SARA suppresses myofibroblast precursor transdifferentiation in fibrogenesis in a mouse model of scleroderma.

We previously reported that Smad anchor for receptor activation (SARA) plays a critical role in maintaining epithelial cell phenotype. Here, we show that SARA suppressed myofibroblast precursor transdifferentiation in a mouse model of scleroderma. Mice overexpressing SARA specifically in PDGFR-ß+ pericytes and pan-leukocytes (SARATg) developed significantly less skin fibrosis in response to bleomycin injection compared with wild-type littermates (SARAWT). Single-cell RNA-Seq analysis of skin PDGFR-ß+ cells implicated pericyte subsets assuming myofibroblast characteristics under fibrotic stimuli, and SARA overexpression blocked the transition. In addition, a cluster that expresses molecules associated with Th2 cells and macrophage activation was enriched in SARAWT mice, but not in SARATg mice, after bleomycin treatment. Th2-specific Il-31 expression was increased in skin of the bleomycin-treated SARAWT mice and patients with scleroderma (or systemic sclerosis, SSc). Receptor-ligand analyses indicated that lymphocytes mediated pericyte transdifferentiation in SARAWT mice, while with SARA overexpression the myofibroblast activity of pericytes was suppressed. Together, these data suggest a potentially novel crosstalk between myofibroblast precursors and immune cells in the pathogenesis of SSc, in which SARA plays a critical role.

Stromal Transcription Factor 21 Regulates Development of the Renal Stroma via Interaction with Wnt/ß-Catenin Signaling.

Background: Kidney formation requires coordinated interactions between multiple cell types. Input from the interstitial progenitor cells is implicated in multiple aspects of kidney development. We previously reported that transcription factor 21 (Tcf21) is required for ureteric bud branching. Here, we show that Tcf21 in Foxd1+ interstitial progenitors regulates stromal formation and differentiation via interaction with ß-catenin. Methods: We utilized the Foxd1Cre;Tcf21f/f murine kidney for morphologic analysis. We used the murine clonal mesenchymal cell lines MK3/M15 to study Tcf21 interaction with Wnt/ß-catenin. Results: Absence of Tcf21 from Foxd1+ stromal progenitors caused a decrease in stromal cell proliferation, leading to marked reduction of the medullary stromal space. Lack of Tcf21 in the Foxd1+ stromal cells also led to defective differentiation of interstitial cells to smooth-muscle cells, perivascular pericytes, and mesangial cells. Foxd1Cre;Tcf21f/f kidney showed an abnormal pattern of the renal vascular tree. The stroma of Foxd1Cre;Tcf21f/f kidney demonstrated marked reduction in ß-catenin protein expression compared with wild type. Tcf21 was bound to ß-catenin both upon ß-catenin stabilization and at basal state as demonstrated by immunoprecipitation in vitro. In MK3/M15 metanephric mesenchymal cells, Tcf21 enhanced TCF/LEF promoter activity upon ß-catenin stabilization, whereas DNA-binding deficient mutated Tcf21 did not enhance TCF/LEF promoter activity. Kidney explants of Foxd1Cre;Tcf21f/f showed low mRNA expression of stromal Wnt target genes. Treatment of the explants with CHIR, a Wnt ligand mimetic, restored Wnt target gene expression. Here, we also corroborated previous evidence that normal development of the kidney stroma is required for normal development of the Six2+ nephron progenitor cells, loop of Henle, and the collecting ducts. Conclusions: These findings suggest that stromal Tcf21 facilitates medullary stroma development by enhancing Wnt/ß-catenin signaling and promotes stromal cell proliferation and differentiation. Stromal Tcf21 is also required for the development of the adjacent nephron epithelia.

Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3.

The function of hypoxia-inducible factor-1α (HIF-1α) in chronic kidney disease is disputed. Here we report that interactions of HIF-1α with transforming growth factor-ß (TGF-ß) signaling may promote its fibrotic effects. Knockout of HIF-1α is protective against glomerulosclerosis and glomerular type-I collagen accumulation in a mouse podocyte ablation model. Transcriptional analysis of cultured renal cells showed that α2(I) collagen expression is directly regulated by HIF-1α binding to a functional hypoxia-responsive element in its promoter at -335 relative to the transcription start site. Activation of COL1A2 transcription by HIF-1α occurred in the absence of hypoxia and is strongly enhanced by TGF-ß signaling. TGF-ß, in addition to increasing HIF-1α levels, increased both HIF-1α binding to the COL1A2 promoter and HIF-1α N-terminal transactivation domain activity. These effects of TGF-ß on HIF-1α were inhibited in Smad3-null mouse embryonic fibroblasts, suggesting a requirement for Smad3. Phosphorylated Smad3 also associated with the -335 hypoxia-responsive element of the COL1A2 promoter independent of a Smad DNA binding sequence. Smad3 binding to the -335 hypoxia-responsive element required HIF-1α both in vitro and in kidney lysate from the disease model, suggesting formation of an HIF-1α-Smad3 transcriptional complex. Thus, HIF-1α-Smad3 has a novel interaction in glomerulosclerosis.

Anti-TGF-ß Antibody, 1D11, Ameliorates Glomerular Fibrosis in Mouse Models after the Onset of Proteinuria.

Fibrosis is a final common pathway leading to loss of kidney function, in which the fibrogenic cytokine, transforming growth factor ß (TGF-ß), plays a central role. While previous studies showed that TGF-ß antagonism by various means prevents fibrosis in mouse models, clinical approaches based on these findings remain elusive. 1D11 is a neutralizing antibody to all three isoforms of TGF-ß. In both adriamycin (ADR)-induced nephropathy and NEP25 podocyte ablation nephropathy, thrice-weekly intraperitoneal administration of 1D11 from the day of disease induction until the mice were sacrificed (day 14 for ADR and day 28 for NEP25), significantly reduced glomerular COL1A2 mRNA accumulation and histological changes. Consistent with our previous findings, proteinuria remained overt in the mice treated with 1D11, suggesting distinct mechanisms for proteinuria and fibrogenesis. Podocyte numbers determined by WT1 staining were significantly reduced in NEP25-model glomeruli as expected, while WT1-positive cells were preserved in mice receiving 1D11. Even when 1D11 was administered after the onset of proteinuria on day 3, 1D11 preserved WT1-positive cell numbers in glomeruli and significantly reduced glomerular scar score (2.5 ± 0.2 [control IgG] vs. 1.8 ± 0.2 [1D11], P < 0.05) and glomerular COL1A2 mRNA expression (19.3 ± 4.4 [control IgG] vs. 8.4 ± 2.4 [1D11] fold increase over the healthy control, P < 0.05). Transmission electron microscopy revealed loss of podocytes and denuded glomerular basement membrane in NEP25 mice with disease, whereas podocytes remained attached to the basement membrane, though effaced and swollen, in those receiving 1D11 from day 3. Together, these data suggest that TGF-ß neutralization by 1D11 prevents glomerular fibrosis even when started after the onset of proteinuria. While overt proteinuria and podocyte effacement persist, 1D11 prevents total podocytes detachment, which might be a key event activating fibrogenic events in glomeruli.

Catalytic activity of thiacalix[4]arenetetrasulfonate metal complexes on modified anion-exchangers for ascorbic acid oxidation.

The catalysis of ascorbic acid (AsA) oxidation by anion-exchangers modified with metal complexes of thiacalix[4]arenetetrasulfonate (Me-TCAS[4]A-500, Me=Mn(3+), Fe(3+), Co(3+), Ce(4+), Cu(2+), Zn(2+), Ni(2+), and H2) were investigated. Me-TCAS[4]A-500 (Me=Mn(3+), Fe(3+), Ce(4+), and Cu(2+)) all exhibited the ability to catalyze the oxidative reaction of AsA to dehydroascorbic acid. However, in the presence of high concentrations of AsA, only Cu(2+)-TCAS[4]A-500 was capable of complete oxidation of the acid. Moreover, after six repeat uses, Cu(2+)-TCAS[4]A-500 maintained high and relatively constant catalytic activity. Prior treatment of glucose solutions with Cu(2+)-TCAS[4]A-500, even in the presence of high AsA concentrations, enabled the satisfactory determination of glucose without interference by AsA. Cu(2+)-TCAS[4]A-500 will therefore be applicable as an artificial substitute for ascorbate oxidase, and may be useful as a means to eliminate AsA interference during the analysis of vital compounds such as glucose and uric acid.

Hypoxia-inducible factor-2α and TGF-ß 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 ß-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)-ß induction of collagen in renal tubular cells. Here, we performed in vitro and in vivo studies of potential glomerular crosstalk between TGF-ß 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-ß1 treatment increased both HIF-1α and HIF-2α expression in normoxia. TGF-ß1 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-ß receptor (ALK5) kinase activity was required for increased, TGF-ß-stimulated HIF-α expression in response to TGF-ß, and inhibiting PI3-kinase markedly decreased HIF-α expression. Blocking HIF-1α/2α expression using siRNA decreased basal and TGF-ß1-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-ß and HIFs to promote renal fibrogenesis in normoxia and suggest that the HIF-2α isoform is more important during glomerulosclerosis.

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

Transforming growth factor (TGF)-ß 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-ß-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-ß-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-ß-induced fibrogenesis. mTORC1 activity was increased in human mesangial cells treated with TGF-ß in a TGF-ß receptor-dependent manner. Short hairpin (sh)RNA to Smad3 decreased, while overexpression of Smad3 increased, the mTORC1 activity, suggesting that TGF-ß stimulation of mTORC1 also requires Smad3. Pretreatment with rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-ß-induced COL1A2-luc activity and collagen I protein expression. mTORC1 inhibition also prevented the TGF-ß-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-ß-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.

Divergent roles of Smad3 and PI3-kinase in murine adriamycin nephropathy indicate distinct mechanisms of proteinuria and fibrogenesis.

Multiple transforming growth factor (TGF)-ß-induced fibrogenic signals have been described in vitro. To evaluate mechanisms in vivo, we used an adriamycin nephropathy model in 129x1/Svj mice that display massive proteinuria by days 5 to 7 and pathological findings similar to human focal segmental glomerulosclerosis by day 14. TGF-ß mRNA expression increased after day 7 along with nuclear translocation of the TGF-ß receptor-specific transcription factor Smad3. Inhibiting TGF-ß prevented both pathological changes and type-I collagen and fibronectin mRNA expression, but proteinuria persisted. Renal Akt was phosphorylated in adriamycin-treated mice, suggesting PI3-kinase activation. Expression of mRNA for the p110γ isozyme of PI3-kinase was specifically increased and p110γ colocalized with nephrin by immunohistochemistry early in disease. Nephrin levels subsequently decreased. Inhibition of p110γ by AS605240 preserved nephrin expression and prevented proteinuria. In cultured podocytes, adriamycin stimulated p110γ expression. AS605240, but not a TGF-ß receptor kinase inhibitor, prevented adriamycin-induced cytoskeletal disorganization and apoptosis, supporting a role for p110γ in podocyte injury. AS605240, at a dose that decreased proteinuria, prevented renal collagen mRNA expression in vivo but did not affect TGF-ß-stimulated collagen induction in vitro. Thus, PI3-kinase p110γ mediates initial podocyte injury and proteinuria, both of which precede TGF-ß-mediated glomerular scarring.

Interdependence of HIF-1α and TGF-ß/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)-ß is known to mediate renal fibrosis, we proposed a profibrotic role for cross talk between the TGF-ß1 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-ß1 treatment further increased HIF-1α expression in cells treated with hypoxia and also increased HIF-1α in normoxic conditions. TGF-ß1 did not increase HIF-1α mRNA levels nor decrease the rate of protein degradation, suggesting that it enhances normoxic HIF-1α translation. TGF-ß receptor (ALK5) kinase activity was required for increased HIF-1α expression in response to TGF-ß1, but not to hypoxia. A dominant negative Smad3 decreased the TGF-ß-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-ß. Finally, blocking HIF-1α transcription with a biochemical inhibitor, a dominant negative construct, or gene-specific knockdown decreased basal and TGF-ß1-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-ß1-stimulated renal cell fibrogenesis.

