Tomoko Hayashida

Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses in human mesangial cells.

Transforming growth factor β (TGF‐β) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF‐β1 activated extracellular signal‐regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant‐negative ERK blockade reduced TGF‐β1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C‐terminal SSPXSP motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 µM) or U0126 (25 µM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK‐dependent activation of Smad2/3 occurs only in certain cell types. TGF‐β stimulated phosphorylation of an expressed Smad3A construct, with a mutated C‐terminal SSPXSP motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced α2(I) collagen promoter activity, an effect blocked by co‐transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF‐β1 on collagen promoter activity were additive. These results indicate that ERK‐dependent R‐Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.

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.

High Ambient Glucose Enhances Sensitivity to TGF-β1 via Extracellular Signal—Regulated Kinase and Protein Kinase Cδ Activities in Human Mesangial Cells

High ambient glucose activates intracellular signaling pathways to induce cytokines such as TGF-beta1 in the extracellular matrix accumulation of diabetic nephropathy. These same pathways also may directly modulate TGF-beta1 signaling. R-Smad phosphorylation, association with Smad4, and nuclear accumulation after TGF-beta1 treatment (1.0 ng/ml) were significantly higher in mesangial cells that were conditioned to 20 mM glucose for 72 h than mesangial cells in 6.5 mM glucose, suggesting that high glucose enhanced responsiveness to TGF-beta1. Neither TGF-beta1 bioactivity nor TGF-beta receptor binding was significantly different between in 6.5 and 20 mM glucose-conditioned cultures. Furthermore, adding a neutralizing anti-TGF-beta1 antibody during glucose conditioning did not affect the enhanced Smad responsiveness, indicating that enhancement likely did not result from increased TGF-beta expression. In contrast, a mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK inhibitor, PD98059, completely abrogated the effect of high glucose. Glucose stimulation of ERK was inhibited by the general protein kinase C (PKC) inhibitor calphostin C and by the PKCdelta-specific inhibitor rottlerin, whereas Gö6976, an inhibitor of conventional PKC, had no effect on ERK activity. Specificity of the PKC inhibitors was further verified by PKCbeta and delta kinase assay. High glucose increased expression of several PKC isozymes, but only PKCdelta showed proportionally increased membrane translocation and kinase activity in cells that were conditioned to 20 mM glucose. Finally, both ERK and PKCdelta inhibition during glucose conditioning abrogated enhanced alpha1(I) collagen mRNA and promoter induction by TGF-beta1. Taken together, these data strongly suggest that heightened ERK and PKCdelta activity in high ambient glucose conditions interact with the Smad pathway, leading to enhanced responsiveness to TGF-beta1 and increased extracellular matrix production in mesangial cells.

Regulation of Vascular Type 1 Angiotensin Receptors by Cytokines

Although various cytokines are known to be expressed in atherosclerotic lesions, it is not known how these cytokines affect receptors for the peptide hormone angiotensin II (Ang II). We therefore examined the effects of interleukin-1 alpha (220 U/mL [10 ng/mL]), tumor necrosis factor-alpha (280 U/mL [100 ng/mL]), and interferon gamma (100 U/mL) on Ang II type 1 (AT1) receptors expressed in rat vascular smooth muscle cells. Treatment with interleukin-1 alpha caused a 1.4- to 1.7-fold increase in AT1 binding after 24 hours (P<.01) and a 2.3-fold increase in AT1 mRNA (P<.05). Tumor necrosis factor-alpha and interferon gamma did not cause a significant change in AT1 binding when administered alone but caused a 30% reduction in binding when administered together (P<.05). The maximal decrease in AT1 binding (60%, P<.01) was seen with the combination of interleukin-1 alpha with tumor necrosis factor-alpha and interferon gamma. Although the upregulation of AT1 by interleukin-1 alpha was unaffected by pretreatment of cells with N-monomethyl-L-arginine or indomethacin, downregulation of AT1 by interleukin-1 alpha combined with tumor necrosis factor-alpha/interferon gamma was inhibited by N-monomethyl-L-arginine (P<.01). Interleukin-1 alpha treatment enhanced Ang II-induced [3H]uridine incorporation, whereas treatment with interleukin-1 alpha combined with tumor necrosis factor-alpha/interferon gamma attenuated Ang II-induced [3H]uridine and [3H]leucine incorporation. These results demonstrate that interleukin-1 alpha upregulates AT1 receptors and enhances Ang II-stimulated hypertrophic responses. However, a combination of interleukin-1 alpha with tumor necrosis factor-alpha and interferon gamma downregulates AT1 receptors by a nitric oxide-dependent mechanism and reduces Ang II-stimulated trophic responses in vascular smooth muscle cells.

MAP-kinase activity necessary for TGFβ1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397

The signals mediating transforming growth factor β (TGFβ)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFβ-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGFβ1-dependent. By immunocytochemistry, TGFβ1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGFβ-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFβ-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGFβ-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFβ-induced responses to regulate collagen production.

