Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 deficiency protects brain from ischemic injury in mice

Mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) is one of several kinases directly regulated by p38 MAP kinase. A role of p38 MAP kinase in ischemic brain injury has been previously suggested by pharmacological means. In the present study, we provide evidence for a role of MK2 in cerebral ischemic injury using MK2-deficient (MK2(-/-)) mice. MK2(-/-) mice subjected to focal ischemia markedly reduced infarct size by 64 and 76% after transient and permanent ischemia, respectively, compared with wild-type mice. Furthermore, MK2(-/-) mice had significant reduction in neurological deficits. Real-time PCR analysis identified a significantly lower expression in interleukin-1beta mRNA (53% reduction) but not in tumor necrosis factor-alpha mRNA in MK2(-/-) mice over wild-type animals after ischemic injury. The significant reduction in interleukin-1beta was also confirmed in MK2(-/-) mice by enzyme-linked immunosorbent assay. The marked neuroprotection from ischemic brain injury in MK2(-/-) mice was not associated with the alteration of hemodynamic or systemic variables, activation of caspase-3, or apoptosis. Our data provide new evidence for the involvement of MAP kinase pathway in focal ischemic brain injury and suggest that this effect might be associated with the expression of interleukin-1beta in the ischemic brain tissue.     

Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes

Transient receptor potential canonical‐6 (TRPC6) ion channels, expressed at high levels in podocytes of the filtration barrier, are recently implicated in the pathogenesis of various forms of proteinuric kidney diseases. Indeed, inherited or acquired up‐regulation of TRPC6 activities are suggested to play a role in podocytopathies. Yet, we possess limited information about the regulation of TRPC6 in human podocytes. Therefore, in this study, we aimed at defining how the protein kinase C (PKC) system, one of the key intracellular signalling pathways, regulates TRPC6 function and expression. On human differentiated podocytes, we identified the molecular expressions of both TRPC6 and several PKC isoforms. We also showed that TRPC6 channels are functional since the TRPC6 activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) induced Ca²⁺‐influx to the cells. By assessing the regulatory roles of the PKCs, we found that inhibitors of the endogenous activities of classical and novel PKC isoforms markedly augmented TRPC6 activities. In contrast, activation of the PKC system by phorbol 12‐myristate 13‐acetate (PMA) exerted inhibitory actions on TRPC6 and suppressed its expression. Importantly, PMA treatment markedly down‐regulated the expression levels of PKCα, PKCβ, and PKCη reflecting their activation. Taken together, these results indicate that the PKC system exhibits a ‘tonic’ inhibition on TRPC6 activity in human podocytes suggesting that pathological conditions altering the expression and/or activation patterns of podocyte‐expressed PKCs may influence TRPC6 activity and hence podocyte functions. Therefore, it is proposed that targeted manipulation of certain PKC isoforms might be beneficial in certain proteinuric kidney diseases with altered TRPC6 functions.     

Cyclin I protects podocytes from apoptosis

The limited regenerative capacity of the glomerular podocyte following injury underlies the development of glomerulosclerosis and progressive renal failure in a diverse range of kidney diseases. We discovered that, in the kidney, cyclin I is uniquely expressed in the glomerular podocyte, and have constructed cyclin I knock-out mice to explore the biological function of cyclin I in these cells. Cyclin I knock-out (-/-) podocytes showed an increased susceptibility to apoptosis both in vitro and in vivo. Following induction of experimental glomerulonephritis, podocyte apoptosis was increased 4-fold in the cyclin I -/- mice, which was associated with dramatically decreased renal function. Our previous data showed that the Cdk inhibitor p21(Cip1/Waf1) protects podocytes from certain apoptotic stimuli. In cultured cyclin I -/- podocytes, the level of p21(Cip1/Waf1) was lower at base line, had a shorter half-life, and declined more rapidly in response to apoptotic stimuli than in wild-type cells. Enforced expression of p21(Cip1/Waf1) reversed the susceptibility of cyclin I -/- podocytes to apoptosis. Cyclin I protects podocytes from apoptosis, and we provide preliminary data to suggest that this is mediated by stabilization of p21(Cip1/Waf1).     

