The essential role of MEKK3 signaling in angiotensin II-induced calcineurin/nuclear factor of activated T-cells activation

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes, such as T-cell activation, apoptosis, skeletal myocyte differentiation, and cardiac hypertrophy. We determined that active MEKK3 was capable of activating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes and reprogramming cardiac gene expression. In contrast, small interference RNA directed against MEKK3 and a dominant negative form of MEKK3 caused the reduction of NFAT activation in response to angiotensin II in cardiac myocytes. Genetic studies showed that MEKK3-deficient mouse embryo fibroblasts failed to activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring MEKK3 to the MEKK3-deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. We determined that angiotensin II induced MEKK3 phosphorylation. Thus, MEKK3 functions downstream of the AT1 receptor and is essential for calcineurin/NFAT activation. Finally, we determined that MEKK3-mediated activation of calcineurin/NFAT signaling was associated with the phosphorylation of modulatory calcineurin-interacting protein 1 at Ser(108) and Ser(112). Taken together, our studies reveal a previously unrecognized novel essential regulatory role of MEKK3 signaling in calcineurin/NFAT activation.     

Effect of dominant-negative epidermal growth factor receptors on cardiomyocyte hypertrophy

Angiotensin II (AngII) induces heart growth via cardiomyocyte hypertrophy, and central to this is the capacity of the type 1 AngII receptor (AT1R) to "transactivate" epidermal growth factor receptors (EGFRs)--a family with four main subtypes (HER1-4)--although the exact molecular mechanism remains unresolved. In this study, the pharmacological inhibition of AngII-stimulated ERK1/2 activation and cardiomyocyte hypertrophy by increasing concentrations of an EGFR inhibitor, AG1478, indicated that other EGFR subtypes, in addition to HER1, may be involved. We constructed expression vectors and adenoviruses expressing truncated mutant versions of HER1, HER2, and HER4 and determined their capacity to act as dominant-negative inhibitors when co-transfected with full-length EGFRs. It is surprising that adenoviral-mediated expression of these truncated EGFRs in cardiomyocytes led to paradoxical, ligand-independent increases in cardiomyocyte hypertrophy and unusual morphological changes. These results challenge our perception of AT1R-mediated EGFR transactivation and imply that truncated EGFRs may affect cell function through unconventional mechanisms.     

肿瘤坏死因子-α诱导心肌肥大中CaN信号通路的作用

目的:研究钙调神经磷酸酶(CaN)信号通路在肿瘤坏死因子-α(TNF-α)诱导心肌细胞肥大中的作用。方法:Lowry法测心肌细胞蛋白含量;计算机图象分析系统测心肌细胞体积;[3H]-亮氨酸掺入法测心肌细胞蛋白合成;Till阳离子测定系统观察胞内[Ca2+]i瞬变;Western blot法测定CaN的表达。结果:①CaN特异性抑制剂CsA(0.2μmol/L)明显抑制TNF-α(100μg/L)诱导的心肌细胞蛋白含量、蛋白合成和细胞体积增大,但对正常心肌细胞生长无影响。②CaN特异性抑制剂CsA(0.2μmol/L)明显降低TNF-α诱导的心肌细胞内钙离子浓度([Ca2+]i)瞬变幅度增高。③TNF-α明显增强心肌细胞内CaN的表达。结论:TNF-α可能通过引起心肌细胞[Ca2+]i升高,促进CaN表达诱导心肌细胞肥大。     

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.     

Estrogen controls embryonic stem cell proliferation via store-operated calcium entry and the nuclear factor of activated T-cells (NFAT)

Embryonic stem cells (ESCs) can self-renew indefinitely and differentiate into all cell lineages. Calcium is a universal second messenger which regulates a number of cellular pathways. Previous studies showed that store-operated calcium channels (SOCCs) but not voltage-operated calcium channels are present in mouse ESCs (mESCs). In this study, store-operated calcium entry (SOCE) was found to exist in mESCs using confocal microscopy. SOCC blockers lanthanum, 2-aminoethoxydiphenyl borate (2-APB) and SKF-96365 reduced mESC proliferation in a concentration-dependent manner, suggesting that SOCE is important for ESC proliferation. Pluripotent markers, Sox-2, Klf-4, and Nanog, were down-regulated by 2-APB, suggesting that self-renewal property of mESCs relies on SOCE. 17β-estradiol (E2) enhanced mESC proliferation. This enhanced proliferation was associated with an increment of SOCE. Both stimulated proliferation and increased SOCE could be reversed by SOCC blockers suggesting that E2 mediates its stimulatory effect on proliferation via enhancing SOCE. Also, cyclosporin A and INCA-6, inhibitors of calcineurin [phosphatase that de-phosphorylates and activates nuclear factor of activated T-cells (NFAT)], reversed the proliferative effect of E2, indicating that NFAT is involved in E2-stimulated proliferation. Interestingly, E2 caused the nuclear translocation of NFATc4, and this could be reversed by 2-APB. These results suggested that NFATc4 is the downstream target of E2-induced SOCE. The present investigation provides the first line of evidence that SOCE and NFAT are crucial for ESCs to maintain their unique characteristics. In addition, the present investigation also provides novel information on the mechanisms of how E2, an important female sex hormone, affects ESC proliferation.     

