A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration.

Smooth muscle cells are exposed to growth factors and cytokines that contribute to pathological states including airway hyperresponsiveness, atherosclerosis, angiogenesis, smooth muscle hypertrophy, and hyperplasia. A common feature of several of these conditions is migration of smooth muscle beyond the initial boundary of the organ. Signal transduction pathways activated by extracellular signals that instigate migration are mostly undefined in smooth muscles. We measured migration of cultured tracheal myocytes in response to platelet-derived growth factor, interleukin-1beta, and transforming growth factor-beta. Cellular migration was blocked by SB203580, an inhibitor of p38(MAPK). Time course experiments demonstrated increased phosphorylation of p38(MAPK). Activation of p38(MAPK) resulted in the phosphorylation of HSP27 (heat shock protein 27), which may modulate F-actin polymerization. Inhibition of p38(MAPK) activity inhibited phosphorylation of HSP27. Adenovirus-mediated expression of activated mutant MAPK kinase 6b(E), an upstream activator for p38(MAPK), increased cell migration, whereas overexpression of p38alpha MAPK dominant negative mutant and an HSP27 phosphorylation mutant blocked cell migration completely. The results indicate that activation of the p38(MAPK) pathway by growth factors and proinflammatory cytokines regulates smooth muscle cell migration and may contribute to pathological states involving smooth muscle dysfunction.     

Insulin and AMPK regulate FA translocase/CD36 plasma membrane recruitment in cardiomyocytes via Rab GAP AS160 and Rab8a Rab GTPase

The FA translocase cluster of differentiation 36 (CD36) facilitates FA uptake by the myocardium, and its surface recruitment in cardiomyocytes is induced by insulin, AMP-dependent protein kinase (AMPK), or contraction. Dysfunction of CD36 trafficking contributes to disordered cardiac FA utilization and promotes progression to disease. The Akt substrate 160 (AS160) Rab GTPase-activating protein (GAP) is a key regulator of vesicular trafficking, and its activity is modulated via phosphorylation. Our study documents that AS160 mediates insulin or AMPK-stimulated surface translocation of CD36 in cardiomyocytes. Knock-down of AS160 redistributes CD36 to the surface and abrogates its translocation by insulin or the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). Conversely, overexpression of a phosphorylation-deficient AS160 mutant (AS160 4P) suppresses the stimulated membrane recruitment of CD36. The AS160 substrate Rab8a GTPase is shown via overexpression and knock-down studies to be specifically involved in insulin/AICAR-induced CD36 membrane recruitment. Our findings directly demonstrate AS160 regulation of CD36 trafficking. In myocytes, the AS160 pathway also mediates the effect of insulin, AMPK, or contraction on surface recruitment of the glucose transporter GLUT4. Thus, AS160 constitutes a point of convergence for coordinating physiological regulation of CD36 and GLUT4 membrane recruitment.     

TLR4 regulates cardiac lipid accumulation and diabetic heart disease in the nonobese diabetic mouse model of type 1 diabetes

Toll-like receptor (TLR)4 regulates inflammation and metabolism and has been linked to the pathogenesis of heart disease. TLR4 is upregulated in diabetic cardiomyocytes, and we examined the role of TLR4 in modulating cardiac fatty acid (FA) metabolism and the pathogenesis of diabetic heart disease in nonobese diabetic (NOD) mice. Both wild-type (WT) NOD and TLR4-deficient NOD animals had increased plasma triglyceride levels after the onset of diabetes. However, by comparison, TLR4-deficient NOD mouse hearts had lower triglyceride accumulation in the early stages of diabetes, which was associated with a reduction in myeloid differentiation primary response gene (88) (MyD88), phosphorylation of p38 MAPK (phospho-p38), lipoprotein lipase (LPL), and JNK levels but increased phospho-AMP-activated protein kinase (AMPK). Oleic acid treatment in H9C2 cardiomyocytes also led to cellular lipid accumulation, which was attenuated by TLR4 small interfering RNA. TLR4 deficiency in the cells decreased FA-induced augmentation of MyD88, phospho-p38, and LPL, suggesting that TLR4 may modulate FA-induced lipid metabolism in cardiomyocytes. In addition, although cardiac function was impaired in both diabetic WT NOD and TLR4-deficient NOD animals compared with control nondiabetic mice, this deficit was less in the diabetic TLR4-deficient NOD mice, which had greater ejection fraction, greater fractional shortening, and increased left ventricular developed pressure in the early stages after the development of diabetes compared with their diabetic WT NOD counterparts. Thus, we conclude that TLR4 plays a role in regulating lipid accumulation in cardiac muscle after the onset of type 1 diabetes, which may contribute to cardiac dysfunction.     

