Michael S. Simonson

Endothelin stimulates phospholipase C, Na+/H+ exchange, c-fos expression, and mitogenesis in rat mesangial cells.

A recently described peptide hormone, endothelin, is a potent vasoconstrictor, but it is unclear whether endothelin has other biological actions. These experiments extend the range of biological actions of endothelin to stimulation of mitogenesis. Endothelin at low concentrations (0.1-10 nM) induced mitogenesis by quiescent rat glomerular mesangial cells in culture. Mitogenesis induced by endothelin was accompanied by activation of phospholipase C with increased inositol phosphate turnover and increments of intracellular [Ca2+]. Endothelin also activated Na+/H+ exchange, causing cytosolic alkalinization, and enhanced transcription of the c-fos protooncogene, additional biochemical signals closely linked to proliferation. In addition to being a vasoconstrictor, endothelin thus also functions as a mitogen, presumably through activation of phospholipase C.

Cellular signaling by peptides of the endothelin gene family.

Endothelins (ET) are a family of regulatory peptides synthesized by selected endothelial and epithelial cells that act in a paracrine fashion on nearby smooth muscle or connective tissue cells. We review the pathways of transmembrane signaling triggered by binding of endothelin peptides to receptors on the plasma membrane. Although our understanding of many components is unclear, endothelin peptides appear to evoke a phosphoinositide‐linked signaling system that bears a striking resemblance to signaling pathways activated by other regulatory peptides. Expression of endothelin receptors and specific pathways stimulated by activated receptors are controlled in a cell‐ and tissue‐specific manner, which perhaps explains the diverse biological actions of endothelin in different tissues. Complex negative feedback pathways regulate endothelin‐induced signaling at the receptor and second messenger levels. Moreover, by regulating the activity of sequence‐specific DNA binding proteins, short‐term signals by ET can be extended to long‐term effects involving gene expression. Regulation of gene expression by ET could account for complex events such as mitogenesis and vascular and tissue remodeling in disease.— Simonson, M. S.; Dunn, M. J. Cellular signaling by peptides of the endothelin gene family. FASEB J. 4: 2989‐3000; 1990.

Endothelin-1 stimulates contraction of rat glomerular mesangial cells and potentiates beta-adrenergic-mediated cyclic adenosine monophosphate accumulation.

The newly isolated peptide, endothelin-1 (ET-1), is a potent pressor agent that reduces GFR and the glomerular ultrafiltration coefficient. Recent evidence demonstrates that ET-1 mobilizes intracellular Ca2+ [( Ca2+]i) in glomerular mesangial cells by activating the phosphoinositide cascade. The present experiments were designed to examine whether ET-1 stimulates mesangial cell contraction and regulates the synthesis of PGE2 and cAMP, which dampen vasoconstrictor-induced mesangial contraction. ET-1 (greater than or equal to 1 nM) reduced the cross-sectional area of rat mesangial cells cultured on three-dimensional gels of collagen type I. ET-1 also caused complex rearrangements of F-actin microfilaments consistent with a motile response. Contraction in response to ET-1 occurred only at concentrations that activate phospholipase C, and contraction was unaffected by blockade of dihydropyridine-sensitive Ca2+ channels. Elevation of [Ca2+]i with ionomycin, to equivalent concentrations of [Ca2+]i achieved with ET-1, also reduced mesangial cell cross-sectional area. ET-1 (0.1 microM) also evoked [3H]arachidonate release and a fivefold increase in PGE2 synthesis as well as increased synthesis of PGF2 alpha and small changes of TXB2. ET-1 caused a minor increase in intracellular cAMP accumulation only in the presence of 3-isobutyl-1-methylxanthine. ET-1 also amplified cAMP production in response to isoproterenol. TPA and ionomycin, alone and in combination, failed to mimic the potentiating effect of ET-1; however, indomethacin blocked ET-1-induced potentiation of isoproterenol-stimulated cAMP, which was restored by addition of exogenous 10 nM PGE2. Thus the present data demonstrate that ET-1 stimulates mesangial cell contraction via pharmacomechanical coupling and activates phospholipase A2 to produce PGE2, PGF2 alpha, and TXB2. ET-1 also amplified beta adrenergic-stimulated cAMP accumulation by a PGE2-dependent mechanism.

Interleukin-1 generates transmembrane signals from phospholipids through novel pathways in cultured rat mesangial cells.