Rac1 promotes TGF-β-stimulated mesangial cell type I collagen expression through a PI3K/Akt-dependent mechanism

Transforming growth factor (TGF)-beta is a central mediator in the progression of glomerulosclerosis, leading to accumulation of aberrant extracellular matrix proteins and inappropriate expression of smooth muscle alpha-actin in the kidney. Previously, we reported that disrupting the cytoskeleton diminished TGF-beta-stimulated type I collagen accumulation in human mesangial cells. As cytoskeletal signaling molecules, including the Rho-family GTPases, have been implicated in fibrogenesis, we sought to determine the respective roles of RhoA and Rac1 in HMC collagen I expression. TGF-beta1 activated both RhoA and Rac1 within 5 min of treatment, and this activation was dependent on the kinase activity of the type I TGF-beta receptor. TGF-beta1-stimulated induction of type I collagen mRNA expression and promoter activity was diminished by inhibiting Rac1 activity and was increased by a constitutively active Rac1 mutant, whereas inhibiting RhoA activity had no such effect. Rac1 activation required phosphatidylinositol-3-kinase (PI3K) activity. Furthermore, the PI3K antagonist, LY294002, reduced TGF-beta1-stimulated COL1A2 promoter activity and Rac1 activation. It also partially blocked active Rac1-stimulated collagen promoter activity, suggesting that PI3K activity contributes to both TGF-beta activation of Rac1 and signal propagation downstream of Rac1. Thus, while both Rac1 and RhoA are rapidly activated in response to TGF-beta1 in human mesangial cells, only Rac1 activation enhances events that contribute to mesangial cell collagen expression, through a positive feedback loop involving PI3K.

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.

TGF-β/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1α expression

Transforming growth factor (TGF)-β is a major mediator of kidney fibrosis. In the past decade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-β-induced fibrogenesis. One such pathway involves mammalian target of rapamycin complex (mTORC)1. We recently reported that the hypoxia-inducible factor (HIF)-1 is essential for TGF-β-induced collagen expression regardless of ambient oxygen tension. A modulator of HIF expression other than oxygen tension is mTORC1. We therefore sought to evaluate a possible role for mTORC1 activity in TGF-β-induced fibrogenesis. mTORC1 activity was increased in human mesangial cells treated with TGF-β in a TGF-β receptor-dependent manner. Short hairpin (sh)RNA to Smad3 decreased, while overexpression of Smad3 increased, the mTORC1 activity, suggesting that TGF-β stimulation of mTORC1 also requires Smad3. Pretreatment with rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-β-induced COL1A2-luc activity and collagen I protein expression. mTORC1 inhibition also prevented the TGF-β-stimulated increase in both hypoxia-responsive element (HRE) activity and HIF-1α protein expression, while activation of mTORC1 by active Rheb increased basal but not TGF-β-induced HRE activity. shRNA to Smad3 reduced HRE activity, while overexpression of Smad3 increased HIF-1α protein expression and activity in an mTORC1-dependent manner. Lastly, overexpression of HIF-1α bypassed the inhibitory effect of mTORC1 blockade on collagen expression. These results suggest that Smad3/mTORC1 interaction to promote HIF-1 expression is a key step in normoxic kidney fibrogenesis.

Hypoxia-inducible factor-2α and TGF-β signaling interact to promote normoxic glomerular fibrogenesis

Hypoxia-inducible factors (HIFs) are transcription factors consisting of an oxygen-sensitive α-subunit binding to a stable β-subunit. HIFs regulate multiple signaling pathways that could contribute to fibrogenesis, supporting their potential role in hypoxia-mediated renal fibrosis. We previously reported that HIF-1 is upregulated and required for transforming growth factor (TGF)-β induction of collagen in renal tubular cells. Here, we performed in vitro and in vivo studies of potential glomerular crosstalk between TGF-β and normoxic HIF signaling. HIF-α has two major isoforms, HIF-1α and HIF-2α with different target gene sets. In cultured human mesangial cells, TGF-β1 treatment increased both HIF-1α and HIF-2α expression in normoxia. TGF-β1 did not increase HIF-1α/2α mRNA levels nor decrease the rate of protein degradation, suggesting that it enhances HIF-1α/2α expression through translation. TGF-β receptor (ALK5) kinase activity was required for increased, TGF-β-stimulated HIF-α expression in response to TGF-β, and inhibiting PI3-kinase markedly decreased HIF-α expression. Blocking HIF-1α/2α expression using siRNA decreased basal and TGF-β1-stimulated type I collagen expression, while overexpressing nondegradable HIF-α increased the collagen response, with HIF-2α being significantly more effective than HIF-1α. In adriamycin-induced mouse glomerulosclerosis, HIF-2α target genes were upregulated in sclerosing glomeruli. Taken together, our data demonstrate potential signaling interaction between TGF-β and HIFs to promote renal fibrogenesis in normoxia and suggest that the HIF-2α isoform is more important during glomerulosclerosis.