Bax translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activated protein kinases

The cytosolic protein Bax plays a key role in apoptosis by migrating to mitochondria and releasing proapoptotic proteins from the mitochondrial intermembrane space. The present study investigates the movement of Bax in isolated rat neonatal cardiomyocytes subjected to simulated ischaemia (minus glucose, plus cyanide), using green fluorescent protein-tagged Bax as a means of imaging Bax movements. Simulated ischaemia induced Bax translocation from the cytosol to mitochondria, commencing within 20 min of simulated ischaemia and progressing for several hours. Under the same conditions, there was an increase in the active, phosphorylated forms of p38 MAPK (mitogen-activated protein kinase) and AMPK (AMP-activated protein kinase). The AMPK activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin also stimulated Bax translocation. Inhibition of p38 MAPK with SB203580 attenuated the phosphorylation of the downstream substrates, MAPK-activated protein kinases 2 and 3, but not that of the upstream MAPK kinase 3, nor of AMPK. Under all conditions (ischaemia, AICAR and metformin), SB203580 blocked Bax translocation completely. It is concluded that Bax translocation to mitochondria is an early step in ischaemia and that it occurs in response to activation of p38 MAPK downstream of AMPK.     

Resistin is a key mediator of glucose-dependent insulinotropic polypeptide (GIP) stimulation of lipoprotein lipase (LPL) activity in adipocytes

Studies on the physiological roles of the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) have largely focused on its insulinotropic action and ability to regulate beta-cell mass. In previous studies on the stimulatory effect of GIP on adipocyte lipoprotein lipase (LPL), a pathway was identified involving increased phosphorylation of protein kinase B (PKB) and reduced phosphorylation of LKB1 and AMP-activated protein kinase (AMPK). The slow time of onset of the responses suggested that GIP may have induced release of an intermediary molecule, and the current studies focused on the possible contribution of the adipokine resistin. In differentiated 3T3-L1 adipocytes, GIP, in the presence of insulin, increased resistin secretion through a pathway involving p38 mitogen-activated protein kinase (p38 MAPK) and the stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK). The other major incretin hormone, glucagon-like peptide-1 (GLP-1), exhibited no significant effects. Chronic elevation of circulating GIP levels in the Vancouver Diabetic Fatty (VDF) Zucker rat resulted in increases in circulating resistin levels and activation of p38 MAPK or SAPK/JNK in epididymal fat tissue, suggesting the existence of identical pathways in vivo as well as in vitro. Administration of resistin to 3T3-L1 adipocytes mimicked the effects of GIP on the PKB/LKB1/AMPK/LPL pathway: increasing phosphorylation of PKB, reducing levels of phosphorylated LKB1 and AMPK, and increasing LPL activity. Knockdown of resistin using RNA interference attenuated the effect of GIP on the PKB/LKB1/AMPK/LPL pathway in 3T3-L1 adipocytes, supporting a role for resistin as a mediator.     

Role of F-actin organization in p38 MAP kinase-mediated apoptosis and necrosis in neonatal rat cardiomyocytes subjected to simulated ischemia and reoxygenation

