Constance E. Runyan

The Phosphatidylinositol 3-Kinase/Akt Pathway Enhances Smad3-stimulated Mesangial Cell Collagen I Expression in Response to Transforming Growth Factor-β1

Transforming growth factor (TGF)-β has been associated with renal glomerular matrix accumulation. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-β1 in human glomerular mesangial cells. Here, we report that the PI3K/Akt pathway also plays a role in TGF-β1-increased collagen I expression. TGF-β1 stimulates the activity of phosphoinositide-dependent kinase (PDK)-1, a downstream target of PI3K, starting at 1 min. Akt, a kinase downstream of PDK-1, is phosphorylated and concentrates in the membrane fraction within 5 min of TGF-β1 treatment. The PI3K inhibitor LY294002 decreases TGF-β1-stimulated α1(I) and α2(I) collagen mRNA expression. Similarly, LY294002 or an Akt dominant negative construct blocks TGF-β1 induction of COL1A2 promoter activity. However, PI3K stimulation alone is not sufficient to increase collagen I expression, since neither a constitutively active p110 PI3K construct nor PDGF, which induces Akt phosphorylation, is able to stimulate COL1A2 promoter activity or mRNA expression, respectively. LY294002 inhibits stimulation of COL1A2 promoter activity by Smad3. In a Gal4-LUC assay system, blockade of the PI3K pathway significantly decreases TGF-β1-induced transcriptional activity of Gal4-Smad3. Activity of SBE-LUC, a Smad3/4-responsive construct, is stimulated by over-expression of Smad3 or Smad3D, in which the three C-terminal serine phospho-acceptor residues are mutated. This induction is blocked by LY294002, suggesting that inhibition of the PI3K pathway decreases Smad3 transcriptional activity independently of C-terminal serine phosphorylation. However, TGF-β1-induced total serine phosphorylation of Smad3 is decreased by LY294002, suggesting that Smad3 is phosphorylated by the PI3K pathway at serine residues other than the direct TGF-β receptor I target site. Thus, although the PI3K-PDK1-Akt pathway alone is insufficient to stimulate COL1A2 gene transcription, its activation by TGF-β1 enhances Smad3 transcriptional activity leading to increased collagen I expression in human mesangial cells. This cross-talk between the Smad and PI3K pathways likely contributes to TGF-β1 induction of glomerular scarring.

TGF-beta signal transduction in chronic kidney disease

Transforming growth factor (TGF)-beta is a central stimulus of the events leading to chronic progressive kidney disease, having been implicated in the regulation of cell proliferation, hypertrophy, apoptosis and fibrogenesis. The fact that it mediates these varied events suggests that multiple mechanisms play a role in determining the outcome of TGF-beta signaling. Regulation begins with the availability and activation of TGF-beta and continues through receptor expression and localization, control of the TGF-beta family-specific Smad signaling proteins, and interaction of the Smads with multiple signaling pathways extending into the nucleus. Studies of these mechanisms in kidney cells and in whole-animal experimental models, reviewed here, are beginning to provide insight into the role of TGF-beta in the pathogenesis of renal dysfunction and its potential treatment.

Interdependence of HIF-1α and TGF-β/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression

Increasing evidence suggests that chronic kidney disease may develop following acute kidney injury and that this may be due, in part, to hypoxia-related phenomena. Hypoxia-inducible factor (HIF) is stabilized in hypoxic conditions and regulates multiple signaling pathways that could contribute to renal fibrosis. As transforming growth factor (TGF)-β is known to mediate renal fibrosis, we proposed a profibrotic role for cross talk between the TGF-β1 and HIF-1α signaling pathways in kidney cells. Hypoxic incubation increased HIF-1α protein expression in cultured human renal tubular epithelial cells and mouse embryonic fibroblasts. TGF-β1 treatment further increased HIF-1α expression in cells treated with hypoxia and also increased HIF-1α in normoxic conditions. TGF-β1 did not increase HIF-1α mRNA levels nor decrease the rate of protein degradation, suggesting that it enhances normoxic HIF-1α translation. TGF-β receptor (ALK5) kinase activity was required for increased HIF-1α expression in response to TGF-β1, but not to hypoxia. A dominant negative Smad3 decreased the TGF-β-stimulated reporter activity of a HIF-1α-sensitive hypoxia response element. Conversely, a dominant negative HIF-1α construct decreased Smad-binding element promoter activity in response to TGF-β. Finally, blocking HIF-1α transcription with a biochemical inhibitor, a dominant negative construct, or gene-specific knockdown decreased basal and TGF-β1-stimulated type I collagen expression, while HIF-1α overexpression increased both. Taken together, our data demonstrate cooperation in signaling between Smad3 and HIF-1α and suggest a new paradigm in which HIF-1α is necessary for normoxic, TGF-β1-stimulated renal cell fibrogenesis.

