Nitric Oxide and cGMP-dependent Protein Kinase Regulation of Glucose-mediated Thrombospondin 1-dependent Transforming Growth Factor-β Activation in Mesangial Cells

Abstract

Excessive transforming growth factor-␤ (TGF-␤) activity in hyperglycemia contributes to the development of diabetic nephropathy. Glucose stimulation of TGF-␤ activity and matrix synthesis are dependent on autocrine thrombospondin 1 (TSP1) to convert latent TGF-␤ to its biologically active form. The mechanisms by which glucose regulates TSP1 are not known. High glucose inhibits nitric oxide (NO) bioavailability and decreased NO increases TGF-␤ activity and extracellular matrix accumulation. Yet, the impact of NO signaling on TSP1 activation of TGF-␤ is unknown. We tested the role of NO signaling in the regulation of TSP1 expression and TSP1-dependent TGF-␤ activity in rat mesangial cells exposed to high glucose. On exposure to 30 mM glucose, NO accumulation in the conditioned media and intracellular cGMP levels were significantly decreased. The addition of an NO donor prevented the glucose-dependent increase in TSP1 mRNA, protein, and TGF-␤ bioactivity. The effects of the NO donor were blocked by ODQ (a soluble guanylate cyclase inhibitor) or Rp-8-pCPTcGMPS (an inhibitor of cGMP-dependent protein kinase). These effects of high glucose were also reversed by the nitric-oxide synthase cofactor tetrahyrobiopterin (BH 4 ). These results show that high glucose mediates increases in TSP1 expression and TSP1-dependent TGF-␤ bioactivity through down-modulation of NO-cGMP-dependent protein kinase signaling.Hyperglycemia is a crucial factor in the development of diabetic renal complications, with multiple lines of evidence suggesting that high glucose stimulates an increase in TGF-␤ 1 activity (1-3). This is important because TGF-␤ is a key mediator of the fibrotic changes in the pathogenesis of diabetic nephropathy (4 -6).TGF-␤ is synthesized and secreted as latent complex (latent TGF-␤). It must be converted to the active state before binding to its receptors and eliciting cellular functions. Latent TGF-␤ can be activated by a number of factors, including heat, extreme pH, plasmin, altered glycosylation, integrin binding, and reactive oxygen species (7-9). However, there is increasing evidence that the extracellular matrix protein, thrombospondin 1 (TSP1), is a major physiologic regulator of TGF-␤ activation (10 -12). TSP1, a disulfide-linked 180-kDa trimer, is a multifunctional protein that is produced by a variety of cells, including mesangial cells. In vitro and in vivo studies show that high glucose up-regulates TSP1 expression (13)(14)(15)(16)31) and that TSP1 protein is increased in the kidneys of diabetic patients (17). Recently, we and others showed that TSP1 is responsible for the activation of TGF-␤ in mesangial cells when exposed to high glucose for either short (2 days) or long term (3 weeks) treatment (18,19). However, the mechanisms involved in regulating TSP1 expression by high glucose concentrations are unclear.Nitric oxide has been implicated in the pathophysiology of diabetic nephropathy (20 -22). In glomerular mesangial cells, decreased nitric oxide (NO) bioavailability is observed at high glucose conce...

Nitric oxide modulates expression of extracellular matrix genes linked to fibrosis in kidney mesangial cells

“…This observation is consistent with the results of Wang et al (2002). Our results further demonstrate that different NO donors act in a time-and concentration-dependent manner to prevent increases in steady-state TSP-1 mRNA.…”

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…Inhibition could be reversed by treating cells with the sGC inhibitor 1H- [1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ, IC 50 = 20 nM). This is consistent with the finding that inhibition of glucose-induced TSP1 expression in mesangial cells by an NO donor was reversed by ODQ and by the inhibitor of cGMP-dependent protein kinase Rp-8-pCPTcGMPS (291). Constitutive expression of cGMP-dependent protein kinase in mesangial cells also prevented induction of TSP1 expression in mesangial cells (293).…”

Blockade of TSP1-Dependent TGF-β Activity Reduces Renal Injury and Proteinuria in a Murine Model of Diabetic Nephropathy

“…7,30,35,36,38,39,53,54 In the present studies, we addressed whether selective targeting of only TGF-␤ activated by TSP1 would be an effective approach to reduce renal dysfunction in a mouse model of type 1 diabetes. These studies showed that i.p.…”

“…The increased oxidative stress under high glucose conditions stimulates increased TSP1 protein expression by mesangial cells due to decreased nitric oxide-protein kinase G-mediated transcriptional repression and increased expression of the transcription factor USF2. 30,31 Glucose also increases TSP1 expression by podocytes, 32 and angiotensin II also increases TSP1 expression by renal mesangial cells and cardiac and renal interstitial cells in vitro, which results in increased TGF-␤ activity. 7,31,33 There is also evidence that TSP1-mediated latent TGF-␤ activation is involved in the development of diabetic nephropathy: TSP1 protein is increased in the glomeruli of patients with types 1 and 2 diabetic nephropathy, which correlates with increased TGF-␤ activity.…”