Activation of p38 mitogen-activated protein (MAP) kinase (MAPK) has been implicated in the mechanism of cardiomyocyte (CMC) protection and injury. The p38 MAPK controversy may be related to differential effects of this kinase on apoptosis and necrosis. We have hypothesized that p38 MAPK-mediated F-actin reorganization promotes apoptotic cell death, whereas it protects from osmotic stress-induced necrotic cell death. Cultured neonatal rat CMCs were subjected to 2 h of simulated ischemia followed by reoxygenation. p38 MAPK activity measured by phosphorylation of MAP kinase-activated protein (MAPKAP) kinase 2 was increased during simulated ischemia and reoxygenation. This was associated with translocation of heat shock protein 27 (HSP27) from the cytosolic to the cytoskeletal fraction and F-actin reorganization. Cytochrome c release from mitochondria, caspase-3 activation, and DNA fragmentation were increased during reoxygenation. Robust lactate dehydrogenase (LDH) release was observed under hyposmotic (140 mosM) reoxygenation. The p38 MAPK inhibitor SB-203580 abrogated activation of p38 MAPK, translocation of HSP27, and F-actin reorganization and prevented cytochrome c release, caspase-3 activation, and DNA fragmentation. Conversely, SB-203580 enhanced LDH release during hyposmotic reoxygenation. The F-actin disrupting agent cytochalasin D inhibited F-actin reorganization and prevented cytochrome c release, caspase-3 activation, and DNA fragmentation, whereas it enhanced LDH release during hyposmotic reoxygenation. When CMCs were incubated under the isosmotic condition for the first 15 min of reoxygenation, SB-203580 and cytochalasin D increased ATP content of CMCs and prevented LDH release after the conversion to the hyposmotic condition. These results suggest that F-actin reorganization mediated by activation of p38 MAPK plays a differential role in apoptosis and protection against osmotic stress-induced necrosis during reoxygenation in neonatal rat CMCs; however, the sarcolemmal fragility caused by p38 MAPK inhibition can be reversed during temporary blockade of physical stress during reoxygenation.     

Adiponectin modulates carnitine palmitoyltransferase-1 through AMPK signaling cascade in rat cardiomyocytes

Adiponectin, an adipocyte-derived polypeptide hormone, plays an important role in regulating fatty acid oxidation. beta-oxidation of fatty acids supplies most of the cardiac energy and carnitine palmitoyltransferase (CPT)-1 serves as a key regulator during this process. To characterize the potential effects of adiponectin on CPT-1, we incubated rat neonatal cardiomyocytes with globular adiponectin (gAd). Results showed that gAd promoted the activity and mRNA expression of CPT-1. The underlying signal pathway involved in this modulatory effect was further investigated. Inhibition of AMP-activated protein kinase (AMPK) with adenine 9-beta-d-arabinofuranoside (AraA) completely abrogated gAd-mediated AMPK and acetyl coenzyme A carboxylase (ACC) phosphorylation and suppressed the promotion of CPT-1 activity. gAd also induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and peroxisome proliferator-activated receptor (PPAR)-alpha, which was inhibited by AraA. SB202190, a p38MAPK inhibitor, blocked gAd-stimulated PPAR-alpha phosphorylation. When AMPK and/or p38MAPK was inhibited, gAd-enhanced mRNA expression of CPT-1 was partially reduced. In conclusion, our study suggests that the activation of AMPK signaling cascade participates in the promotion effect of gAd on CPT-1.     

p38 MAPK-dependent small HSP27 and αB-crystallin phosphorylation in regulation of myocardial function following cardioplegic arrest

We previously demonstrated that myocardial p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27) are phosphorylated following cardioplegic arrest in patients undergoing cardiac surgery and correlate with reduced cardiac function. The following studies were performed to determine whether inhibition of p38 MAPK and/or overexpression of nonphosphorylatable HSP27 improves cardiac function following cardioplegic arrest. Langendorff-perfused isolated rat hearts were subjected to 2 h of intermittent cold cardioplegia followed by 30 min of reperfusion. Hearts were treated with (CP+SB) or without (CP) the p38 MAPK inhibitor SB-203580 (5 μM) supplied in the cardioplegia. Sham-treated hearts served as controls. In separate experiments, isolated rat ventricular myocytes infected with either green fluorescent protein (GFP) or a nonphosphorylatable HSP27 mutant (3A-HSP27) were subjected to 3 h of cold hypoxic cardioplegia and simulated reperfusion (CP) followed by video microscopy and length change measurements. Baseline parameters of cardiac function were similar between groups [left ventricular developed pressure (LVDP), 119 ± 4.9 mmHg; positive and negative first derivatives of LV pressure (± dP/dt), 3,139 ± 245 and 2, 314 ± 110 mmHg/s]. CP resulted in reduced cardiac function (LVDP, 72.2 ± 5.8 mmHg; ± dP/dt, 2,076 ± 231 and -1,317 ± 156 mmHg/s) compared with baseline. Treatment with 5 μM SB-203580 significantly improved CP-induced cardiac function (LVDP, 101.9 ± 0 mmHg; ± dP/dt, 2,836 ± 163 and -2,108 ± 120 mmHg/s; P = 0.03, 0.01, and 0.04, CP+SB vs. CP). Inhibition of p38 MAPK significantly lowered CP-induced p38 MAPK, HSP27, and αB-crystallin (cryAB) phosphorylation. In vitro CP decreased myocyte length changes from 10.3 ± 1.5% (GFP) to 5.7 ± 0.8% (GFP+CP). Infection with 3A-HSP27 completely rescued CP-induced decreased myocyte contraction (11.1 ± 1.0%). However, infection with 3A-HSP27 did not block the endogenous HSP27 response. We conclude that inhibition of p38 MAPK and subsequent HSP27 and cryAB phosphorylation and/or overexpression of nonphosphorylatable HSP27 significantly improves cardiac performance following cardioplegic arrest. Modulation of HSP27 phosphorylation may improve myocardial stunning following cardiac surgery.     

The metabolic "switch" AMPK regulates cardiac heparin-releasable lipoprotein lipase

The "fuel gauge" AMP-activated protein kinase (AMPK) facilitates ATP production to meet energy demands during metabolic stress. Given the importance of lipoprotein lipase (LPL) in providing hearts with fatty acids (FA), the preferred substrate consumed by the heart, the objective of the present study was to investigate whether activation of AMPK influences LPL at its functionally relevant location, the coronary lumen. Hearts from overnight-fasted rats were first perfused with heparin to release LPL, and homogenates from these hearts were then used to measure total and phospho-AMPK-alpha by Western blotting. Manipulation of AMPK activity [with drugs like adenine 9-beta-D-arabinofuranoside (Ara-A) and insulin (that inhibit) or perhexiline and oligomycin (that stimulate)] and its influence on LPL was also determined. Fasting augmented the activity of both AMPK and luminal LPL on immediate removal of hearts, effects that still remained even after in vitro perfusion of hearts for 1 h. Inhibition of AMPK in fasted hearts using an inhibitor like Ara-A or through provision of insulin markedly lowered the enhanced luminal LPL activity. In contrast, AMPK activators, like perhexiline and oligomycin, produced a significant elevation in heparin-releasable LPL activity. Thus, with fasting or drugs that influence AMPK, a strong correlation between this metabolic switch and cardiac LPL activity was established. Our data suggest that, in addition to its direct role in promoting FA oxidation, AMPK-mediated recruitment of LPL to the coronary lumen could represent an immediate compensatory response by the heart to guarantee FA supply.     

Capsaicin stimulates glucose uptake in C2C12 muscle cells via the reactive oxygen species (ROS)/AMPK/p38 MAPK pathway

Capsaicin has been reported to regulate blood glucose levels and to ameliorate insulin resistance in obese mice. This study demonstrates that capsaicin increases glucose uptake directly by activating AMP-activated protein kinase (AMPK) in C2C12 muscle cells, which manifested as an attenuation of glucose uptake when compound C, an AMPK inhibitor, was co-administered with capsaicin. However, the insulin signaling molecules insulin receptor substrate-1 (IRS-1) and Akt were not affected by capsaicin. Additional results showed that p38 mitogen-activated protein kinase (MAPK) is also involved in capsaicin-induced glucose transport downstream of AMPK because capsaicin increased p38 MAPK phosphorylation significantly and its specific inhibitor SB203580 inhibited capsaicin-mediated glucose uptake. Treatment with an AMPK inhibitor reduced p38 MAPK phosphorylation, but the p38 MAPK inhibitor had no effect on AMPK. Capsaicin stimulated ROS generation in C2C12 muscle cells, and when ROS were captured using the nonspecific antioxidant NAC, the increase in both capsaicin-induced AMPK phosphorylation and capsaicin-induced glucose uptake was attenuated, suggesting that ROS function as an upstream activator of AMPK. Taken together, these results suggest that capsaicin, independent of insulin, increases glucose uptake via ROS generation and consequent AMPK and p38 MAPK activations.     

Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis

Insulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin.     

Possible involvement of heat shock protein 25 in the angiotensin II-induced glomerular mesangial cell contraction via p38 MAP kinase.