Although IL-1 stimulates cellular responses in both lymphoid and nonlymphoid cells, the second messengers by which IL-1 activates cells are unknown. Recombinant IL-1 alpha (rIL-1) is a comitogen for glomerular mesangial cells. Using this model we explored potential transmembrane signals by which IL-1 stimulates cellular responses. Certain mitogens hydrolyze inositol phospholipids by phospholipase C to generate 1,2-diacylglycerol, a cofactor for protein kinase C, and inositol (1,4,5)-trisphosphate, which mobilizes intracellular calcium. rIL-1 induced a peak increase in [3H]1,2-diacylglycerol formation at 1 min. Production of 1,2-diacylglycerol often parallels the generation of phosphatidic acid; however, rIL-1 stimulated [32P]phosphatidate formation only after 60 min. rIL-1 did not change the inositol phosphate or cytosolic free calcium concentrations, demonstrating that rIL-1 does not activate an inositol phospholipid-specific phospholipase C. [3H]Phosphorylethanolamine, but not [3H]phosphorylserine or [3H]phosphorylcholine, was maximally elevated at 1 min in mesangial cells incubated with rIL-1. Radioactivity incorporated into phosphatidylethanolamine but not phosphatidylcholine was also decreased in IL-1-stimulated mesangial cells compared with control at 1 min. These data suggest that rIL-1 activates a phospholipase C predominantly linked to phosphatidylethanolamine. In contrast to other mitogens, rIL-1 did not alter intracellular pH. Both 12-0-tetradecanoyl-phorbol-13-acetate, a homologue of 1,2-diacylglycerol, and phosphatidate but not phosphatidylcholine in the presence of 0.5% fetal bovine serum stimulated mesangial cell proliferation. rIL-1-induced cellular activation may be mediated, at least in part, by phospholipid-derived second messengers generated through novel pathways.

GATA-6 induces p21(Cip1) expression and G1 cell cycle arrest.

GATA transcription factors represent a family of highly conserved zinc finger proteins with tissue-specific expression patterns. Previous studies have shown that GATA-6 is expressed in vascular smooth muscle cells (VSMCs) and rapidly down-regulated when VSMCs are induced to proliferate. Here we investigated whether the GATA-6 transcription factor can modulate cellular proliferation. Transient transfection with a GATA-6 expression vector inhibited S-phase entry in VSMCs and in mouse embryonic fibroblasts (MEFs) lacking both p53 alleles. The GATA-6-induced growth arrest correlated with a marked increase in the expression of the general cyclin-dependent kinase (Cdk) inhibitor p21. In contrast to p53-deficient MEFs and VSMCs, MEFs null for both p21 alleles were refractory to the GATA-6-induced growth inhibition. These data demonstrate that elevated GATA-6 expression can promote the quiescent phenotype in VSMCs.

AMP-activated Protein Kinase Inhibits Transforming Growth Factor-β-induced Smad3-dependent Transcription and Myofibroblast Transdifferentiation

In wound healing, myofibroblast transdifferentiation (MFT) is a metaplastic change in phenotype producing profibrotic effector cells that secrete and remodel the extracellular matrix. Unlike pathways that induce MFT, the molecular mechanisms that negatively regulate MFT are poorly understood. Here, we report that AMP-activated protein kinase (AMPK) blocks MFT in response to transforming growth factor-β (TGFβ). Pharmacological activation of AMPK inhibited TGFβ-induced secretion of extracellular matrix proteins collagen types I and IV and fibronectin. AMPK activation also prevented induction of the myofibroblast phenotype markers α-smooth muscle actin and the ED-A fibronectin splice variant. AMPK activators did not prevent MFT in cells transduced with an adenovirus expressing dominant negative, kinase-dead AMPKα2. Moreover, AMPK activators did not inhibit MFT induction in AMPKα1,2–/– fibroblasts, demonstrating a requirement for AMPKα expression. Adenoviral transduction of constitutively active AMPKα2 was sufficient to prevent TGFβ-induced collagen I, α-smooth muscle actin, and ED-A fibronectin. AMPK did not reduce TGFβ-stimulated Smad3 COOH-terminal phosphorylation and nuclear translocation, which are necessary for MFT. However, AMPK activation inhibited TGFβ-induced transcription driven by Smad3-binding cis-elements. Consistent with a role for AMPK in transcriptional regulation, nuclear translocation of AMPKα2 correlated with the appearance of active AMPKα in the nucleus. Collectively, these results demonstrate that AMPK inhibits TGFβ-induced transcription downstream of Smad3 COOH-terminal phosphorylation and nuclear translocation. Furthermore, activation of AMPK is sufficient to negatively regulate MFT in vitro.

