Association of TGF-beta signaling in angiotensin II-induced PAI-1 mRNA upregulation in mesangial cells: role of PKC

This study aimed to identify the intracellular signaling pathway in angiotensin II (Ang II)-induced upregulation of plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in cultured rat glomerular mesangial cells, and to examine the interaction between Ang II and TGF-beta signaling. Ang II-induced upregulation of PAI-1 mRNA expression was prevented by a protein kinase C (PKC) inhibitor, bisindorylmaleimide I. While phorbol 12-myristate 13-acetate (PMA) upregulated the PAI-1 mRNA expression, a calcium ionophore, ionomycin, had little effect. Mesangial cells pretreated with PMA for 24 h to downregulate PKC demonstrated attenuated response to Ang II. A protein tyrosine kinase inhibitor, genistein, completely blocked both Ang II- and PMA-induced PAI-1 mRNA expression. Transforming growth factor-beta1 (TGF-beta1) alone induced the expression, and in the presence of Ang II, TGF-beta1 superinduced PAI-1 mRNA expression to a higher extent. Both bisindorylmaleimide I and genistein suppressed the Ang II plus TGF-beta1-induced PAI-1 mRNA upregulation to the basal level, while downregulation of PKC attenuated the synergistic upregulation of PAI-1 mRNA expression to the level comparable to TGF-beta1 alone. These data suggest that, in rat mesangial cells, (1) PKC and protein tyrosine kinase(s) are involved in the Ang II signaling cascade, (2) protein tyrosine kinase(s) works downstream from PKC in the cascade, and (3) there is an interaction between the Ang II and TGF-beta signal pathways downstream from PKC. In in vivo settings, local activation of renin-angiotensin and TGF-beta systems in the glomeruli may synergistically augment PAI-1 expression, promote mesangial matrix accumulation and progression of glomerular injury.     

Aldosterone promotes fibronectin production through a Smad2-dependent TGF-beta1 pathway in mesangial cells

Accumulating evidence demonstrates that aldosterone can cause extra-cellular matrix (ECM) accumulation, in addition to regulating sodium and potassium homeostasis. Increased extra-cellular matrix production by renal glomerular mesangial cells has been suggested to be involved in pathogenesis of glomerular sclerosis. The present studies examine whether aldosterone is also produced in renal mesangial cells, and the effect of aldosterone on ECM accumulation in these cells. In cultured renal mesangial cells, aldosterone synthase (CYP11B2), mineralocorticoid receptor (MR), and 11beta-HSD2 mRNA expressions were detected by RT-PCR. The ability of renal mesangial cells to produce aldosterone was confirmed by directly detecting aldosterone in culture medium via radioimmunoassay. Real-time RT-PCR showed that the expression of CYP11B2 mRNA in mesangial cells was significantly enhanced by AngII (P<0.001) and by potassium (P<0.05). Exposure of the cultured mesangial cells to aldosterone significantly increased fibronectin production from 12.4+/-1.9 to 74.6+/-16.8ng/ml (P<0.05). The aldosterone induced fibronectin production was abolished by aldosterone receptor antagonist spironolactone. Aldosterone also increased the TGF-beta1 reporter luciferase activity from 0.8+/-0.1 to 1.7+/-0.1 (P<0.05). Immunoblot showed TGF-beta1 protein expression was increased following aldosterone treatment. Blocking TGF-beta1 signaling pathway by knocking down Smad2 significantly blunted the aldosterone induced fibronectin production. The present studies indicate that renal mesangial cell is a target of local aldosterone action, which promotes ECM protein fibronectin production via TGF-beta1/Smad2 signaling pathway.     

Rapid effects of aldosterone in primary cultures of cardiomyocytes – do they suggest the existence of a membrane-bound receptor?

Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone?+?spironolactone?+?BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca²⁺. Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure.     

