Inhibition of glycogen synthase kinase 3beta during heart failure is protective

Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3beta in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3beta (Tg-GSK-3beta-DN) and tetracycline-regulatable wild-type GSK-3beta. GSK-3beta-DN significantly reduced the kinase activity of endogenous GSK-3beta, inhibited phosphorylation of eukaryotic translation initiation factor 2B epsilon, and induced accumulation of beta-catenin and myeloid cell leukemia-1, confirming that GSK-3beta-DN acts as a dominant negative in vivo. Tg-GSK-3beta-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the alpha-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3beta induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3beta-DN and nontransgenic mice, Tg-GSK-3beta-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3beta transgene in tetracycline-regulatable wild-type GSK-3beta mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3beta-DN in cardiac myocytes inhibited tumor necrosis factor-alpha-induced apoptosis, and the antiapoptotic effect of GSK-3beta-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3beta induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3beta inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3beta during heart failure could be compensatory.     

Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A

Glycogen synthase kinase 3 (GSK-3) is implicated in multiple biological processes including metabolism, gene expression, cell fate determination, proliferation, and survival. GSK-3 activity is inhibited through phosphorylation of serine 21 in GSK-3 alpha and serine 9 in GSK-3 beta. These serine residues of GSK-3 have been previously identified as targets of protein kinase B (PKB/Akt), a serine/threonine kinase located downstream of phosphatidylinositol 3-kinase. Here, we show that serine 21 in GSK-3 alpha and serine 9 in GSK-3 beta are also physiological substrates of cAMP-dependent protein kinase A. Protein kinase A physically associates with, phosphorylates, and inactivates both isoforms of GSK-3. The results indicate that depending on the stimulatory context, the activity of GSK-3 can be modulated either by growth factors that work through the phosphatidylinositol 3-kinase-protein kinase B cascade or by hormonal stimulation of G protein-coupled receptors that link to changes in intracellular cAMP levels.     

糖原合成酶激酶-3β与心血管疾病

糖原合成酶激酶-3β(GSK-3β)是一种丝氨酸/苏氨酸蛋白激酶,大量的实验研究证明GSK-3β与缺血-再灌注损伤密切相关,缺血预处理、后处理及许多心肌保护药物被认为都是通过调节GSK-3β来发挥他们的心肌保护作用,而且GSK-3β被认为是一个心肌肥大的负调节因子。鉴于GSK-3β与心血管疾病的密切相关性。该文将GSK-3β在缺血-再灌注损伤及心肌肥大中的作用及其机制作一综述。     

Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain.

According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation. In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death. To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria. PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death. In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta. Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis. These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease.     

Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation

Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca^2 +-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca^2 +-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca^2 +. These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β.     

Inhibition of insulin-stimulated glycogen synthesis by 5-aminoimidasole-4-carboxamide-1-beta-d-ribofuranoside-induced adenosine 5'-monophosphate-activated protein kinase activation: interactions with Akt, glycogen synthase kinase 3-3alpha/beta, and glycogen synthase in isolated rat soleus muscle

The aim of this study was to investigate the effects of 5-aminoimidasole-4-carboxamide-1-beta-d-ribofuranoside (AICAR)-induced AMP-activated protein kinase activation on glycogen metabolism in soleus (slow twitch, oxidative) and epitrochlearis (fast twitch, glycolytic) skeletal muscles. Isolated soleus and epitrochlearis muscles were incubated in the absence or presence of insulin (100 nM), AICAR (2 mM), and AICAR plus insulin. In soleus muscles exposed to insulin, glycogen synthesis and glycogen content increased 6.4- and 1.3-fold, respectively. AICAR treatment significantly suppressed ( approximately 60%) insulin-stimulated glycogen synthesis and completely prevented the increase in glycogen content induced by insulin. AICAR did not affect either basal or insulin-stimulated glucose uptake but significantly increased insulin-stimulated ( approximately 20%) lactate production in soleus muscles. Interestingly, basal glucose uptake was significantly increased ( approximately 1.4-fold) in the epitrochlearis muscle, even though neither basal nor insulin-stimulated rates of glycogen synthesis, glycogen content, and lactate production were affected by AICAR. We also report the novel evidence that AICAR markedly reduced insulin-induced Akt-Thr308 phosphorylation after 15 and 30 min exposure to insulin, which coincided with a marked reduction in glycogen synthase kinase 3 (GSK)-3alpha/beta phosphorylation. Importantly, phosphorylation of glycogen synthase was increased by AICAR treatment 45 min after insulin stimulation. Our results indicate that AICAR-induced AMP-activated protein kinase activation caused a time-dependent reduction in Akt308 phosphorylation, activation of glycogen synthase kinase-3alpha/beta, and the inactivation of glycogen synthase, which are compatible with the acute reduction in insulin-stimulated glycogen synthesis in oxidative but not glycolytic skeletal muscles.     

Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation.

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.     

Homocysteine thiolactone inhibits insulin-stimulated DNA and protein synthesis: possible role of mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation

Hyperhomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of homocysteine are related to the formation of homocysteine thiolactone and the consequent increase in oxidative stress. We have recently found that homocysteine thiolactone inhibits insulin receptor tyrosine kinase activity, which results in decreased phosphatidylinositol 3-kinase (PI3K) activity and inhibition of glycogen synthesis. Oxidative stress seemed to be the mechanism underlying these effects, since glutathione was able to restore the insulin signaling as well as the insulin-mediated glycogen synthesis. In the present work we have further investigated insulin receptor signaling studying mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation. Again, homocysteine thiolactone (50 microM) prevented insulin-mediated MAPK, GSK-3 and p70 S6K phosphorylation and these effects were blocked by glutathione (250 microM). Since MAPK and PI3K pathways, including GSK3 and S6K, seem to mediate insulin-mediated growth and proliferation, we measured DNA and protein synthesis. We have found that homocysteine thiolactone (50 microM) inhibits insulin-mediated growth and proliferation, as previously shown for glycogen synthesis. Again, these effects seem to be mediated by oxidative stress, since 250 microM glutathione completely abolished the effects of homocysteine thiolactone on insulin-stimulated DNA and protein synthesis. In conclusion, these data suggest that homocysteine thiolactone impairs insulin signaling by a mechanism involving oxidative stress, leading to a defect in the action of insulin on growth and proliferation.     

Bradykinin prevents reperfusion injury by targeting mitochondrial permeability transition pore through glycogen synthase kinase 3beta

Although bradykinin has been demonstrated to protect the heart at reperfusion, the detailed cellular and molecular mechanisms that mediate the protection remain elusive. Here we aimed to determine whether bradykinin protects the heart at reperfusion by modulating the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3beta (GSK-3beta). Bradykinin given at reperfusion reduced infarct size in isolated rat hearts subjected to 30 min regional ischemia followed by 2 h of reperfusion. The infarct-limiting effect of bradykinin was reversed by atractyloside, an opener of the mPTP, suggesting that bradykinin may protect the heart at reperfusion by modulating the mPTP opening. In support of this observation, bradykinin prevented the collapse of mitochondrial membrane potential (DeltaPsi(m)), an index of the mPTP opening. Bradykinin increased GSK-3beta phosphorylation at reperfusion, and the selective inhibitor of GSK-3beta SB216763 reduced infarct size and prevented the loss of DeltaPsi(m) by mimicking the effect of bradykinin. The effect of bradykinin on GSK-3beta phosphorylation was blocked by wortmannin and LY294002, and bradykinin increased Akt phosphorylation at reperfusion. Further experiments showed that the MEK inhibitor PD98059 prevented the effect of bradykinin on GSK-3beta. However, the mTOR/p70s6K pathway inhibitor rapamycin did not alter bradykinin-induced GSK-3beta phosphorylation and bradykinin failed to alter phosphorylation of either mTOR or p70s6K at reperfusion. Taken together, these data suggest that bradykinin protects the heart at reperfusion by modulating the mPTP opening through inhibition of GSK-3beta. The PI3-kinase/Akt pathway and ERK, but not the mTOR/p70s6K pathway account for the suppression of GSK-3beta by bradykinin.     

The epsilon subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation

Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the epsilon-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2-induced Cx43 phosphorylation. In nonstimulated myocytes, PKCepsilon displayed a discontinuous pattern of localization at intercellular contact sites and partial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKCepsilon distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCepsilon. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCepsilon but not PKCalpha coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCepsilon, suggesting increased association between PKCepsilon and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCepsilon decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2-induced effects on cardiac GJs and that PKCepsilon likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.