Cytoskeletal Rearrangement and Signal Transduction in TGF-β1–Stimulated Mesangial Cell Collagen Accumulation

TGF-beta1 has been implicated in glomerular extracellular matrix accumulation, although the precise cellular mechanism(s) by which this occurs is not fully understood. The authors have previously shown that the Smad signaling pathway is present and functional in human glomerular mesangial cells and plays a role in activating type I collagen gene expression. It also was determined that TGF-beta1 activates ERK mitogen-activated protein kinase in mesangial cells to enhance Smad activation and collagen expression. Here, it was shown that TGF-beta1 rapidly induces cytoskeletal rearrangement in human mesangial cells, stimulating smooth muscle alpha-actin detection in stress fibers and promoting focal adhesion complex assembly and redistribution. Disrupting the actin cytoskeleton with cytochalasin D (Cyto D) selectively decreased basal and TGF-beta1-induced cell-layer collagen I and IV accumulation. The balance of matrix metalloproteinases (MMP) and inhibitors was altered by Cyto D or TGF-beta1 alone, increasing MMP activity, increasing MMP-1 expression, and decreasing tissue inhibitor of matrix metalloproteinase-2 expression. Cyto D also decreased basal and TGF-beta1-stimulated alpha1(I) collagen mRNA but did not inhibit TGF-beta-stimulated alpha1(IV) mRNA expression. A similar decrease in alpha1(I) mRNA expression caused by the actin polymerization inhibitor latrunculin B was partially blocked by the addition of jasplakinolide, which promotes actin assembly. The Rho-family GTPase inhibitor C. difficile toxin B or the Rho-associated kinase inhibitor Y-27632 also blocked TGF-beta1-stimulated alpha1(I) mRNA expression. Cytoskeletal disruption reduced Smad2 phosphorylation but had little effect on mRNA stability, TGF-beta receptor number, or receptor affinity. Thus, TGF-beta1-mediated collagen I accumulation is associated with cytoskeletal rearrangement and Rho-GTPase signaling.

Cell Phenotype-specific Down-regulation of Smad3 Involves Decreased Gene Activation as Well as Protein Degradation

Signaling by transforming growth factor-β (TGF-β), a regulator of several biological processes, including renal fibrosis, is mediated, in part, by the Smad proteins. Tight control of Smad level and activity is critical for proper TGF-β biological functions. Here, we have investigated the mechanisms involved in regulating Smad3 expression. In human glomerular mesangial cells, Smad3 protein levels were specifically reduced by 24 h of TGF-β1 treatment, whereas Smad2 and Smad4 levels were not. TGF-β1 increased endogenous Smad3 ubiquitination, and proteasome inhibitor treatment blocked TGF-β1-mediated Smad3 down-regulation resulting in accumulation of ubiquitinated Smad3. These data support the concept that Smad3 down-regulation occurs via degradation by the ubiquitin/proteasome machinery. However, changes in Smad3 protein levels were also paralleled by changes in Smad3 mRNA expression. TGF-β1 did not decrease Smad3 mRNA stability, but it significantly inhibited Smad3 promoter activity. In renal tubular epithelial cells, decreased Smad3 levels were observed only after exposure to TGF-β1 for longer time periods (5–7 days) that paralleled epithelial-to-mesenchymal transition, as determined by increased expression of smooth muscle α-actin and decreased expression of E-cadherin. Decline in Smad3 expression also occurred in kidneys after unilateral ureteral obstruction, a model of tubulointerstitial fibrosis associated with TGF-β up-regulation and epithelial-to-mesenchymal transition. Our data show for the first time that TGF-β1 modulates the expression of a receptor-activated Smad at both the protein and transcriptional level. Smad3 down-regulation could represent a feedback loop controlling TGF-β signaling in a cell phenotype-specific manner.