Integrins modulate cellular fibrogenesis at multiple levels; Regulation of TGF-ß signaling.

Fibrosis could occur in virtually any organ or tissue. The fibrotic lesion indolently disrupts the structure of the healthy organ, thereby hampering its proper function, consequence of which is devastating. Among the myriad factors that modulate fibrogenesis, transforming growth factor ß (TGF-ß) is one of the most studied and its central role for fibrotic disorders has been strongly suggested. Due to the pleiotropic nature of this cytokine, TGF-ß modulates multiple cellular responses throughout fibrogenesis. The complexity is supported by the TGF-ß receptor-specific phosphorylation of both the canonical, Smad-, and non-canonical, "non-Smad," pathways. The latter modulates Smad activity either independent of Smad or by phosphorylating the Smad linker region, distinct from those receptor-regulated. Despite the commodity of this mediator, the mechanism by which TGF-ß signaling causes specific pathogenesis and disease varies depending on the nature of the organ and the cells that compose that organ. Cells express a specific series of integrins that act as cellular sensors for the extracellular environment, determining subsequent cellular signals in a cell-type specific manner. Integrins may change their expression pattern under pathological conditions and contribute to the regulation of fibrogenesis via modulating ambient TGF-ß activity. This regulation includes release of active TGF-ß from its latent form and modulation of various signals downstream of integrin-engagement, which participate in the non-canonical regulation of TGF-ß signaling. TGF-ß also induces expression of integrins, as well as their ligand extracellular matrix, generating an amplification loop. Furthermore, myriads of intracellular signaling molecules that associate with integrin engagement could non-canonically modulate TGF-ß signals. The entire picture of this mutual regulation between integrin and the TGF-ß pathways might be difficult to draw. Instead, this review intends to depict several critical aspects of this regulation, with examples from various types of fibrosis in different tissues to help understanding the integrin-modulation of fibrogenesis, a critical clue for therapeutic approaches to fibrosis.

Loss of beta1-integrin enhances TGF-beta1-induced collagen expression in epithelial cells via increased alphavbeta3-integrin and Rac1 activity.

Transforming growth factor ß (TGF-ß) promotes tissue fibrosis via the receptor-specific Smad pathway and non-canonical pathways. We recently reported that TGF-ß1-stimulated collagen expression by cultured kidney cells requires integrin-dependent activation of focal adhesion kinase (FAK) and consequent ERK MAP kinase activity leading to Smad3 linker region phosphorylation. Here, we defined a role for αvß3-integrin in this non-canonical pathway. A human kidney tubular cell line in which ß1-integrin was knocked down (ß1-k/d) demonstrated enhanced type I collagen mRNA expression and promoter activity. A second shRNA to either αv-integrin or ß3-integrin, but not to another αv-binding partner, ß6-integrin, abrogated the enhanced COL1A2 promoter activity in ß1-k/d cells. Although αvß3-integrin surface expression levels were not different, αvß3-integrins colocalized with sites of focal adhesion significantly more in ß1-k/d cells, and activated αvß3-integrin was detected only in ß1-k/d cells. Further, the collagen response was decreased by a function-blocking antibody or a peptide inhibitor of αvß3-integrin. In cells lacking αvß3-integrin, the responses were attenuated, whereas the response was enhanced in αvß3-overexpressing cells. Rac1 and ERK, previously defined mediators for this non-canonical pathway, showed increased activities in ß1-k/d cells. Finally, inhibition of αvß3-integrin decreased Rac1 activity and COL1A2 promoter activity in ß1-k/d cells. Together, our results indicate that decreasing ß1 chain causes αvß3-integrin to become functionally dominant and promotes renal cell fibrogenesis via Rac1-mediated ERK activity.