In the rat kidney, mesangial cells (MCs), especially those in the extraglomerular mesangium (EGM) region of the juxtagomerular apparatus, express high amounts of heat shock protein 25 (HSP25). Because MCs are contractile in vivo and HSP25 is known to modulate polymerization/depolymerization of F-actin and to be involved in smooth muscle contraction, it is possible that HSP25 participates in the contraction process of MCs. We analyzed a permanent mouse MC line using Northern and Western blot analyses, and observed that similar to the MCs in the glomerulus, these cells also express high amounts of HSP25 constitutively. Exposure of these cells to angiotensin II (ANG II: 2 x 10(-7) M) evoked contraction and a concomitant increase in HSP25 phosphorylation, while the cytoplasmic fraction of HSP25 was transiently reduced. Because phosphorylation of HSP25 is essential for its actin-modulating function, we suppressed the activity of p38 MAP kinase, the major upstream activator of HSP25 phosphorylation, with the specific inhibitor SB 203580. This maneuver reduced HSP25 phosphorylation dramatically, abolished cell contraction, and prevented the decrease of the cytoplasmic HSP25 content. This suggests that HSP25 might be a component of the contraction machinery in MCs and that this process depends on p38 MAP kinase-mediated HSP25 phosphorylation. The decrease of cytoplasmic HSP25 content observed after ANG II exposure is probably the result of a transient redistribution of HSP25 into a buffer-insoluble fraction, because the whole cell content of HSP25 did not change, a phenomenon known to be related to the actin-modulating activity of HSP25. The fact that this function requires phosphorylation of HSP25 would explain the observation that HSP25 does not redistribute in SB 203580-pretreated cells.     

Berberine-stimulated glucose uptake in L6 myotubes involves both AMPK and p38 MAPK

Berberine is a plant alkaloid used in traditional Chinese medicine and has been reported to have antihyperglycemic activity in NIDDM patients. However, the molecular basis for this action is yet to be elucidated. Here we investigate the effects and signaling pathways of berberine on L6 rat skeletal muscles. Our study demonstrates that berberine stimulates glucose uptake in a time- and dose-dependent manner. Intriguingly, berberine-stimulated glucose uptake does not vary as insulin concentration increases, and could not be blocked by the PI 3-kinase inhibitor wortmannin. Berberine only weakly stimulates the phosphorylation of Akt/PKB, a key molecule in the insulin signaling pathway, but strongly promotes the phosphorylation of AMPK and p38 MAPK. The effects of berberine are not a result of pro-oxidant action, but a consequence of an increased cellular AMP:ATP ratio. Moreover, berberine-stimulated glucose uptake is inhibited by the AMPK inhibitor Compound C and the p38 MAPK inhibitor SB202190. Inhibition of AMPK reduces p38 MAPK phosphorylation, suggesting that AMPK lies upstream of p38 MAPK. These results suggest that berberine circumvents insulin signaling pathways and stimulates glucose uptake through the AMP-AMPK-p38 MAPK pathway, which may account for the antihyperglycemic effects of this drug.     

Acute p38 MAPK activation decreases force development in ventricular myocytes

Evidence suggests that p38 mitogen-activated protein kinase (MAPK) activation influences cardiac function on an acute basis. The characterization and mechanisms by which this occurs were investigated in the present study. Adult rat ventricular myocytes treated with 1 mM arsenite for 30 min had a 16-fold increase in p38 MAPK phosphorylation that was attenuated by SB-203580 (a p38 MAPK inhibitor). Extracellular signal-regulated protein kinase (ERK) and c-Jun NH2-terminal kinase (JNK) were also minimally activated, but this activation was not sensitive to SB-203580. In addition, arsenite caused a p38 MAPK-independent translocation/activation of protein phosphatase 2a (PP2a) and decrease in phosphorylation of myosin light chain 2 (LC2). Arsenite-p38 MAPK activation led to translocation of heat shock protein 27 but not alpha B-crystallin to the myofilaments. Using isolated cardiomyocytes, we determined that arsenite reduces isometric tension without a change in Ca2+ sensitivity of tension via p38 MAPK and lowers myofibrillar actomyosin Mg2+-ATPase activity in a p38 MAPK-independent manner. Thus arsenite induces a p38 MAPK-independent change in PP2a and LC2 that may account for the arsenite-dependent decrease in ATPase and a p38 MAPK-dependent modification of the myofilaments that decreases myocardial force development.     

Role of p38 mitogen-activated protein kinase pathway in estrogen-mediated cardioprotection following trauma-hemorrhage

p38 mitogen-activated protein kinase (MAPK) activates a number of heat shock proteins (HSPs), including HSP27 and alpha(B)-crystallin, in response to stress. Activation of HSP27 or alpha(B)-crystallin is known to protect organs/cells by increasing the stability of actin microfilaments. Although our previous studies showed that 17beta-estradiol (E(2)) improves cardiovascular function after trauma-hemorrhage, whether the salutary effects of E(2) under those conditions are mediated via p38 MAPK remains unknown. Male rats (275-325 g body wt) were subjected to soft tissue trauma and hemorrhage (35-40 mmHg mean blood pressure for approximately 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were injected intravenously with vehicle, E(2) (1 mg/kg body wt), E(2) + the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt), or SB-203580 alone, and various parameters were measured 2 h thereafter. Cardiac functions that were depressed after trauma-hemorrhage were returned to normal levels by E(2) administration, and phosphorylation of cardiac p38 MAPK, HSP27, and alpha(B)-crystallin was increased. The E(2)-mediated improvement of cardiac function and increase in p38 MAPK, HSP27, and alpha(B)-crystallin phosphorylation were abolished with coadministration of SB-203580. These results suggest that the salutary effect of E(2) on cardiac function after trauma-hemorrhage is in part mediated via upregulation of p38 MAPK and subsequent phosphorylation of HSP27 and alpha(B)-crystallin.     

Distinct roles of MK2 and MK5 in cAMP/PKA- and stress/p38MAPK-induced heat shock protein 27 phosphorylation

Background

Classical mammalian mitogen-activated protein kinase (MAPK) pathways consist of a cascade of three successive phosphorylation events resulting in the phosphorylation of a variety of substrates, including another class of protein kinases referred to as MAPK-activating protein kinases (MAPKAPKs). The MAPKAPKs MK2, MK3 and MK5 are closely related, but MK2 and MK3 are the major downstream targets of the p38^MAPK pathway, while MK5 can be activated by the atypical MAPK ERK3 and ERK4, protein kinase A (PKA), and maybe p38^MAPK. MK2, MK3, and MK5 can phosphorylate the common substrate small heat shock protein 27 (HSP27), a modification that regulates the role of HSP27 in actin polymerization. Both stress and cAMP elevating stimuli can cause F-actin remodeling, but whereas the in vivo role of p38^MAPK-MK2 in stress-triggered HSP27 phosphorylation and actin reorganization is well established, it is not known whether MK2 is involved in cAMP/PKA-induced F-actin rearrangements. On the other hand, MK5 can phosphorylate HSP27 and cause cytoskeletal changes in a cAMP/PKA-dependent manner, but its role as HSP27 kinase in stress-induced F-actin remodeling is disputed. Therefore, we wanted to investigate the implication of MK2 and MK5 in stress- and PKA-induced HSP27 phosphorylation.

Results

Using HEK293 cells, we show that MK2, MK3, and MK5 are expressed in these cells, but MK3 protein levels are very moderate. Stress- and cAMP-elevating stimuli, as well as ectopic expression of active MKK6 plus p38^MAPK or the catalytic subunit of PKA trigger HSP27 phosphorylation, and specific inhibitors of p38^MAPK and PKA prevent this phosphorylation. Depletion of MK2, but not MK3 and MK5 diminished stress-induced HSP27 phosphorylation, while only knockdown of MK5 reduced PKA-induced phosphoHSP27 levels. Stimulation of the p38^MAPK, but not the PKA pathway, caused activation of MK2.

Conclusion

Our results suggest that in HEK293 cells MK2 is the HSP27 kinase engaged in stress-induced, but not cAMP-induced phosphorylation of HSP27, while MK5 seems to be the sole MK to mediate HSP27 phosphorylation in response to stimulation of the PKA pathway. Thus, despite the same substrate specificity towards HSP27, MK2 and MK5 are implicated in different signaling pathways causing actin reorganization.     