FK506, transforming growth factor-beta1 and mesangial matrix synthesis: parallels and differences compared with cyclosporine A

Cyclosporine A (CsA)-induced glomerulosclerosis is a well-described side effect of CsA treatment. Current evidence indicates that FK506 causes similar morphologic changes. Recently, we demonstrated that CsA up-regulates the expression of transforming growth factor-beta1 (TGF-beta1), its receptors type I (TbetaR-I) and type II (TbetaR-II), as well as related matrix protein synthesis in mesangial cells (MCs). Here, we assessed the effect of FK506 on the expression of TGF-beta1, TbetaR-I, TbetaR-II, fibronectin (FN) and plasminogen activator inhibitor type-1 (PAI-1) in MCs. Resting MCs were incubated with/without FK506. Time- and concentration-dependent expression was measured at the mRNA and protein level. Compared to untreated controls, FK506 stimulated TGF-beta1 mRNA (maximum at 8 h, 100 ng/mL: 2.13+/-0.15-fold, P<0.005) and protein expression (maximum at 96 h, 100 ng/mL: 1.96+/-0.29-fold, P<0.005). In contrast, TbetaR-I and TbetaR-II protein expression remained unchanged. Concerning matrix protein synthesis, FK506 slightly increased FN production (96 h, 100 ng/mL: 1.38+/-0.28-fold, P<0.05), but not PAI-1 production. These results indicate that, comparable to CsA, FK506 induced glomerulosclerosis is also due to a direct effect on mesangial matrix production, which is at least in part mediated via up-regulation of TGF-beta1 expression. The fact that, unlike CsA, FK506 does not increase the expression of TbetaR-I, TbetaR-II, and PAI-1, deserves further investigation.     

Aldosterone induces myofibroblastic transdifferentiation and collagen gene expression through the Rho-kinase dependent signaling pathway in rat mesangial cells

There is accumulating evidence indicating the role of aldosterone in the pathogenesis of hypertension and renal injury. In this study, we investigated the role of the Rho-kinase dependent signaling pathway in aldosterone-induced myofibroblastic transdifferentiation and collagen gene expression in rat mesangial cells (RMCs). Stimulation with aldosterone (1 nmol/L) significantly increased phosphorylation of myosin phosphatase target subunit-1 (MYPT-1), a marker of Rho-kinase activity, with a peak at 20 min in RMCs. Pre-incubation with a selective mineralocorticoid receptor antagonist, eplerenone (10 µmol/L), or a specific Rho-kinase inhibitor, Y27632 (10 µmol/L), attenuated the aldosterone-induced increase in MYPT-1 phosphorylation. Aldosterone also induced hypertrophy in RMCs, accompanied by an increase in actin polymerization and expression of α-smooth muscle actin (α-SMA), a myofibroblastic transdifferentiation marker. Collagen type I, III and IV mRNA levels were also increased with aldosterone stimulation. Pre-treatment with eplerenone or Y27632 prevented the aldosterone-induced cell hypertrophy, actin polymerization, the increase in α-SMA expression and the increases of collagen type I, III, IV mRNA levels in RMCs. These results suggest that aldosterone-induced mesangial cell hypertrophy is associated with cell transformation, leading to an increase in collagen gene expression via the Rho-kinase dependent signaling pathway.     

Contrasting effects of eplerenone and spironolactone on adrenal cell steroidogenesis

Spironolactone and eplerenone are widely used as mineralocorticoid antagonists. Spironolactone has several nonspecific actions including inhibition of androgen receptor and steroid hormone biosynthesis. While studies have shown that eplerenone does not exhibit nonspecific actions on androgen receptor, its effects on steroid hormone production have not been reported. Herein, the effects of eplerenone (0.1-30 microM) and spironolactone (0.1-30 microM) on steroid production were examined in human adrenocortical H295R cells. Spironolactone inhibited basal production of cortisol (91%) and aldosterone (53%). Treatment of H295R cells with angiotensin II (Ang II) for 24 h increased aldosterone production by 11-fold. Spironolactone inhibited Ang II stimulation of aldosterone production by 80%. Addition of pregnenolone increased aldosterone (9-fold) and cortisol (3-fold) production. Spironolactone inhibited pregnenolone metabolism to aldosterone (67%) and cortisol (74%). The inhibitory effects of spironolactone occurred at concentrations far higher than those needed to block mineralocorticoid receptor, suggesting an action directly on the enzymes involved in steroid production. In contrast, eplerenone did not inhibit basal, Ang II, forskolin, pregnenolone-stimulated cortisol, or aldosterone production. Together, these data demonstrate that opposed to spironolactone, pharmacologic concentrations of eplerenone do not inhibit adrenal cell aldosterone or cortisol production.     

