CD2-associated protein/phosphoinositide 3-kinase signaling has a preventive role in angiotensin II-induced podocyte apoptosis

Angiotensin II (Ang II) works as a paracrine or autocrine cytokine agent to regulate renal functions and promotes podocytes dysfunction directly or indirectly, causing proteinuria. The glomerular slit diaphragm (SD) serves as a size-selective barrier and is linked to the actin-based cytoskeleton by adaptor proteins, including CD2-associated protein (CD2AP). Therefore, damages to CD2AP affect not only the function of the SD, but also directly disrupt the podocyte cytoskeleton, leading to proteinuria. In addition, CD2AP can facilitate the nephrin-induced phosphoinositide 3-kinase (PI3-K)/Akt signaling, which protects podocytes from apoptosis. Here we found that CD2AP staining was located diffusely but predominantly in the peripheral cytoplasm and CD2AP co-localized with nephrin in mouse podocytes; however, Ang II decreased CD2AP staining diffusely and induced a separation from concentrated nephrin. Ang II notably reduced CD2AP expression in time- and concentration-dependent manners, and this was significantly recovered by losartan. Ang II induced podocyte apoptosis in time- and concentration-dependent manners in TUNEL and FACS assays. LY294002, a PI3-K inhibitor, further reduced CD2AP expression and increased podocyte apoptosis, which was augmented by siRNA for CD2AP. Thus, Ang II induces the relocalization and reduction of CD2AP via AT1R, which would cause podocyte apoptosis by the suppression of CD2AP/PI3-K signaling.     

CD147 inhibits the nuclear factor of activated T-cells by impairing Vav1 and Rac1 downstream signaling

CD147 is a transmembrane protein that plays crucial roles in the development and function of the reproductive, visual, and nervous systems. CD147 also exerts positive and negative actions in T-cells by still obscure mechanisms. In this study, we have analyzed the expression, localization, and function of CD147 during T-cell receptor signaling responses. We show here that CD147 is an integral component of the T-cell immune synapse and that its overexpression leads to the inhibition of NF-AT (nuclear factor of activated T-cells) activity induced by Vav1, a Rac1 exchange factor. This inhibitory activity is mediated by the CD147 intracellular tail and is totally independent of its extracellular or transmembrane regions. The molecular dissection of the influence of CD147 on the Vav1 pathway indicates that its inhibitory action takes place downstream of Vav1 and Rac1 but upstream of the serine/threonine kinases JNK and Pak1. The interference of CD147 with these pathways is highly specific because the overexpression of CD147 does not affect the activity of other GDP/GTP exchange factors or the stimulation of the ERK cascade. Finally, we show that the CD147 knockdown in Jurkat cells promotes higher levels of NF-AT stimulation and Pak1 phosphorylation upon T-cell receptor cross-linking. Instead, the lack of CD147 does not affect other signaling cascades that participate in the same cellular response. Taken together, these results indicate that CD147, via the selective inhibition of specific downstream elements of the Vav1/Rac1 route, contributes to the negative regulation of T-cell responses.     

Fluvastatin inhibits angiotensin II-induced nuclear factor kappa B activation in renal tubular epithelial cells through the p38 MAPK pathway

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has been shown to reduce the progression of renal disease independent of cholesterol-lowering effect, but the mechanism of potential protective effect remains unclear. Here, we investigate the effect of fluvastatin on activation of nuclear factor-κB (NF-κB) induced by angiotensin II (AngII) in rat kidney tubule epithelial cells (NRK-52E). Electrophoretic mobility shift assays (EMSA) was used to detect NF-κB activation. Phosphorylation of cellular p38 mitogen-activated protein kinase (p38MAPK) was determined by western blot analysis. AngII stimulated the DNA-binding activity of NF-κB and phosphorylation of p38MAPK in cultured NRK-52E cells in a dose-dependent (10⁻⁹–10⁻⁶ mol/l) manner (P < 0.01). AngII (10⁻⁶ mol/l) induced a rapid (5 min) increase of the p38MAPK phosphorylation. NF-κB DNA-binding activity was increased at as early as 30 min, peaked at 2 h after AngII treatment. This stimulatory effect of AngII on NF-κB was blocked by SB203580 (a specific inhibitor of p38MAPK). Incubation of cells with fluvastatin significantly inhibited the AngII-induced NF-κB activation in a dose-dependent (10⁻⁷–10⁻⁵ mol/l) manner (P < 0.05). Exogenous mevalonate (10⁻⁴mol/l) prevented the effect of fluvastatin on NF-κB activation. These results suggest the fluvastatin reduced AngII-induced NF-κB activation via the p38MAPK pathway in NRK-52E cells. The effect is at least partly due to blocking the biosynthesis of mevalonate.     