Hsp25 and the p38 MAPK pathway are involved in differentiation of cardiomyocytes

The small heat-shock protein HSP25 is expressed in the heart early during development, and although multiple roles for HSP25 have been proposed, its specific role during development and differentiation is not known. P19 is an embryonal carcinoma cell line which can be induced to differentiate in vitro into either cardiomyocytes or neurons. We have used P19 to examine the role of HSP25 in differentiation. We found that HSP25 expression is strongly increased in P19 cardiomyocytes. Antisense HSP25 expression reduced the extent of cardiomyocyte differentiation and resulted in reduced expression of cardiac actin and the intermediate filament desmin and reduced level of cardiac mRNAs. Thus, HSP25 is necessary for differentiation of P19 into cardiomyocytes. In contrast, P19 neurons did not express HSP25 and antisense HSP25 expression had no effect on neuronal differentiation. The phosphorylation of HSP25 by the p38/SAPK2 pathway is known to be important for certain of its functions. Inhibition of this pathway by the specific inhibitor SB203580 prevented cardiomyocyte differentiation of P19 cells. In contrast, PD90589, which inhibits the ERK1/2 pathway, had no effect. Surprisingly, cardiogenesis was only sensitive to SB203580 during the first 2 days of differentiation, before HSP25 expression increases. In contrast to the effect of antisense HSP25, SB203580 reduced the level of expression of the mesodermal marker Brachyury-T during differentiation. Therefore, we propose that the p38 pathway acts on an essential target during early cardiogenesis. Once this initial step is complete, HSP25 is necessary for the functional differentiation of P19 cardiomyocytes, but its phosphorylation by p38/SAPK2 is not required.     

Absence of a human DnaJ protein hTid-1S correlates with aberrant actin cytoskeleton organization in lesional psoriatic skin

The biochemical mechanism by which the human tumorous imaginal disc1(S) (hTid-1(S)) interferes with actin cytoskeleton organization in keratinocytes of human skin epidermis was investigated. We found that hTid-1, specifically hTid-1(S), interacts with MK5, a p38-regulated/activated protein kinase, and inhibits the protein kinase activity of MK5 that phosphorylates heat shock protein HSP27 in cultured HeLa cells. Thus, hTid-1(S) expression inhibits the phosphorylation of HSP27 known to play important roles in F-actin polymerization and actin cytoskeleton organization. The interplay between MK5/HSP27 signaling and hTid-1(S) expression was supported by the inhibition of HSP27 phosphorylation and MK5 activity in HeLa cells in response to hypoxia during which hTid-1(S) expression was down-regulated. We also found that overexpression of hTid-1(S) results in the inhibition of HSP27 phosphorylation, F-actin polymerization, and actin cytoskeleton organization in transduced HaCaT keratinocytes. This study further proposes that the loss of hTid-1(S) expression in the basal layer of skin epidermis correlates with the enhanced HSP27 phosphorylation, keratinocyte hyperproliferation, and excess actin cytoskeleton organization in lesional psoriatic skin.     

Ketone bodies alter dinitrophenol-induced glucose uptake through AMPK inhibition and oxidative stress generation in adult cardiomyocytes

In aerobic conditions, the heart preferentially oxidizes fatty acids. However, during metabolic stress, glucose becomes the major energy source, and enhanced glucose uptake has a protective effect on heart function and cardiomyocyte survival. Thus abnormal regulation of glucose uptake may contribute to the development of cardiac disease in diabetics. Ketone bodies are often elevated in poorly controlled diabetics and are associated with increased cellular oxidative stress. Thus we sought to determine the effect of the ketone body beta-hydroxybutyrate (OHB) on cardiac glucose uptake during metabolic stress. We used 2,4-dinitrophenol (DNP), an uncoupler of the mitochondrial oxidative chain, to mimic hypoxia in cardiomyocytes. Our data demonstrated that chronic exposure to OHB provoked a concentration-dependent decrease of DNP action, resulting in 56% inhibition of DNP-mediated glucose uptake at 5 mM OHB. This was paralleled by a diminution of DNP-mediated AMP-activated protein kinase (AMPK) and p38 MAPK phosphorylation. Chronic exposure to OHB also increased reactive oxygen species (ROS) production by 1.9-fold compared with control cells. To further understand the role of ROS in OHB action, cardiomyocytes were incubated with H(2)O(2). Our results demonstrated that this treatment diminished DNP-induced glucose uptake without altering activation of the AMPK/p38 MAPK signaling pathway. Incubation with the antioxidant N-acetylcysteine partially restored DNP-mediated glucose but not AMPK/p38 MAPK activation. In conclusion, these results suggest that ketone bodies, through inhibition of the AMPK/p38 MAPK signaling pathway and ROS overproduction, regulate DNP action and thus cardiac glucose uptake. Altered glucose uptake in hyperketonemic states during metabolic stress may contribute to diabetic cardiomyopathy.