Candesartan attenuates Angiotensin II-induced mesangial cell apoptosis via TLR4/MyD88 pathway

Angiotensin II (Ang II) can stimulate Toll-like receptor 4 (TLR4) expression in mesangial cells (MCs), but the role of TLR4 in the Ang II-induced apoptosis and the effect of candesartan on TLR4 expression remain unclear. Here, we report that Ang II-induced MC apoptosis in a time-dependent manner and up-regulated TLR4/MyD88 expression, and that the intracellular ROS was subsequently increased. We also show that candesartan attenuated the Ang II-induced MC apoptosis, and that this protective effect was dependent on decreased TLR4/MyD88 expression as well as reduced intracellular ROS formation. Furthermore, Ang II increased the apoptosis inducing factor protein level, while candesartan markedly reduced this increase. These results demonstrate that TLR4/MyD88 pathway was involved in the Ang II promoted MC apoptosis, which was related to TLR4/MyD88 mediated oxidative stress. These data also suggest that candesartan exerted anti-apoptotic effect as an antioxidant by modulating this pathway.     

Aldosterone-induced brain MAPK signaling and sympathetic excitation are angiotensin II type-1 receptor dependent

Angiotensin II (ANG II)-induced mitogen-activated protein kinase (MAPK) signaling upregulates angiotensin II type-1 receptors (AT(1)R) in hypothalamic paraventricular nucleus (PVN) and contributes to AT(1)R-mediated sympathetic excitation in heart failure. Aldosterone has similar effects to increase AT(1)R expression in the PVN and sympathetic drive. The present study was undertaken to determine whether aldosterone also activates the sympathetic nervous system via MAPK signaling and, if so, whether its effect is independent of ANG II and AT(1)R. In anesthetized rats, a 4-h intravenous infusion of aldosterone induced increases (P < 0.05) in phosphorylated (p-) p44/42 MAPK in PVN, PVN neuronal excitation, renal sympathetic nerve activity (RSNA), mean blood pressure (MBP), and heart rate (HR). Intracerebroventricular or bilateral PVN microinjection of the p44/42 MAPK inhibitor PD-98059 reduced the aldosterone-induced RSNA, HR, and MBP responses. Intracerebroventricular pretreatment (5 days earlier) with pooled small interfering RNAs targeting p44/42 MAPK reduced total and p-p44/42 MAPK, aldosterone-induced c-Fos expression in the PVN, and the aldosterone-induced increases in RSNA, HR, and MBP. Intracerebroventricular infusion of either the mineralocorticoid receptor antagonist RU-28318 or the AT(1)R antagonist losartan blocked aldosterone-induced phosphorylation of p44/42 MAPK and prevented the increases in RSNA, HR, and MBP. These data suggest that aldosterone-induced sympathetic excitation depends upon that AT(1)R-induced MAPK signaling in the brain. The short time course of this interaction suggests a nongenomic mechanism, perhaps via an aldosterone-induced transactivation of the AT(1)R as described in peripheral tissues.     

A vitamin D analog regulates mesangial cell smooth muscle phenotypes in a transforming growth factor-beta type II receptor-mediated manner.