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.

Rac1 promotes TGF-beta-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-beta (TGF-beta) promotes cancer progression and fibrosis. Here we show that expression of the TGF-beta receptor-associated protein, SARA (Smad anchor for receptor activation), decreases within 72 h of exposure to TGF-beta 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-beta 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 alpha-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-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.

MAP-kinase activity necessary for TGFbeta1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397.

The signals mediating transforming growth factor beta (TGFbeta)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFbeta-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 TGFbeta1-dependent. By immunocytochemistry, TGFbeta1 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 TGFbeta-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFbeta-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 TGFbeta-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFbeta-induced responses to regulate collagen production.

RACK1 binds to Smad3 to modulate transforming growth factor-beta1-stimulated alpha2(I) collagen transcription in renal tubular epithelial cells.

Although it is clear that transforming growth factor-beta1 (TGF-beta1) is critical for renal fibrogenesis, the complexity of the involved mechanisms is increasingly apparent. TGF-beta1 stimulates phosphorylation of Smad2/3 and activates other signaling molecules as well. The molecular link between these other kinases and Smads is not known. We sought new binding partners for Smad3 in renal cells and identified receptor for activated protein kinase C 1 (RACK1) as a novel binding partner of Smad3. The linker region of Smad3 and the tryptophan-aspartic acid repeat 6 and 7 of RACK1 are sufficient for the association. RACK1 also interacts with Smad3 in the human kidney epithelial cell line, HKC. Silencing RACK1 increases transcriptional activity of TGF-beta1-responsive promoter sequences of the Smad binding element (SBE), p3TP-Lux, and alpha2(I) collagen. Conversely, overexpressed RACK1 negatively modulates alpha2(I) collagen transcriptional activity in TGF-beta1-stimulated cells. RACK1 did not affect phosphorylation of Smad3 at the C terminus or in the linker region. However, RACK1 reduced direct binding of Smad3 to the SBE motif. Mutating a RACK1 tyrosine at residue 246, but not at 228, decreased the inhibitory effect of RACK1 on both alpha2(I) collagen promoter activity and Smad binding to SBE induced by TGF-beta1. These results suggest that RACK1 modulates transcription of alpha2(I) collagen by TGF-beta1 through interference with Smad3 binding to the gene promoter.

High ambient glucose enhances sensitivity to TGF-beta1 via extracellular signal--regulated kinase and protein kinase Cdelta 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.

Intrahepatic porto-hepatic venous shunts in Rendu-Osler-Weber disease: imaging demonstration.

This study describes the imaging features of the intrahepatic portohepatic venous (PHV) shunt, which is a potential cause of portosystemic encephalopathy in Rendu-Osler-Weber disease. Six patients with Rendu-Osler-Weber disease (two men, four women; age range 42-73 years) were retrospectively studied. There were two from one family and three from another family. Of these patients, one was diagnosed with definitive portosystemic encephalopathy because of a psychiatric disorder. We retrospectively reviewed the radiological examinations, including abdominal angiography (n=6), three-phase dynamic helical computed tomography (CT; n=3), and conventional enhanced CT (n=1). In one patient, CT during angiography and CT angioportography were also performed. Evaluation was placed on the imaging features of intrahepatic PHV shunts. On angiography, intrahepatic PHV shunts showing multiple and small shunts <5 mm in diameter in an apparent network were detected in all patents. In two patients, a large shunt with a size of either 7 or 10 mm was associated. These intrahepatic PHV shunts were predominantly distributed in the peripheral parenchyma. Intrahepatic PHV shunts would be characterized by small and multiple shunts in an apparent network on the periphery with or without a large shunt.

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

Transforming growth factor beta (TGF-beta) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF-beta1 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-beta1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C-terminal SS(P)XS(P) motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 microM) or U0126 (25 microM). 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-beta stimulated phosphorylation of an expressed Smad3A construct, with a mutated C-terminal SS(P)XS(P) 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 alpha2(I) collagen promoter activity, an effect blocked by co-transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF-beta1 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.