CHP2 activates the calcineurin/nuclear factor of activated T cells signaling pathway and enhances the oncogenic potential of HEK293 cells

CHP2 (calcineurin B homologous protein 2) was initially identified as a tumor-associated antigen highly expressed in hepatocellular carcinoma. Its biological function remains largely unknown except for a potential role in transmembrane Na(+)/H(+) exchange. In the present study, we observed that ectopic expression of CHP2 promoted the proliferation of HEK293 cells, whereas knockdown of endogenous CHP2 expression in HepG2 inhibited cell proliferation. When inoculated into nude mice, CHP2 transfected HEK293 cells displayed markedly increased oncogenic potential. In analysis of the underlying molecular mechanisms, we found that like calcineurin B, CHP2 was able to bind to and stimulate the phosphatase activity of calcineurin A. In accord with this, CHP2-transfected cells showed increased nuclear presence of NFATc3 (nuclear factor of activated T cells) and enhanced NFAT activity. Finally, both accelerated cell proliferation and NFAT activation following CHP2 transfection could be suppressed by the calcineurin inhibitor cyclosporine A, suggesting an intrinsic connection between these events. Taken together, our results highlighted a potential role of CHP2 in tumorigenesis and revealed a novel function of CHP2 as an activator of the calcineurin/NFAT signaling pathway.     

Autocrine/Paracrine secretion of IL-6 family cytokines causes angiotensin II-induced delayed STAT3 activation

We recently reported that angiotensin II (AngII) biphasically activates the JAK/STAT pathway and induces delayed phosphorylation of STAT3 in the late stage (120 min) in cardiomyocytes. This study was designed to determine the mechanism of delayed phosphorylation of STAT3. Conditioned medium prepared from AngII-stimulated cardiomyocytes could reproduce the tyrosine phosphorylation of STAT3 at 5 min. This delayed phosphorylation was almost completely inhibited by anti-gp130 blocking antibody RX435, but not by TAK044 (ET-A/B-R antagonist), prazosin, or propranolol. AngII induced phosphorylation of gp130 in the late stage, which was temporally in parallel with the delayed phosphorylation of STAT3. AngII augmented IL-6, CT-1, and LIF mRNA expression at 30-60 min, but not CNTF expression. AngII increased IL-6 protein levels by 3-fold in the conditioned media at 2 h compared with the control. These findings indicated that AngII-induced delayed activation of STAT3 is caused by autocrine/paracrine secreted IL-6 family cytokines.     

Troglitazone inhibits angiotensin II-induced extracellular signal-regulated kinase 1/2 nuclear translocation and activation in vascular smooth muscle cells.

The thiazolidinedione troglitazone inhibits angiotensin II-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activity in vascular smooth muscle cells. Activation of extracellular signal-regulated kinase 1/2 by angiotensin II is a multistep process involving both its phosphorylation by mitogen-activated protein kinase extracellular signal-regulated kinase kinase in the cytoplasm and a subsequent translocation to the nucleus. The cytoplasmic activation of extracellular signal-regulated kinase 1/2 in vascular smooth muscle cells proceeds through the protein kinase Czeta --> mitogen-activated protein kinase extracellular signal-regulated kinase kinase --> extracellular signal-regulated kinase pathway. Troglitazone did not affect the angiotensin II-induced activation of protein kinase Czeta or its downstream signaling kinases extracellular signal-regulated kinase 1/2 in the cytosol. In contrast, angiotensin II-induced activation of protein kinase Czeta and extracellular signal-regulated kinase 1/2 in the nucleus were both inhibited by troglitazone. Nuclear translocation of extracellular signal-regulated kinase 1/2 induced by angiotensin II was completely blocked by troglitazone. Protein kinase Czeta, however, did not translocate upon angiotensin II stimulation. Troglitazone, therefore, inhibits both angiotensin II-induced nuclear translocation of extracellular signal-regulated kinase 1/2 and the nuclear activity of its upstream signaling kinase protein kinase Czeta. Since extracellular signal-regulated kinase 1/2 nuclear translocation may be a critical signaling step for multiple growth factors that stimulate vascular smooth muscle cells proliferation and migration, troglitazone may provide a new therapeutical approach for the prevention and treatment of atherosclerosis and restenosis.     

Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway.     

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.     

一氧化氮在血管紧张素Ⅱ诱导心肌细胞肥大中的作用

在培养新生大鼠心肌细胞上,探讨一氧化氮（ＮＯ）在血管紧张素Ⅱ诱导的心肌细胞以大中的作用。结果显示,血管紧张素Ⅱ可使心肌细胞蛋白质含量显著增加,心肌细胞一氧化氮合酶（ＮＯＳ）活性和培养液ＮＯ浓度明显降低。血管紧张素Ⅱ可明显降低心肌细胞ｅＮＯＳｍＲＮＡ水平。Ｓａｒａｌｓｉｎ和百日咳毒素（ＰＴＸ）可抑制血管紧张素Ⅱ诱导的蛋白质含量增加、心肌细胞ＮＯＳ活性减弱和培养液ＮＯ浓度降低。硝普钠提高心肌细胞培养