Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor ␤1 (TGF-␤1), and addition of TGF-␤1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-␤1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGF␤1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGF␤1-driven matrix production within a diabetic milieu.

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, Ras S35 and Ras G37 , which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while Ras G37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.Ras proteins are important signaling intermediates that convey signals initiated at the cell surface to effector pathways in the cytoplasm. Ras exerts effects on cell transformation and proliferation, the actin cytoskeleton, differentiation, and apoptosis. It is likely that these effects are mediated by multiple effectors, including Raf-1, RalGDS (2), phosphatidylinositol 3-kinase (PI3K) (48), and other Ras-binding proteins (reviewed in reference 38), as well as members of the Rho family (26,44,45).The elucidation of multiple Ras effector pathways was greatly facilitated by the isolation of Ras mutants which interact with single downstream effectors (60). Ras S35 binds preferentially to Raf, while Ras G37 binds to RalGDS (49,53,60 While the differential effects of Ras S35 and Ras G37 on Raf and MAPK activity are consistently observed (23,25,27,46,49,60), other effects of these mutants vary. Both Ras S35 and Ras G37 transform some NIH 3T3 strains, inducing growth in medium with low serum levels, anchorage-independent proliferation, and tumor formation in nude mice (27). The activity of Ras G37 in these assays suggests that Ras stimulates some of the same biological effects through Raf-independent pathways and that the effector pathways used by Ras vary in different cell types. Consistently, Ras V12 stimulates transformation in most cells, while activated forms of Raf transform fibroblasts but not epithelial cells (41). If Ras signals through multiple effectors, the selection of a particular effector pathway should be regulated. One potential way to achieve such regulation is through cross talk between Ras and other signaling pathways.One of the best-studied examples of cross talk is that between Ras and protein kinase A (PKA). In many cells, cyclic AMP (cAMP) inhibits Ras signaling through Raf and the MAPK cascade (reviewed in reference 6). cAMP activates PKA, which phosphorylates Raf-1 at multiple serine residues. PKA-mediated phosphorylation reduces the affinity of Raf for Ras (17,63) and decreases Raf kinase activity (10,17,57), effects which may inhibit Ras-mediated proliferation. Ras and cAMP also collaborate to produce similar effects. In PC12 cells, Ras and cAMP induce neurite extension (13, 54) and promote cell survival (64). Ras and cAMP stimulate proliferation in thyroid cells (7,36,39), and Ras activity is required for the mitogenic effects of thyrotropin (TSH) (32). Despite the requirement for Ras, TSH downregulates signaling through Raf and the MAPK cascade...