Ca2+ signaling by distinct endothelin peptides in glomerular mesangial cells

Ca2+ signaling by peptides of the endothelin (ET) gene family was studied in cultured glomerular mesangial cells. In addition to the increase in cytosolic free [Ca2+] ([Ca2+]i) previously described for ET-1, we also observed that ET-2, ET-3, and sarafotoxin S6b generate similar [Ca2+]i waveforms but with dissimilar potencies and kinetics. The prepro form of ET-1 was inactive, suggesting that mature ET peptides are constrained in an inactive conformation within the preproET species. ET isopeptides caused both release of Ca2+ from intracellular stores and Ca2+ influx via a voltage- and dihydropyridine-insensitive pathway. ET-mediated Ca2+ influx was independent of the increase in [Ca2+]i. Activation of protein kinase C inhibited ET-induced Ca2+ signaling, whereas addition of ET to protein kinase C-depleted cells resulted in enhanced [Ca2+]i waveforms. Mesangial cells also demonstrated a marked adaptive desensitization response to ET. These data demonstrate that Ca2+ signaling is a common response to different ET peptides in glomerular mesangial cells and that activation of protein kinase C down-regulates these Ca2+ signals.

Voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases propagate signals from endothelin-1 receptors to the c-fos promoter.

Endothelin-1 (ET-1) triggers poorly understood nuclear signaling cascades that control gene expression, cell growth, and differentiation. To better understand how ET-1 regulates gene expression, we asked whether voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases (CaMKs) propagate signals from ET-1 receptors to the c-fos promoter in mesangial cells. Ca2+ influx through voltage-insensitive Ca2+ channels, one of the earliest postreceptor events in ET-1 signaling, mediated induction of c-fos mRNA and activation of the c-fos promoter by ET-1. A CaMK inhibitor (KN-93) blocked activation of the c-fos promoter by ET-1. Ectopic expression of CaMKII potentiated stimulation by ET-1, providing further evidence that CaMKs contribute to c-fos promoter activation by ET-1. The c-fos serum response element was necessary but not sufficient for CaMKII to activate the c-fos promoter. Activation of the c-fos promoter by ET-1 and CaMKII also required the FAP cis element, an AP-1-like sequence adjacent to the serum response element. Thus, voltage-insensitive Ca2+ channels and CaMKs apparently propagate ET-1 signals to the c-fos promoter that require multiple, interdependent cis elements. Moreover, these experiments suggest an important role for voltage-insensitive Ca2+ channels in nuclear signal transduction in nonexcitable cells.

Urine neutrophil gelatinase–associated lipocalin levels do not improve risk prediction of progressive chronic kidney disease

Novel biomarkers may improve our ability to predict which patients with chronic kidney disease (CKD) are at higher risk for progressive loss of renal function. Here we assessed the performance of urine neutrophil gelatinase-associated lipocalin (NGAL) for outcome prediction in a diverse cohort of 3386 patients with CKD in the CRIC study. In this cohort, the baseline mean estimated glomerular filtration rate (eGFR) was 42.4 ml/min/1.73m2; the median 24-hour urine protein was 0.2 gm/day; and the median urine NGAL concentration was 17.2 ng/mL. Over an average follow-up of 3.2 years, there were 689 cases in which the eGFR was decreased by half or incident end-stage renal disease developed. Even after accounting for eGFR, proteinuria and other known CKD progression risk factors, urine NGAL remained a significant independent risk factor (Cox model hazard ratio 1.70 highest to lowest quartile). The association between baseline urine NGAL levels and risk of CKD progression was strongest in the first two years of biomarker measurement. Within this time frame, adding urine NGAL to a model which included eGFR, proteinuria and other CKD progression risk factors led to net reclassification improvement of 24.7%; but the C-statistic remained nearly identical. Thus, while urine NGAL was an independent risk factor of progression among patients with established CKD of diverse etiology, it did not substantially improve prediction of outcome events.

Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G

BackgroundIn type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved.MethodsSaturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated.ResultsThe saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial β-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid β-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-indpendent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis.ConclusionsTaken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA.