Fibronectin Synthesis In Mesangial Cells Research Articles

Berberine suppresses high glucose-induced TGF-β1 and fibronectin synthesis in mesangial cells through inhibition of sphingosine kinase 1/AP-1 pathway

Sphingosine kinase 1 (SphK1) pathway is critical in the pathogenesis of diabetic nephropathy. Recently, we found that berberine suppressed the activation of SphK1 pathway in diabetic kidney, protecting against diabetic nephropathy. The potential molecular mechanism, however, is still unknown. Here, we showed that berberine prevented the expression of α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) and fibronectin (FN) in mesangial cells cultured by high glucose. Furthermore, berberine suppressed the activation of SphK1 pathway via inhibition of the activity and expression of SphK1 in mesangial cells cultured by high glucose. Surprisingly, berberine blocked the increased activity and expression of SphK1 in mesangial cells transfected by wild type SphK1 under normal glucose condition. However, berberine had no inhibitory effect on the recombinant human SphK1 protein. Finally, berberine markedly attenuated the high glucose-induced activator protein-1 (AP-1) activity in mesangial cells. Altogether, these data not only demonstrate that berberine is an important agent against diabetic nephropathy through inhibition of SphK1/AP-1 pathway, but also indicate that the inhibition of SphK1/AP-1 by berberine is independent of ambient glucose concentration.

Inhibition of the Jak/STAT signaling pathway prevents the high glucose-induced increase in tgf-beta and fibronectin synthesis in mesangial cells.

High glucose (HG) causes glomerular mesangial cell (GMC) growth, production of transforming growth factor (TGF)-beta, and increased synthesis of matrix proteins such as fibronectin, contributing to diabetic nephropathy. We recently found that exposure of cells to HG also activates the growth-promoting enzyme janus kinase 2 (JAK2) and its latent signal transducers and activators of transcription (STAT) transcription factors (STAT1, STAT3, and STAT5). Our purpose was to determine the effect that inhibition of JAK2 and these STAT transcription factors has on the HG-induced increase in TGF-beta and fibronectin synthesis in GMC. Exposure of GMC to 25 mmol/l glucose caused the activation of JAK2, STAT1, STAT3, and STAT5 plus an increase in TGF-beta and fibronectin synthesis, as compared with 5.5 mmol/l glucose. This HG-induced increase in synthesis of TGF-beta and fibronectin was prevented by concomitant incubation with AG-490, a specific JAK2 inhibitor. The HG-induced JAK2, STAT1, and STAT3 tyrosine phosphorylations in GMC were also abolished by AG-490. Preincubation of GMC cultured in 25 mmol/l glucose with a specific JAK2 or STAT1 antisense oligonucleotide also prevented both TGF-beta and fibronectin synthesis. These results provide direct evidence for linkages between JAK2, STAT1, and the glucose-induced overproduction of TGF-beta and fibronectin in GMC.

ATHEROSCLEROSIS-INDUCED CHRONIC ISCHEMIA CAUSES BLADDER FIBROSIS AND NON-COMPLIANCE IN THE RABBIT

The overall goal was to determine whether chronic ischemia and hypercholesterolemia interfere with bladder function and structure. The roles of atherosclerosis-induced chronic ischemia and hypercholesterolemia in bladder fibrosis and non-compliance were studied in the rabbit. The relationship between ischemia-induced changes in the expression of transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) and the severity of bladder fibrosis was also investigated. Male New Zealand White rabbits were divided into chronic bladder ischemia (CBI, n = 11), hypercholesterolemia (Hch, n = 8) and control (n = 8) groups. The CBI group underwent balloon endothelial injury of the iliac arteries and received a 0.5% cholesterol diet. The Hch group received a 0.5% cholesterol diet alone. The control group was placed on a regular diet. After 16 weeks, iliac artery and bladder wall blood flow measurements, cystometrograms (CMG) and aorto-iliac arteriograms were obtained in all animals. Iliac arteries and bladder tissues were processed for histological staining and computer-assisted histomorphometric image analysis. The expressions of TGF-beta1 and bFGF in bladder tissue were determined by immunohistochemical staining utilizing monoclonal antibodies. At 16 weeks, arteriography and histology showed significant diffuse atherosclerotic occlusive disease of the aorto-iliac arteries in the CBI group. Iliac artery and bladder wall blood flows were significantly decreased in the CBI group compared with the Hch and control groups. Atherosclerosis-induced CBI shifted the volume-pressure curve to the left and caused severe bladder fibrosis. Hypercholesterolemia also caused fibrosis and non-compliance but to a much lesser extent compared with those caused by CBI. In histomorphometry, the percentage of detrusor smooth muscle was moderately decreased in the Hch group and severely decreased in the CBI group compared with the control group. In immunohistochemical stains of bladder tissues, bFGF expression was similar in the three groups of animals. TGF-beta1 expression was significantly greater in bladder tissues from the CBI group compared with the Hch and control groups. Our studies show that atherosclerosis-induced chronic ischemia increases TGF-beta1 expression in the bladder leading to fibrosis, smooth muscle atrophy and non-compliance. Hypercholesterolemia also interferes with bladder structure and compliance but to a significantly lesser extent compared with CBI. Our studies suggest that arterial insufficiency and hypercholesterolemia, common aging-associated disorders, may play important roles in the pathophysiology of voiding dysfunction in the elderly.

Nitric oxide suppresses increases in mesangial cell protein kinase C, transforming growth factor beta, and fibronectin synthesis induced by thromboxane.

Thromboxane (TX) stimulation of fibronectin (FN) synthesis in mesangial cells (MC) is dependent on protein kinase C (PKC)-mediated increases in transforming growth factor beta (TGF beta), and is suppressed by increases in cellular cGMP. The current studies evaluate the role of cGMP-dependent and -independent actions of nitric oxide (NO) in modulating the responses of MC to the TX analogue U46619. TX-stimulated increases in PKC activity, TGF beta, and FN synthesis in MC were suppressed by either 8-Br-PET-cGMP or the NO donor S-nitroso-N-acetylpenicillamine (SNAP). By contrast, NO, but not cGMP, inhibited basal PKC activity, TGF beta bioactivity and FN synthesis. The cGMP-dependent protein kinase 1-alpha inhibitor 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate (Rp) restored the PKC, TGF beta, and the FN synthetic responses to TX when added to MC before exposure of the cells to either cGMP or SNAP. However, neither Rp nor the guanylate cyclase inhibitor Ly83583 significantly altered SNAP inhibition of basal PKC. In addition, Rp failed to alter the decreases in basal TGF beta bioactivity and FN synthesis seen in the presence of SNAP. In contrast to the FN response to U46619, cGMP and SNAP did not affect the stimulation of FN synthesis by exogenous TGF beta. The later findings are consistent with inhibitory actions of NO and cGMP at, or proximal to, U46619-induced increases in TGF beta in the suppression of TX-signaled increases in FN synthesis. Thus, NO depresses basal PKC and TGF beta bioactivity in MC by mechanisms that are largely independent of cGMP, whereas NO inhibition of these MC responses to TX is mediated primarily by increases in cGMP and activation of protein kinase 1-alpha.

Role for Transforming Growth Factor β in Thromboxane-Induced Increases in Mesangial Cell Fibronectin Synthesis

Thromboxane A2 (TXA2) has been implicated in the pathogenesis of progressive glomerulosclerosis and stimulates the synthesis of matrix protein by mesangial cells (MCs). This study examined the role of transforming growth factor beta (TGF-beta) in the mediation of the action of the stable TXA2/prostaglandin (PG) endoperoxide analog U-46619 to stimulate fibronectin (Fn) synthesis in cultured rat MC. Exogenous TGF-beta increased Fn synthesis by MC in a concentration- and time-dependent fashion, as reflected by incorporation of [35S]methionine into immunoprecipitable Fn. Submaximal concentrations of TGF-beta (1-2.5 pmol/l) increased Fn synthesis two- to threefold, a response comparable in magnitude to that observed with a maximal stimulatory concentration (1 mumol/l) of U-46619. Anti-TGF-beta antibody, but not isotypic IgG, blocked the increases in Fn synthesis induced by both U-46619 and exogenous TGF-beta. Endogenous TGF-beta bioactivity in MC culture media, assessed by the mink lung epithelial cell system, was significantly increased by 1 mumol/l U-46619 (1.7 +/- 0.3 pmol/l) compared with that of control media (0.6 +/- 0.1 pmol/l, P < 0.05). Total (active plus latent) TGF-beta bioactivity, assayed after heat activation of latent TGF-beta, was also significantly higher in media of MCs cultured with U-46619 (45 +/- 4 pmol/l) compared with control (24 +/- 4 pmol/l). Thus, U-46619 increased endogenous TGF-beta bioactivity to a level sufficient to account for the enhancement of Fn synthesis observed with U-46619, as reflected by the Fn synthetic response to exogenous TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Role for transforming growth factor beta in thromboxane-induced increases in mesangial cell fibronectin synthesis

Role for transforming growth factor beta in thromboxane-induced increases in mesangial cell fibronectin synthesis

Thromboxane stimulation of mesangial cell fibronectin synthesis is signalled by protein kinase C and modulated by cGMP

Thromboxane (TX) has been implicated in the pathogenesis of glomerulosclerosis in several models of glomerular injury. In the present study, we examined the role of the protein kinase C (PKC) signalling system in expression of the action of the TXA2/PGH2 analogue U-46619 to stimulate fibronectin (Fn) synthesis in cultured rat mesangial cells (MC), and the influence of cGMP on this MC response. U-46619 activated PKC and enhanced Fn synthesis in MC in a time and concentration dependent fashion. Both responses to U-46619 were blocked by GF 109203X, a selective inhibitor of PKC activity, as well as by calphostin C and staurosporine, PKC inhibitors structurally distinct from GFX. Down-regulation of PKC by prior sustained exposure of MC to 0.5 microM phorbol myristate acetate similarly blocked increases in Fn synthesis induced by U-46619. The TXA2/PGH2 receptor antagonist Sq-29548 also prevented activation of PKC and stimulation of Fn synthesis by U-46619, consistent with transduction of these responses via specific high affinity TXA2/PGH2 receptors on MC. Addition of exogenous 8-Br-cGMP or stimulation of endogenous cGMP generation with atrial natriuretic peptide (ANP) suppressed both U-46619 activation of PKC and stimulation of Fn synthesis. cGMP did not alter TXA2/PGH2 receptor number of affinity in MC, but significantly suppressed phorbol ester activation of PKC. Thus, cGMP inhibition of U-46619 actions is expressed at steps distal to TX receptor binding and may involve effects at and proximal to activation of PKC. Interactions between the PKC and cGMP cellular signalling systems may be important determinants of MC matrix protein production in response to TX.

Effects of glycated protein on the expression of very late antigen 5 (VLA5), a fibronectin receptor, of cultured rat mesangial cells.

Advanced glycosylation end products are believed to play a role in the increase in extracellular matrix in diabetic glomerulosclerosis via a pathway involving a mesangial cell fibronectin receptor. In the present study, I assessed cultured rat mesangial cells for the presence of mRNA for VLA5, one of the fibronectin receptors. Using these cells, I also evaluated the effects of glycated proteins on the expression of VLA5 and fibronectin. Mesangial cells of Wistar rats were cultured in RPMI 1640 containing 20% FCS. The cells were incubated for 72 hr in a medium containing 50 mg/ml of nonglycated BSA or in a medium containing 50 mg/ml glycated BSA (AGE-BSA). Immunostaining and Northern blot analyses showed that the cells incubated with AGE-BSA exhibited a decrease in VLA5 protein and related mRNA, but showed an increase in the synthesis of fibronectin and related mRNA. On the other hand, non-glycated BSA did not induce these changes in the expression of VLA5 or fibronectin in the mesanginal cells. Northern blot analysis for VLA5 mRNA was also conducted using mesangial cells cultured in a medium with a high glucose concentration (30 mM). However, the high glucose condition did not have any effect on the expression of VLA5 protein or related mRNA. These results suggest that glycated proteins may regulate fibronectin synthesis in mesangial cells by modifying the expression of fibronectin receptors, such as the inhibition of VLA5 synthesis that acts as negative feedback of fibronectin synthesis.

The Effect of Transforming Growth Factor β 1 on Mesangial Cell Fibronectin Synthesis: Increased Incorporation into the Extracellular Matrix and Reduced p I but No Effect on Alternative Splicing

Fibronectin is a multidomain glycoprotein which accumulates in mesangial proliferative glomerulonephritis (MPGN). Recent evidence has implicated transforming growth factor β (TGF-β) in the pathogenesis of experimental MPGN. We have, therefore, examined the influence of TGF-β 1 on mesangial cell fibronectin synthesis. Considering, first, levels of mRNA, TGF-β 1 increased steady-state fibronectin RNA in cultured mesangial cells by 1.9 times 24 hr after treatment of cycling mesangial cells and by 11.8 times in growth-arrested cells. There was, however, no alteration in fibronectin pre-mRNA splicing in either the EIIIA or IIICS regions. Fibronectin protein concentrations in cell culture supernatants, determined by immunoprecipitation of supernatants from cells labeled with [ 35S]methionine and by ELISA, were not increased by treatment with TGF-β 1. Western blots and immunoprecipitation of metabolically labeled cells showed that fibronectin was increased, however, in the deoxycholate-insoluble extracellular matrix (ECM) of cells stimulated with TGF-β 1. TGF-β 1 altered the physicochemical properties of fibronectin in ECM and supernatant such that the isoelectric point of fibronectin, determined from Western blots of 2D SDS-PAGE gels, was reduced so that both became more acidic. These studies demonstrate, therefore, that in addition to increasing its synthesis, TGF-β 1 increases incorporation of fibronectin into the ECM. Because fibronectin possesses binding sites for other ECM proteins, greater incorporation of fibronectin following TGF-β 1 treatment may be an important pathogenetic mechanism in mesangial sclerosis. Moreover, the altered charge of fibronectin may increase localization of serum immunoglobulins to the mesangium.

Role for protein kinase C in the mediation of increased fibronectin accumulation by mesangial cells grown in high-glucose medium.

The fibronectin content of RMC cultures grown for 8-14 days in medium containing 30 mM (540 mg/dl) D-glucose was increased 30-60% relative to that of control cells cultured in 10 mM (180 mg/dl) glucose. Addition of equiosmolar concentrations (20 mM, 360 mg/dl) of L-glucose, 3-O-methylglucose, or mannitol to 10 mM glucose media did not alter fibronectin accumulation compared with values observed in 10 mM glucose alone. The basal phosphorylation of the 80,000-M(r) MARKS protein, which is a substrate for PKC, and the phosphorylation induced by acute (15-min) exposure of cells to 15% FCS or to 0.1 microM (50 ng/ml) PDBu were all increased in cells grown in 30 mM compared with 10 mM glucose. By contrast, phosphorylation of the 80,000-M(r) protein in response to a maximal concentration of PDBu (1 microM, 500 ng/ml) was not different in cells grown in 30 mM compared with 10 mM glucose. The acute increases in phosphorylation of the 80,000-M(r) protein were blocked by the PKC inhibitor calphostin C. Chronic (7-day) exposure of mesangial cells grown in 10 mM glucose to 0.1 microM of the PKC agonist PDBu also resulted in a sustained 40% increase in 80,000-M(r) phosphorylation and a 20-30% increase in fibronectin accumulation. As assessed by [35S]methionine incorporation, mesangial cell fibronectin synthesis was increased by exposure to PDBu, a finding consistent with earlier observations with 30 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of high glucose on cellular proliferation and fibronectin production by cultured human mesangial cells.

Diabetic glomerulosclerosis is characterized by the accumulation of the matrix protein fibronectin in the glomerular mesangium and could result from increased mesangial cell fibronectin synthesis induced by hyperglycemia. To test this hypothesis, we cultured human mesangial cells for up to 14 days in media containing normal (5 mM) or high glucose (20 to 115 mM) concentrations and assessed cellular proliferation and fibronectin synthesis. When compared to 5 mM glucose, high glucose levels significantly inhibited cellular proliferation in a dose dependent fashion, as assessed by direct cell counting and thymidine incorporation. After eight days in culture, tissue culture supernatant fibronectin levels, as assessed by ELISA, were significantly higher from cells cultured under high glucose conditions than cells exposed to normal glucose levels. After 14 days and when compared to 5 mM glucose, matrix fibronectin levels and fibronectin mRNA expression (by Northern analysis) were also increased by 20 mM glucose. To control for the osmotic effects of high glucose, mesangial cells were also cultured in the presence of 20 mM or 50 mM mannitol. Mannitol had no effect on cellular proliferation but significantly increased tissue culture supernatant fibronectin levels and fibronectin gene expression. These studies demonstrate that, in vitro, high glucose suppresses human mesangial cell proliferation and stimulates fibronectin synthesis. The increase in fibronectin synthesis may in part result from changes in osmolality induced by high glucose. These data suggest that increased mesangial cell fibronectin synthesis may play a role in the accumulation of glomerular fibronectin common to diabetic glomerulosclerosis.