Adenosine kinase regulation of cardiomyocyte hypertrophy

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKT(Ser??3) phosphorylation but did attenuate sustained phosphorylation of Raf(Ser33?) (24-48 h), mTOR(Ser2???) (24-48 h), p70S6k(Thr3??) (2.5-48 h), and ERK(Thr2?2/Tyr2??) (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6k(Thr3??) phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERK(Thr202/Tyr204) and AKT(Ser??3). Reduction of Raf-induced p70S6k(Thr3??) phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of Raf signaling to mTOR/p70S6k.     

The effects of sarpogrelate on cardiomyocyte hypertrophy

Sarpogrelate was developed as an antiplatelet agent antagonizing 5-hydroxytryptamine (5-HT) receptors. It had been reported that 5-HT receptors were expressed in cardiovascular system, and that sarpogrelate had antihypertrophic effects in vascular smooth muscle cells. Cardiac hypertrophy is a major problem in cardiac diseases, so the present study was designed to elucidate the effects of sarpogrelate on cardiac hypertrophy. Cultured rat cardiomyocytes (MCs) and cardiac nonmyocytes (NMCs) were prepared by Percoll gradient and adhesion method and MCs were incubated with (MCs/NMCs) or without NMCs. As an index of protein synthesis of MCs, [3H]-leucine uptake into MCs and MCs/NMCs was measured. Sarpogrelate decreased [3H]-leucine uptake into MCs (maximum 62.6+/-20.6% of control at 10(-4)M, p<0.05 vs. control). Sarpogrelate also significantly attenuated angiotensin-II- and endothelin-1-induced [3H]-leucine uptake. These results indicated that sarpogrelate might have antihypertrophic effects and could be a useful aid for cardiovascular disease.     

Propofol depresses angiotensin II-induced cardiomyocyte hypertrophy in vitro

Cardiomyocyte hypertrophy is formed in response to pressure or volume overload, injury, or neurohormonal activation. The most important vascular hormone that contributes to the development of hypertrophy is angiotensin II (Ang II). Accumulating studies have suggested that reactive oxygen species (ROS) may play an important role in cardiac hypertrophy. Propofol is a general anesthetic that possesses antioxidant action. We therefore examined whether propofol inhibited Ang II-induced cardiomyocyte hypertrophy. Our results showed that both ROS formation and hypertrophic responses induced by Ang II in cardiomyocytes were partially blocked by propofol. Further studies showed that propofol inhibited the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2) induced by Ang II via a decrease in ROS production. In addition, propofol also markedly attenuated Ang II-stimulated nuclear factor-kappaB (NF-kappaB) activation via a decrease in ROS production. In conclusion, propofol prevents cardiomyocyte hypertrophy by interfering with the generation of ROS and involves the inhibition of the MEK/ERK signaling transduction pathway and NF-kappaB activation.     

Cardiac hypertrophy and changes in contractile function of cardiomyocyte

To investigate the cellular mechanisms of pressure-overload cardiac hypertrophy transition to heart failure, we observed time course of changes in morphology and contractile function of cardiomyocytes in transverse abdominal aortic constriction (TAC) rats. Since TAC rats suffered higher stress, body weight had a slower growth rate compared with that of synchronous control rats. Therefore, the left ventricular to body weight ratio produced experimental bias to evaluate the degree of cardiac hypertrophy. Length and width of collagenase-isolated cardiomyocyte were directly measured. Length, width and calculated surface area of cardiomyocyte showed a progressive increase in 8-, 16-, and 20-week TAC rats. The increasing rate of surface area in cardiomyocytes was higher at the middle stage of TAC (from the eighth to sixteenth week). Due to the constraint of fibrosis formation, the increasing rate of surface area in cardiomyocytes was slower at the late stage of TAC (from the sixteenth to twentieth week). The sarcomere length of cardiomyocytes was unchanged, whereas sarcomere numbers were significantly increased in 8-, 16-, and 20-week TAC rats. Shortening amplitude of unloaded contraction in single cardiomyocyte was significantly enhanced in 1-week TAC rats, but not altered in 8-week TAC rats compared with that in the synchronous control rats. On the contrary, unloaded shortening amplitude of single cardiomyocyte was significantly reduced in 16- and 20-week TAC rats. The above results suggest that the reduced shortening amplitude may be associated with intrinsic molecular alterations in hypertrophied cardiomyocytes.