Howard Goldberg

Extracellular signal-regulated kinase and the small GTP-binding protein, Rac, contribute to the effects of transforming growth factor-β1 on gene expression

The kinases and regulatory proteins that convey signals initiated by transforming growth factor-β (TGF-β) to the nucleus are poorly characterized. To study the role of the extracellular signal-regulated kinase (ERK) pathway in this process, we transiently transfected NIH 3T3 fibroblasts with TGF-β-responsive luciferase reporter genes and expression vectors designed to interrupt this kinase cascade. Mitogen-activated protein (MAP) kinase phosphatase-1 and a dominant negative MAP/ERK kinase 1 mutant reduced stimulation of plasminogen activator inhibitor-1 (PAI-1) promoter activity by TGF-β1 from 11.5- to 4-fold and 4.9-fold, respectively. Similar results were observed with the type I collagen promoters. TGF-β1 increased ERK1 activity 4.5-fold at 5 min and 3.1-fold at 3 h, while Jun kinase and p38 activity were not affected. Cotransfection of a dominant negative mutant of the small G protein, Rac, but not dominant negative Ras, Cdc42, or Rho mutants, reduced the effects of TGF-β1 on the PAI-1 promoter by approximately half. In support of a role for Rac in signaling by TGF-β, GTP binding to Rac was increased 3.7-fold following exposure of NIH 3T3 cells to TGF-β1 for 3 min. These findings indicate that TGF-β1 modulates gene expression partly through ERK and Rac in NIH 3T3 cells.

Reduced cyclosporin accumulation in multidrug-resistant cells.

Cyclosporin accumulation was reduced by 50% or more in multidrug- resistant CHRC5 CHO cells with high levels of P-glycoprotein expression compared to drug sensitive AuxB1 CHO cells. This difference could be overcome by verapamil which is known to interact with P-glycoprotein and reverse multidrug resistance. The difference in cyclosporin accumulation between sensitive and resistant cells decreased with increasing cyclosporin concentrations suggesting that cyclosporine itself regulated its own accumulation through interaction with P-glycoprotein. Indeed, cyclosporin also reversed differences in vinblastine accumulation between resistant and sensitive cell lines. Since P-glycoprotein is highly expressed in the kidney which is also a target for cyclosporin toxicity, the effects of verapamil on cyclosporin accumulation were studied in two renal cell lines, rat mesangial cells and LLCPK1, cells. Verapamil increased cyclosporin accumulation by approximately 70%. These results suggest that cellular cyclosporine accumulation is regulated at least in part by its interaction with P-glycoprotein.

High Glucose-suppressed Endothelin-1 Ca2+ Signaling via NADPH Oxidase and Diacylglycerol-sensitive Protein Kinase C Isozymes in Mesangial Cells

High glucose (HG) is the underlying factor contributing to long term complications of diabetes mellitus. The molecular mechanisms transforming the glomerular mesangial cell phenotype to cause nephropathy including diacylglycerol-sensitive protein kinase C (PKC) are still being defined. Reactive oxygen species (ROS) have been postulated as a unifying mechanism for HG-induced complications. We hypothesized that in HG an interaction between ROS generation, from NADPH oxidase, and PKC suppresses mesangial Ca2+ signaling in response to endothelin-1 (ET-1). In primary rat mesangial cells, growth-arrested (48 h) in 5.6 mm (NG) or 30 mm (HG) glucose, the total cell peak [Ca2+]i response to ET-1 (50 nm) was 630 ± 102 nm in NG and was reduced to 159 ± 15 nm in HG, measured by confocal imaging. Inhibition of PKC with phorbol ester down-regulation in HG normalized the ET-1-stimulated [Ca2+]i response to 541 ± 74 nm. Conversely, an inhibitory peptide specific for PKC-ζ did not alter Ca2+ signaling in HG. Furthermore, overexpression of conventional PKC-β or novel PKC-δ in NG diminished the [Ca2+]i response to ET-1, reflecting the condition observed in HG. Likewise, catalase or p47phox antisense oligonucleotide normalized the [Ca2+]i response to ET-1 in HG to 521 ± 58 nm and 514 ± 48 nm, respectively. Pretreatment with carbonyl cyanide m-chlorophenylhydrazone or rotenone did not restore Ca2+ signaling in HG. Detection of increased intracellular ROS in HG by dichlorofluorescein was inhibited by catalase, diphenyleneiodonium, or p47phox antisense oligonucleotide. HG increased p47phox mRNA by 1.7 ± 0.1-fold as measured by reverse transcriptase-PCR. In NG, H2O2 increased membrane-enriched PKC-β and -δ, suggesting activation of these isozymes. HG-enhanced immunoreactivity of PKC-δ visualized by confocal imaging was attenuated by diphenyleneiodium chloride. Thus, mesangial cell [Ca2+]i signaling in response to ET-1 in HG is attenuated through an interaction mechanism between NADPH oxidase ROS production and diacylglycerol-sensitive PKC.