Inhibition of mitochondrial fission protects podocytes from albumin-induced cell damage in diabetic kidney disease

AimsIdentifying the mechanisms that underlie progression from endothelial damage to podocyte damage, which leads to massive proteinuria, is an urgent issue that must be clarified to improve renal outcome in diabetic kidney disease (DKD). We aimed to examine the role of dynamin-related protein 1 (Drp1)-mediated regulation of mitochondrial fission in podocytes in the pathogenesis of massive proteinuria in DKD.MethodsDiabetes- or albuminuria-associated changes in mitochondrial morphology in podocytes were examined by electron microscopy. The effects of albumin and other diabetes-related stimuli, including high glucose (HG), on mitochondrial morphology were examined in cultured podocytes. The role of Drp1 in podocyte damage was examined using diabetic podocyte-specific Drp1-deficient mice treated with neuraminidase, which removes endothelial glycocalyx.ResultsNeuraminidase-induced removal of glomerular endothelial glycocalyx in nondiabetic mice led to microalbuminuria without podocyte damage, accompanied by reduced Drp1 expression and mitochondrial elongation in podocytes. In contrast, streptozotocin-induced diabetes significantly exacerbated neuraminidase-induced podocyte damage and albuminuria, and was accompanied by increased Drp1 expression and enhanced mitochondrial fission in podocytes. Cell culture experiments showed that albumin stimulation decreased Drp1 expression and elongated mitochondria, although HG inhibited albumin-associated changes in mitochondrial dynamics, resulting in apoptosis. Podocyte-specific Drp1-deficiency in mice prevented diabetes-related exacerbation of podocyte damage and neuraminidase-induced development of albuminuria. Endothelial dysfunction-induced albumin exposure is cytotoxic to podocytes. Inhibition of mitochondrial fission in podocytes is a cytoprotective mechanism against albumin stimulation, which is impaired under diabetic condition. Inhibition of mitochondrial fission in podocytes may represent a new therapeutic strategy for massive proteinuria in DKD.     

Aminocyanopyridine inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)

A class of inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2) was discovered. These compounds have demonstrated activity against the enzyme with IC50 values as low as 130 nM and suppress the expression of TNFalpha in U937 cells. These represent the first small molecule inhibitors of MK-2 to be reported.     

Inhibition of the Ca2+/calmodulin-dependent protein kinase I cascade by cAMP-dependent protein kinase

Several recent studies have shown that Ca2+/calmodulin-dependent protein kinase I (CaMKI) is phosphorylated and activated by a protein kinase (CaMKK) that is itself subject to regulation by Ca2+/calmodulin. In the present study, we demonstrate that this enzyme cascade is regulated by cAMP-mediated activation of cAMP-dependent protein kinase (PKA). In vitro, CaMKK is phosphorylated by PKA and this is associated with inhibition of enzyme activity. The major site of phosphorylation is threonine 108, although additional sites are phosphorylated with lower efficiency. In vitro, CaMKK is also phosphorylated by CaMKI at the same sites as PKA, suggesting that this regulatory phosphorylation might play a role as a negative-feedback mechanism. In intact PC12 cells, activation of PKA with forskolin resulted in a rapid inhibition of both CaMKK and CaMKI activity. In hippocampal slices CaMKK was phosphorylated under basal conditions, and activation of PKA led to an increase in phosphorylation. Two-dimensional phosphopeptide mapping indicated that activation of PKA led to increased phosphorylation of multiple sites including threonine 108. These results indicate that in vitro and in intact cells the CaMKK/CaMKI cascade is subject to inhibition by PKA-mediated phosphorylation of CaMKK. The phosphorylation and inhibition of CaMKK by PKA is likely to be involved in modulating the balance between cAMP- and Ca2+-dependent signal transduction pathways.     

