ABSTRACT
Diabetic nephropathy (DN) is a progressive fibrotic condition that may lead to end-stage renal disease and kidney failure. Transforming growth factor-ß1 and bone morphogenetic protein-7 (BMP7) have been shown to induce DN-like changes in the kidney and protect the kidney from such changes, respectively. Recent data identified insulin action at the level of the nephron as a crucial factor in the development and progression of DN. Insulin requires a family of insulin receptor substrate (IRS) proteins for its physiological effects, and many reports have highlighted the role of insulin and IRS proteins in kidney physiology and disease. Here, we observed IRS2 expression predominantly in the developing and adult kidney epithelium in mouse and human. BMP7 treatment of human kidney proximal tubule epithelial cells (HK-2 cells) increases IRS2 transcription. In addition, BMP7 treatment of HK-2 cells induces an electrophoretic shift in IRS2 migration on SDS/PAGE, and increased association with phosphatidylinositol-3-kinase, probably due to increased tyrosine/serine phosphorylation. In a cohort of DN patients with a range of chronic kidney disease severity, IRS2 mRNA levels were elevated approximately ninefold, with the majority of IRS2 staining evident in the kidney tubules in DN patients. These data show that IRS2 is expressed in the kidney epithelium and may play a role in the downstream protective events triggered by BMP7 in the kidney. The specific up-regulation of IRS2 in the kidney tubules of DN patients also indicates a novel role for IRS2 as a marker and/or mediator of human DN progression.
Subject(s)
Diabetic Nephropathies/metabolism , Gene Expression , Insulin Receptor Substrate Proteins/metabolism , Kidney Tubules/metabolism , Adolescent , Adult , Animals , Base Sequence , Binding Sites , Bone Morphogenetic Protein 7/physiology , Case-Control Studies , Cell Line , Child , Epithelium/metabolism , Female , Humans , Insulin Receptor Substrate Proteins/genetics , Kidney Tubules/pathology , Male , Mice , Middle Aged , Phosphorylation , Protein Processing, Post-Translational , Signal Transduction , Smad4 Protein/genetics , Transcriptional Activation , Young AdultABSTRACT
The critical involvement of TGF-ß1 (transforming growth factor-ß1) in DN (diabetic nephropathy) is well established. However, the role of CTGF (connective tissue growth factor) in regulating the complex interplay of TGF-ß1 signalling networks is poorly understood. The purpose of the present study was to investigate co-operative signalling between CTGF and TGF-ß1 and its physiological significance. CTGF was determined to bind directly to the TßRIII (TGF-ß type III receptor) and antagonize TGF-ß1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-ß1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-ß1 signalling and expression of a unique profile of differentially regulated genes was observed in CTGF/TGF-ß1-treated mesangial cells. Decreased levels of Smad2/3 phosphorylation were evident in STZ (streptozotocin)-induced diabetic mice, concomitant with increased levels of CTGF. Knockdown of TßRIII restored TGF-ß1-mediated Smad signalling and cell contractility, suggesting that TßRIII is key for CTGF-mediated regulation of TGF-ß1. Comparison of gene expression profiles from CTGF/TGF-ß1-treated mesangial cells and human renal biopsy material with histological diagnosis of DN revealed significant correlation among gene clusters. In summary, mesangial cell responses to TGF-ß1 are regulated by cross-talk with CTGF, emphasizing the potential utility of targeting CTGF in DN.
Subject(s)
Connective Tissue Growth Factor/pharmacology , Gene Expression Regulation/physiology , Mesangial Cells/metabolism , Smad Proteins/metabolism , Transforming Growth Factor beta1/pharmacology , Animals , Cell Line , Cell Movement , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Diabetes Mellitus, Experimental , Humans , Mice , Phosphorylation , Proteoglycans/genetics , Proteoglycans/metabolism , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction , Smad Proteins/genetics , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolismABSTRACT
RACK1 can act as a scaffolding protein to integrate IGF-IR and integrin signalling in transformed cells but its actions in regulating IGF-IR signalling in non-transformed cells are less well understood. Here, we investigated the function of RACK1 in the non-transformed cardiomyocyte cell line H9c2. Overexpression of RACK1 in H9c2 cells was sufficient to increase cell size, increase adhesion to collagen 1, enhance protection from hydrogen peroxide-induced cell death, and increase cell migration. However, cell proliferation was decreased in these cells. Small interfering RNA (siRNA)-mediated suppression of RACK1 in H9c2 cells resulted in decreased cell adhesion and migration, but had no effect on cell proliferation or size. Increased basal and IGF-I-mediated Erk phosphorylation was observed in RACK1-overexpressing H9c2 cells. Interestingly, contrary to observations in transformed cells, RACK1 was not observed to interact with the IGF-IR in H9c2 cells. Also in contrast to observations in transformed cells, IGF-I promoted recruitment of Src to RACK1 as well as recruitment of PKCalpha, and PKCepsilon to RACK1. Overall, the data indicate that in H9c2 cells RACK1 can influence cell size, cell survival, adhesion, migration, but its responses to IGF-I are independent of an association with the IGF-IR. Thus, the composition of the RACK1 scaffolding complex and its effects on IGF-I signalling may be different in transformed and non-transformed cells.