Thioredoxin-interacting Protein Mediates High Glucose-induced Reactive Oxygen Species Generation by Mitochondria and the NADPH Oxidase, Nox4, in Mesangial Cells

Background: Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose (HG), inhibits the antioxidant, thioredoxin, and thereby is implicated in oxidative stress. Results: TxNIP deficiency protects mesangial cells from HG-induced oxidative stress and increased collagen by blocking mitochondrial glucose metabolism, NADPH oxidase, and Nox4. Conclusion: TxNIP controls ROS generation by regulating the TCA cycle versus glycolytic glucose flux. Significance: Inhibition of TxNIP is a promising approach to treat glucose toxicity. Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced β-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN.

High glucose activates PKC-ζ and NADPH oxidase through autocrine TGF-β1 signaling in mesangial cells

Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-beta(1) is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC) -zeta activation. We investigated the role of TGF-beta(1) in this action of high glucose on primary rat mesangial cells within 1-48 h. Both high glucose and exogenous TGF-beta(1) stimulated PKC-zeta kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-beta(1) antibodies. Neutralizing TGF-beta(1) antibodies, or a TGF-beta receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-zeta activation by high glucose. TGF-beta(1) also stimulated cellular membrane translocation of PKC-alpha, -beta(1), -delta, and -epsilon, similar to high glucose. High glucose and TGF-beta(1) enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-beta(1) antibodies, LY364947, or a specific PKC-zeta pseudosubstrate peptide inhibitor. Expression of constitutively active PKC-zeta in normal glucose caused upregulation of p22(phox), a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-beta(1) stimulation of PKC isozymes including PI3 kinase activation of PKC-zeta and consequent generation of ROS by NADPH oxidase.

Inhibition of Src Kinase Blocks High Glucose–Induced EGFR Transactivation and Collagen Synthesis in Mesangial Cells and Prevents Diabetic Nephropathy in Mice

Chronic exposure to high glucose leads to diabetic nephropathy characterized by increased mesangial matrix protein (e.g., collagen) accumulation. Altered cell signaling and gene expression accompanied by oxidative stress have been documented. The contribution of the tyrosine kinase, c-Src (Src), which is sensitive to oxidative stress, was examined. Cultured rat mesangial cells were exposed to high glucose (25 mmol/L) in the presence and absence of Src inhibitors (PP2, SU6656), Src small interfering RNA (siRNA), and the tumor necrosis factor-α–converting enzyme (TACE) inhibitor, TAPI-2. Src was investigated in vivo by administration of PP2 to streptozotocin (STZ)-induced diabetic DBA2/J mice. High glucose stimulated Src, TACE, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), extracellular signal–regulated kinase (ERK1/2, p38), and collagen IV accumulation in mesangial cells. PP2 and SU6656 blocked high glucose–stimulated phosphorylation of Src Tyr-416, EGFR, and MAPKs. These inhibitors and Src knockdown by siRNA, as well as TAPI-2, also abrogated high glucose–induced phosphorylation of these targets and collagen IV accumulation. In STZ-diabetic mice, albuminuria, increased Src pTyr-416, TACE activation, ERK and EGFR phosphorylation, glomerular collagen accumulation, and podocyte loss were inhibited by PP2. These data indicate a role for Src in a high glucose-Src-TACE-heparin-binding epidermal growth factor-EGFR-MAPK–signaling pathway to collagen accumulation. Thus, Src may provide a novel therapeutic target for diabetic nephropathy.

O-linked β-N-acetylglucosamine supports p38 MAPK activation by high glucose in glomerular mesangial cells

Hyperglycemia augments flux through the hexosamine biosynthetic pathway and subsequent O-linkage of single β-N-acetyl-d-glucosamine moieties to serine and threonine residues on cytoplasmic and nuclear proteins (O-GlcNAcylation). Perturbations in this posttranslational modification have been proposed to promote glomerular matrix accumulation in diabetic nephropathy, but clear evidence and mechanism are lacking. We tested the hypothesis that O-GlcNAcylation enhances profibrotic signaling in rat mesangial cells. An adenovirus expressing shRNA directed against O-GlcNAc transferase (OGT) markedly reduced basal and high-glucose-stimulated O-GlcNAcylation. Interestingly, O-GlcNAc depletion prevented high-glucose-induced p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase phosphorylation. Downstream of p38, O-GlcNAc controlled the expression of plasminogen activator inhibitor-1, fibronectin, and transforming growth factor-β, important factors in matrix accumulation in diabetic nephropathy. Treating mesangial cells with thiamet-G, a highly selective inhibitor of O-GlcNAc-specific hexosaminidase (O-GlcNAcase), increased O-GlcNAcylation and p38 phosphorylation. The high-glucose-stimulated kinase activity of apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase for p38 that is negatively regulated by Akt, was inhibited by OGT shRNA. Akt Thr(308) and Ser(473) phosphorylation were enhanced following OGT shRNA expression in high-glucose-exposed mesangial cells, but high-glucose-induced p38 phosphorylation was not attenuated by OGT shRNA in cells pretreated with the phosphatidylinositol 3-kinase inhibitor LY-294002. OGT shRNA also reduced high-glucose-stimulated reactive oxygen species (ROS) formation. In contrast, diminished O-GlcNAcylation caused elevated ERK phosphorylation and PKCδ membrane translocation. Thus, O-GlcNAcylation is coupled to profibrotic p38 MAPK signaling by high glucose in part through Akt and possibly through ROS.

