⅠ, Ⅳ, FN,MMP-2/TIMP-2 in cultured rat MsC transfected with R-Smads and Smad4 genes.Methods Primary cultured rat MsC were transfected transiently with recombinant eukaryotic expression plasmids of pcDNA-Smad2, 3 and Smad4 separatetly by LipofectamineTM 2000. The effects of transfection were determined by immunofluorescence, semiquantitative RT-PCR and Western blot analysis, respectively. The Smads-transfected MsCs were treated with recombinant human cytokine TGFβ1, then the expression of FN, ColⅠ, Ⅳ, MMP-2 and TIMP-2 were evaluated by Western blot analysis and the activity of MMP-2 or TIMP-2 was investigated by gelatin zymography or reverse gelatin zymography. Results MsC transfected with Smad2, Smad3 vectors stimulated by TGFβ1showed increased expressions of ColⅠ(fold induction: 1.6 and 1.7 respectively)and Col Ⅳ(fold induction: 2.1 and 2.3 respectively)(P<0.01), while the expression of FN showed no change. MsC transfected with Smad4 vector showed the increase of ColⅠand Col Ⅳexpressions reached 1.8 and 3.3 fold respectively (P<0.01) after TGFβ1 stimulation. MsC transfected with Smad2 gene showed slightly increased expression and enzymatic activity of both MMP-2 and TIMP-2, which were more obviously when stimulated by TGFβ1 (fold induction: 2.8 and 3.0 or 3.0 and 1.7, respectively) (P<0.01). MsC...