Role of SARA (SMAD Anchor for Receptor Activation) in Maintenance of Epithelial Cell Phenotype

By inducing epithelial-to-mesenchymal transition (EMT), transforming growth factor-β (TGF-β) promotes cancer progression and fibrosis. Here we show that expression of the TGF-β receptor-associated protein, SARA (Smad anchor for receptor activation), decreases within 72 h of exposure to TGF-β and that this decline is both required and sufficient for the induction of several markers of EMT. It has been suggested recently that expression of the TGF-β signaling mediators, Smad2 and Smad3, may have different functional effects, with Smad2 loss being more permissive for EMT progression. We find that the loss of SARA expression leads to a concomitant decrease in Smad2 expression and a disruption of Smad2-specific transcriptional activity, with no effect on Smad3 signaling or expression. Further, the effects of inducing the loss of Smad2 mimic those of the loss of SARA, enhancing expression of the EMT marker, smooth muscle α-actin. Smad2 mRNA levels are not affected by the loss of SARA. However, the ubiquitination of Smad2 is increased in SARA-deficient cells. We therefore examined the E3 ubiquitin ligase Smurf2 and found that although Smurf2 expression was unaltered in SARA-deficient cells, the interaction of Smad2 and Smurf2 was enhanced. These results describe a significant role for SARA in regulating cell phenotype and suggest that its effects are mediated through modification of the balance between Smad2 and Smad3 signaling. In part, this is achieved by enhancing the association of Smad2 with Smurf2, leading to Smad2 degradation.

Sex Steroids and the Endothelium

Gonadal steroids clearly influence the course of atherosclerotic cardiovascular disease in women. This observation has suggested that these hormones have beneficial effects on the physiology of the vascular wall. Increased arterial vascular caliber after estrogen treatment, decreased lipid levels in subjects receiving hormone replacement therapy, and the markedly decreased extent of atherosclerotic plaque formation in young women as compared with young men support a cardioprotective effect of ovarian steroids. Generally, it appears that the effects of 17beta-estradiol are particularly beneficial, and the mechanism of action is targeted largely to the endothelial cell. This review describes the evidence for positive effects of estrogens on endothelial cell biology and considers potential mechanisms for estrogen actions on endothelial cell signal transduction.

A conceptual framework for the molecular pathogenesis of progressive kidney disease.

The data regarding the pathogenesis of progressive kidney disease implicate cytokine effects, physiological factors, and myriad examples of relatively nonspecific cellular dysfunction. The sheer volume of information being generated on this topic threatens to overwhelm our efforts to understand progression in chronic kidney disease or to derive rational strategies to treat it. Here, a conceptual framework is offered for organizing and considering these data. Disease is initiated by an injury that evokes a tissue-specific cellular response. Subsequent structural repair may be effective, or the new structure may be sufficiently changed that it requires an adaptive physiological response. If this adaptation is not successful, subsequent cycles of misdirected repair or maladaptation may lead to progressive nephron loss. To illustrate how this framework can be used to organize our approach to disease pathogenesis, the role of cytokines in proteinuria and progressive glomerular disease is discussed. Finally, this theoretical framework is reconsidered to examine its implications for the diagnosis and treatment of clinical conditions. Application of this schema could have significant relevance to both research inquiry and clinical practice.

Phosphatidylinositol 3-Kinase and Rab5 GTPase Inversely Regulate the Smad Anchor for Receptor Activation (SARA) Protein Independently of Transforming Growth Factor-β1

Background: SARA promotes an epithelial cell phenotype, whereas its down-regulation is permissive for EMT. Results: PI3K inhibition decreases SARA protein expression, likely through alterations in Rab5-containing endosomes. Conclusion: PI3K signaling supports an epithelial phenotype. Significance: PI3K has complex effects in fibrogenesis. Our data suggest an antifibrotic action of PI3K that involves maintaining SARA expression. SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-β1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-β1 in that, unlike TGF-β1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85α action, and co-immunoprecipitation of SARA and p85α confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either LY294002 or p85α knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, we expressed GTPase-deficient Rab5 and observed endosomal enlargement and reduced SARA protein expression, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via LY294002 or p85α knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle α-actin. Together, these data suggest that although TGF-β1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K.

Hormonal regulation of gonadal angiogenesis

In the reproductive system, the rapid growth and regression of decidual tissue depends on concomitant increases and decreases in the extent of the vascular network supporting that tissue. This suggests that, like reproductive organ function, angiogenesis and vessel regression are regulated by gonadal steroids. This chapter considers the evidence supporting this hypothesis and discusses potential mechanisms by which these hormones may modulate vascular cell function.