High β-cell mass prevents streptozotocin-induced diabetes in thioredoxin-interacting protein-deficient mice

Thioredoxin-interacting protein (TxNIP) is an endogenous inhibitor of thioredoxin, a ubiquitous thiol oxidoreductase, that regulates cellular redox status. Diabetic mice exhibit increased expression of TxNIP in pancreatic islets, and recent studies suggest that TxNIP is a proapoptotic factor in beta-cells that may contribute to the development of diabetes. Here, we examined the role of TxNIP deficiency in vivo in the development of insulin-deficient diabetes and whether it impacted on pancreatic beta-cell mass and/or insulin secretion. TxNIP-deficient (Hcb-19/TxNIP(-/-)) mice had lower baseline glycemia, higher circulating insulin concentrations, and higher total pancreatic insulin content and beta-cell mass than control mice (C3H). Hcb-19/TxNIP(-/-) did not develop hyperglycemia when injected with standard multiple low doses of streptozotocin (STZ), in contrast to C3H controls. Surprisingly, although beta-cell mass remained higher in Hcb-19/TxNIP(-/-) mice compared with C3H after STZ exposure, the relative decrease induced by STZ was as great or even greater in the TxNIP-deficient animals. Consistently, cultured pancreatic INS-1 cells transfected with small-interfering RNA against TxNIP were more sensitive to cell death induced by direct exposure to STZ or to the combination of inflammatory cytokines interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha. Furthermore, when corrected for insulin content, isolated pancreatic islets from TxNIP(-/-) mice exhibited reduced glucose-induced insulin secretion. These data indicate that TxNIP functions as a regulator of beta-cell mass and influences insulin secretion. In conclusion, the relative resistance of TxNIP-deficient mice to STZ-induced diabetes appears to be because of an increase in beta-cell mass. However, TxNIP deficiency is associated with sensitization to STZ- and cytokine-induced beta-cell death, indicating complex regulatory roles of TxNIP under different physiological and pathological conditions.

Rosiglitazone Prevents High Glucose-Induced Vascular Endothelial Growth Factor and Collagen IV Expression in Cultured Mesangial Cells

Peroxisome proliferator-activated receptor (PPARγ), a ligand-dependent transcription factor, negatively modulates high glucose effects. We postulated that rosiglitazone (RSG), an activator of PPARγ prevents the upregulation of vascular endothelial growth factor (VEGF) and collagen IV by mesangial cells exposed to high glucose. Primary cultured rat mesangial cells were growth-arrested in 5.6 mM (NG) or 25 mM D-glucose (HG) for up to 48 hours. In HG, PPARγ mRNA and protein were reduced within 3 h, and enhanced ROS generation, expression of p22phox, VEGF and collagen IV, and PKC-ζ membrane association were prevented by RSG. In NG, inhibition of PPARγ caused ROS generation and VEGF expression that were unchanged by RSG. Reduced AMP-activated protein kinase (AMPK) phosphorylation in HG was unchanged with RSG, and VEGF expression was unaffected by AMPK inhibition. Hence, PPARγ is a negative modulator of HG-induced signaling that acts through PKC-ζ but not AMPK and regulates VEGF and collagen IV expression by mesangial cells.

Overexpression of the type II transforming growth factor-β receptor inhibits fibroblast proliferation and activates extracellular signal regulated kinase and C-Jun N-terminal kinase

Transforming growth factor‐β (TGF‐β) is a bimodal regulator of cellular growth. The cellular effects of TGF‐β depend on the intensity of signals emanating from TGF‐β receptors. Low levels of receptor activity are sufficient to stimulate cell proliferation, while higher degrees of receptor activation are associated with growth inhibition. To study the mechanisms of these effects, a tetracycline‐inducible expression system was used to overexpress type II TGF‐β receptors in NIH 3T3 fibroblasts. Overexpressed type II TGF‐β receptors suppressed fibroblast proliferation elicited by TGF‐β1, fibroblast growth factor (FGF) or platelet‐derived growth factor (PDGF). Accompanying these anti‐proliferative effects, increases in extracellular‐signal regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) activity were detected. Furthermore, PDGF α‐, but not PDGF β‐receptor protein levels were reduced by type II TGF‐β receptor overexpression. In conclusion, our system is an excellent tool to study the molecular mechanisms of growth inhibition by TGF‐β in fibroblasts. Activation of JNK and ERK, or modulation of PDGF receptor expression may be involved in this process.