Mesangial cells share features with contractile smooth muscle cells and mechanically support the capillary wall. The role of vitamin D compounds and the transforming growth factor-beta (TGF-beta) type II receptor in modulating the smooth muscle phenotype of cultured mesangial cells was examined. Cell proliferation was significantly inhibited by the vitamin D analog 22-oxa-1,25-dihydroxyvitamin D(3) (22-oxacalcitriol; OCT) rather than by 1,25-dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)) in a dose-dependent manner. OCT-treated early passage mesangial cells (MC-E cells) had increased expression levels of type IV collagen and smooth muscle alpha actin mRNA, but 1, 25(OH)(2)D(3)-treated MC-E cells did not. The addition of a TGF-beta(1)-neutralizing antibody to the OCT-treated MC-E cells blocked this inhibitory effect for cell proliferation and attenuated the up-regulated mRNA levels. However, after exposure to 1, 25(OH)(2)D(3) or OCT, there was no significant difference in the secretion of active TGF-beta. We next investigated whether TGF-beta type II receptor (RII) was involved in this regulation. OCT treatment significantly increased the expression of the RII mRNA in MC-E cells. These results suggest that the vitamin D analog OCT induces smooth muscle phenotypic alterations and that this phenomenon was mediated through the induction of RII in cultured mesangial cells.     

Angiotensin II stimulates intercellular adhesion molecule-1 via an AT1 receptor/nuclear factor-kappaB pathway in brain microvascular endothelial cells

Microvascular changes in the brain are significant causes of cerebral edema and ischemia injury. A number of studies suggest that angiotensin (Ang) II may be involved in the initiation and regulation of processes occurring in brain ischemia. We recently reported that Ang II injures brain microvascular endothelial cells (BMEC) partially via stimulating intercellular adhesion molecule-1 (ICAM-1) expression. However, the signaling cascade leading to Ang II-induced ICAM-1 expression in BMEC was unclear. The present study tested the hypothesis that Ang II induces ICAM-1 expression via an AT1 receptor/nuclear factor-kappaB (NF-kappaB) pathway in BMEC. Ang II directly stimulated the expression of ICAM-1 mRNA and protein in primary cultured BMEC. Ang II treatment also resulted in the degradation of IkappaBalpha and increase of NF-kappaB p65 subunit in the nucleus as well as the DNA binding activity of nuclear NF-kappaB. These effects were abolished by pretreatment with the selective AT1 receptor antagonists, losartan and compound EXP-2528, or losartan plus the AT2 receptor antagonist PD123319, but not by PD123319 alone. Moreover, there were no significant differences between the losartan and losartan plus PD123319 groups. These findings indicate that Ang II-induced ICAM-1 upregulation in brain microvascular endothelial cells may be mediated via an AT1 receptor/NF-kappaB pathway.     

Angiotensin II induces the production of MMP-3 and MMP-13 through the MAPK signaling pathways via the AT1 receptor in osteoblasts

Angiotensin II (Ang II) plays an important role in the maintenance of bone mass and integrity by activation of the mitogen-activated protein kinases (MAPKs) and by modulation of balance between resorption by osteoclasts and formation by osteoblasts. However, the role of Ang II in the turnover of extracellular matrix (ECM) in osteoid by osteoblasts remains unclear. Therefore, we examined the effect of Ang II on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors [i.e., tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1)] using osteoblastic ROS17/2.8 cells. Treatment with Ang II strikingly increased the expressions of MMP-3 and -13 and promoted cell proliferation associated with reduced alkaline phosphatase activity as well as enhanced phosphorylated expression of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) in ROS17/2.8 cells. However, Ang II had no effect on the expression of MMP-2, -9, -14, urokinase-type PA, tissue-type PA, TIMP-1, -2, -3, and PAI-1 in cells. Losartan (AT₁ receptor blocker) blocked Ang II-induced expression of MMP-3 and -13, whereas PD123319 (AT₂ receptor blocker) did not completely block these responses. Losartan also blocked the Ang II-induced phosphorylation of ERK1/2, p38 MAPK, and SAPK/JNK. MAPK kinase 1/2 inhibitor PD98059 and JNK inhibitor SP600125 suppressed Ang II-induced expression of MMP-3 and -13. These results suggested that Ang II stimulated the degradation process that occurs during ECM turnover in osteoid by increasing the production of MMP-3 and -13 through MAPK signaling pathways via the AT₁ receptor in osteoblasts. Furthermore, our findings suggest that Ang II does not influence the plasminogen/plasmin pathway in osteoblasts.     

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-beta1 inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-beta1-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.