MAP kinase protects G protein-coupled receptor kinase 2 from proteasomal degradation

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and shuts down signaling from 7-transmembrane receptors (7TMs). Although, receptor activity controls GRK2 expression levels, the underlying molecular mechanisms are poorly understood. We have previously shown that extracellular signal-regulated kinase (ERK1/2) activation increases GRK2 expression [J. Theilade, J. Lerche Hansen, S. Haunso, S.P. Sheikh, Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2), FEBS Lett. 518 (2002) 195-199]. In the present study, we found that ERK1/2 regulates GRK2 degradation rather than synthesis. ERK1/2 blockade using PD98059 decreased GRK2 cellular levels to 0.25-fold of control in Cos7 cells. This effect was due to enhanced degradation of the GRK2 protein, since proteasome blockade prevented down-regulation of GRK2 protein levels in the presence of PD98059. Further, ERK blockade had no effect on GRK2 synthesis as probed using a reporter construct carrying the GRK2 promoter upstream of the luciferase gene. We predict ERK1/2 mediated GRK2 protection could be a general phenomenon as proteasome inhibition increased GRK2 expression in two other cell lines, HEK293 and NIH3T3.     

Ghrelin protects heart against ERS-induced injury and apoptosis by activating AMP-activated protein kinase

Ghrelin, the endogenous ligand of growth hormone secretagogue receptor (GHS-R), is a cardioprotective peptide. In our previous work, we have revealed that ghrelin could protect heart against ischemia/reperfusion (I/R) injury by inhibiting endoplasmic reticulum stress (ERS), which contributes to many heart diseases. In current study, using both in vivo and in vitro models, we investigated how ghrelin inhibits myocardial ERS. In the in vivo rat heart injury model induced by isoproterenol (ISO), we found that exogenous ghrelin could alleviate heart dysfunction, reduce myocardial injury and apoptosis and inhibit the excessive myocardial ERS induced by ISO. More importantly, the activation of AMP-activated protein kinase (AMPK) was observed. To explore the role of AMPK activation in ERS inhibition by ghrelin, we set up two in vitro ERS models by exposing cultured rat cardiomyocytes to tunicamycin(Tm) or dithiothreitol (DTT). In both models, compared with Tm or DTT treatment alone, pre-incubation cardiomyocytes with ghrelin significantly activated AMPK, reversed the upregulation of the ERS markers, C/EBP-homologous protein (CHOP) and cleaved caspase-12, and reduced apoptosis of cardiomyocytes. Further, we found that the ERS inhibitory and anti-apoptotic actions induced by ghrelin were blocked by an AMPK inhibitor. To investigate how ghrelin activates AMPK, selective antagonist of GHS-R1a and inhibitor of Ca²⁺/Calmodulin-dependent protein kinase kinase (CaMKK) were added, respectively, before ghrelin pre-incubation, and we found that AMPK activation was prevented and the ERS inhibitory and anti-apoptotic actions of ghrelin were blocked. In conclusion, ghrelin could protect heart against ERS-induced injury and apoptosis, at least partially through a GHS-R1a/CaMKK/AMPK pathway.     

Identification and SAR of squarate inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)

A novel series of inhibitors for mitogen activated protein kinase-activated protein kinase 2 (MK-2) are reported. These squarate based inhibitors were identified via a high-throughput screen. An MK2 co-structure with the starting ligand was obtained and a structure based approach was followed to optimize potency and selectivity.     

AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress

Oxygen deprivation leads to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), causing ER stress. Under conditions of ER stress, inhibition of protein synthesis and up-regulation of ER chaperone expression reduce the misfolded proteins in the ER. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in energy homeostasis during hypoxia. It has been shown that AMPK activation is associated with inhibition of protein synthesis via phosphorylation of elongation factor 2 (eEF2) in cardiomyocytes. We therefore examined whether AMPK attenuates hypoxia-induced ER stress in neonatal rat cardiomyocytes. We found that hypoxia induced ER stress, as assessed by the expression of CHOP and BiP and cleavage of caspase 12. Knockdown of CHOP or caspase 12 through small interfering RNA (siRNA) resulted in decreased expression of cleaved poly(ADP-ribose) polymerase following exposure to hypoxia. We also found that hypoxia-induced CHOP expression and cleavage of caspase 12 were significantly inhibited by pretreatment with 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), a pharmacological activator of AMPK. In parallel, adenovirus expressing dominant-negative AMPK significantly attenuated the cardioprotective effects of AICAR. Knockdown of eEF2 phosphorylation using eEF2 kinase siRNA abolished these cardioprotective effects of AICAR. Taken together, these findings demonstrate that activation of AMPK contributes to protection of the heart against hypoxic injury through attenuation of ER stress and that attenuation of protein synthesis via eEF2 inactivation may be the mechanism of cardioprotection by AMPK.     

Novel tetrahydro-beta-carboline-1-carboxylic acids as inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)

A structure-activity relationship study was conducted on a series of tetrahydro-beta-carboline-1-carboxylic acid analogs in order to identify the key functionality responsible for activity against the mitogen-activated protein kinase-activated protein kinase 2 enzyme (MK-2). The compounds were further evaluated for their ability to inhibit TNFalpha production in U937 cells and in vivo. These compounds represent a novel structural class of compounds capable of inhibiting MK-2 with remarkable selectivity.     

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis. Gossypol inhibited Ca2+-dependent activity of the enzyme without affecting its basal activity. The IC50 value (concentration causing 50% inhibition) was 31 microM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (Ki = 31 microM) or lysine-rich histone (Ki = 30 microM), and competitively with respect to phosphatidylserine (Ki = 2.1 microM). With Ca2+, irrespective of the presence or absence of 1,3-diolein, the compound lowered Vmax and increased the apparent Ka for Ca2+. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrates in the testis for the enzyme located in nucleosome), with an IC50 value of 88 microM. These results suggested that a phospholipid-sensitive Ca2+-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.     

Intermittent hypoxia protects the rat heart against ischemia/reperfusion injury by activating protein kinase C

The aim of this study was to investigate whether and how protein kinase C (PKC) was involved in the protection afforded by intermittent hypoxia (IH) and the subcellular distribution of different PKC isozymes in rat left ventricle. Post-ischemic recovery of left ventricular developed pressure and +/-dP/dtmax in IH hearts were higher than those of normoxic hearts. Chelerythrine (CHE, 5 microM), a PKC antagonist, significantly inhibited the protective effects of IH, but had no influence on normoxic hearts. CHE significantly reduced the effect of IH on the time to maximal contracture (Tmc), but had no significant effect on the amplitude of maximal contracture (Amc) in IH group. In isolated normoxic cardiomyocytes, [Ca(2+)](i), measured as arbitrary units of fluorescence ratio (340 nm/380 nm) of fura-2, gradually increased during 20 min simulated ischemia and kept at high level during 30 min reperfusion. However, [Ca(2+)](i) kept at normal level during simulated ischemia and reperfusion in isolated IH cardiomyocytes. In normoxic myocytes, [Na(+)](i), indicated as actual concentration undergone calibration, gradually increased during 20 min simulated ischemia and quickly declined to almost the same level as that of pre-ischemia during 30 min simulated reperfusion. However, in IH myocytes, [Na(+)](i) increased to a level lower than the corresponding of normoxic myocytes during simulated ischemia and gradually reduced to the similar level as that of normoxic myocytes after simulated reperfusion. 5 microM CHE greatly increased the levels of [Ca(2+)](i) and [Na(+)](i) during ischemia and reperfusion in normoxic and IH myocytes. In addition, we demonstrated that IH up-regulated the baseline protein expression of particulate fraction of PKC-alpha, epsilon, delta isozymes. There is no significant difference of protein expression of PKC-alpha, epsilon, delta isozymes in cytosolic fraction between IH and normoxic group. The above results suggested that PKC contributed to the cardioprotection afforded by IH against ischemia/reperfusion (I/R) injury; the basal up-regulation of the particulate fraction of PKC-alpha, epsilon, delta isozymes in IH rat hearts and the contribution of PKC to the elimination of calcium and sodium overload might underlie the mechanisms of cardioprotection by IH.     

CTRP9 protein protects against myocardial injury following ischemia-reperfusion through AMP-activated protein kinase (AMPK)-dependent mechanism

Ischemic heart disease is the major cause of death in Western countries. CTRP9 (C1q/TNF-related protein 9) is a fat-derived plasma protein that has salutary effects on glucose metabolism and vascular function. However, the functional role of CTRP9 in ischemic heart disease has not been clarified. Here, we examined the regulation of CTRP9 in response to acute cardiac injury and investigated whether CTRP9 modulates cardiac damage after ischemia and reperfusion. Myocardial ischemia-reperfusion injury resulted in reduced plasma CTRP9 levels and increased plasma free fatty acid levels, which were accompanied by a decrease in CTRP9 expression and an increase in NADPH oxidase component expression in fat tissue. Treatment of cultured adipocytes with palmitic acid or hydrogen peroxide reduced CTRP9 expression. Systemic administration of CTRP9 to wild-type mice, before the induction of ischemia or at the time of reperfusion, led to a reduction in myocardial infarct size following ischemia-reperfusion. Administration of CTRP9 also attenuated myocyte apoptosis in ischemic heart, which was accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK). Treatment of cardiac myocytes with CTRP9 protein reduced apoptosis in response to hypoxia/reoxygenation and stimulated AMPK phosphorylation. Blockade of AMPK activity reversed the suppressive actions of CTRP9 on cardiomyocyte apoptosis. Knockdown of adiponectin receptor 1 diminished CTRP9-induced increases in AMPK phosphorylation and survival of cardiac myocytes. Our data suggest that CTRP9 protects against acute cardiac injury following ischemia-reperfusion via an AMPK-dependent mechanism.     

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H₂O₂ and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 µM), an inhibitor of O-GlcNAcase, was less protective against I/R and H₂O₂ and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation. mitochondria; apoptosis; necrosis, O-linked-N-acetylglucosamine     

Inhibition of Kir2.1 (KCNJ2) by the AMP-activated protein kinase

The inward rectifier K⁺ channel Kir2.1 participates in the maintenance of the cell membrane potential in a variety of cells including neurons and cardiac myocytes. Mutations of KCNJ2 encoding Kir2.1 underlie the Andersen–Tawil syndrome, a rare disorder clinically characterized by periodic paralysis, cardiac arrhythmia and skeletal abnormalities. The maintenance of the cardiac cell membrane potential is decreased in ischaemia, which is known to stimulate the AMP-activated serine/threonine protein kinase (AMPK). This energy-sensing kinase stimulates energy production and limits energy utilization. The present study explored whether AMPK regulates Kir2.1. To this end, cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), of the kinase dead mutant ^αK45RAMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. Kir2.1 activity was determined in two-electrode voltage-clamp experiments. Moreover, Kir2.1 protein abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced Kir2.1-mediated currents and Kir2.1 protein abundance in the cell membrane. Expression of wild type Nedd4-2 or of Nedd4-2^S795A lacking an AMPK phosphorylation consensus sequence downregulated Kir2.1 currents. The effect of wild type Nedd4-2 but not of Nedd4-2^S795A was significantly augmented by additional coexpression of AMPK. In conclusion, AMPK is a potent regulator of Kir2.1. AMPK is at least partially effective through phosphorylation of the ubiquitin ligase Nedd4-2.