(-)-Epigallocatechin gallate attenuates the induction of HSP27 stimulated by sphingosine 1-phosphate via suppression of phosphatidylinositol 3-kinase/Akt pathway in osteoblasts.

We previously reported that sphingosine 1-phosphate stimulates the induction of heat shock protein 27 (HSP27) through p38 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the induction of HSP27 in these cells and its mechanism, since it was previously reported that catechin, including EGCG, suppresses bone resorption. EGCG significantly reduced the induction of HSP27 stimulated by sphingosine 1-phosphate in a dose-dependent manner between 10 and 30 microM. Immunofluorescence microscopy studies revealed that sphingosine 1-phosphate certainly stimulated the induction of HSP27 in the cytosol of these cells, and that EGCG clearly suppressed its induction. However, sphingosine 1-phosphate-stimulated phosphorylation of p38 MAP kinase or MAPKAP-2 was not affected by EGCG. By contrast, EGCG markedly suppressed the phosphorylations of both Akt and glycogen synthase kinase-3beta stimulated by sphingosine 1-phosphate. These results strongly suggest that EGCG suppresses the induction of HSP27 stimulated by sphingosine 1-phosphate via attenuation of, not the p38 MAP kinase pathway, but of the phosphatidylinositol 3-kinase/Akt pathway in osteoblasts.

MAPKAPK-2 modulates p38-MAPK localization and small heat shock protein phosphorylation but does not mediate the injury associated with p38-MAPK activation during myocardial ischemia.

MAPKAPK-2 (MK2) is a protein kinase activated downstream of p38-MAPK which phosphorylates the small heat shock proteins HSP27 and alphaB crystallin and modulates p38-MAPK cellular distribution. p38-MAPK activation is thought to contribute to myocardial ischemic injury; therefore, we investigated MK2 effects on ischemic injury and p38 cellular localization using MK2-deficient mice (KO). Immunoblotting of extracts from Langendorff-perfused hearts subjected to aerobic perfusion or global ischemia or reperfusion showed that the total and phosphorylated p38 levels were significantly lower in MK2(-/-) compared to MK2(+/+) hearts at baseline, but the ratio of phosphorylated/total p38 was similar. These results were confirmed by cellular fractionation and immunoblotting for both cytosolic and nuclear compartments. Furthermore, HSP27 and alphaB crsytallin phosphorylation were reduced to baseline in MK2(-/-) hearts. On semiquantitative immunofluorescence laser confocal microscopy of hearts during aerobic perfusion, the mean total p38 fluorescence was significantly higher in the nuclear compared to extranuclear (cytoplasmic, sarcomeric, and sarcolemmal compartments) in MK2(+/+) hearts. However, although the increase in phosphorylated p38 fluorescence intensity in all compartments following ischemia in MK2(+/+) hearts was lost in MK2(-/-) hearts, it was basally elevated in nuclei of MK2(-/-) hearts and was similar to that seen during ischemia in MK2(+/+) hearts. Despite these differences, similar infarct volumes were recorded in wild-type MK2(+/+) and MK2(-/-) hearts, which were decreased by the p38 inhibitor SB203580 (1 microM) in both genotypes. In conclusion, p38 MAPK-induced myocardial ischemic injury is not modulated by MK2. However, the absence of MK2 perturbs the cellular distribution of p38. The preserved nuclear distribution of active p38 MAPK in MK2(-/-) hearts and the conserved response to SB203580 suggests that activation of p38 MAPK may contribute to injury independently of MK2.

Phosphorylated heat shock protein 27 represses growth of hepatocellular carcinoma via inhibition of extracellular signal-regulated kinase.

Heat shock protein 27, one of the low molecular weight stress proteins, is recognized as a molecular chaperone; however, other functions have not yet been well established. Phosphorylated heat shock protein 27 levels inversely correlate with the progression of human hepatocellular carcinoma. This study shows that phosphorylated heat shock protein 27 interferes with cell growth of the hepatocellular carcinoma-derived HuH7 cells in the presence of the proinflammatory cytokine, tumor necrosis factor-alpha, via inhibition of the sustained activation of the extracellular signal-regulated kinase signal pathway. The activities of Raf/extracellular signal-regulated kinase and subsequent activator protein-1 transactivation and the induction levels of cyclin D1 were lower in HuH7 cells transfected with phosphorylated heat shock protein 27 than those with unphosphorylated heat shock protein 27. Moreover, phosphorylated heat shock protein 27 up-regulated the levels of p38 mitogen-activated protein kinase and mitogen-activated protein kinase phosphatase-1, an inhibitory protein of extracellular signal-regulated kinase. These results indicate that phosphorylated heat shock protein 27 might suppress the extracellular signal-regulated kinase activity in the hepatocellular carcinoma cells via two separate pathways in an inflammatory state. The extracellular signal-regulated kinase activity is inversely correlated with phosphorylated heat shock protein 27 at serine 15 and also in human hepatocellular carcinoma tissues in vivo. Because the extracellular signal-regulated kinase signal pathway is a major proliferation signal of hepatocellular carcinoma, activator protein-1 activation is an early event in hepatocarcinogenesis. These findings strongly suggest that the control of the phosphorylated heat shock protein 27 levels could be a new therapeutic strategy especially to counter the recurrence of hepatocellular carcinoma.

Alpha2 adrenoreceptor agonist regulates protein kinase C-induced heat shock protein 27 phosphorylation in C6 glioma cells.

Dexmedetomidine (Dexmd), a potent and highly specific alpha(2) adrenoreceptor agonist, is an efficient therapeutic agent for sedation. Dexmd has been recently reported to have a neuroprotective effect. Heat shock protein (HSP) 27, a low-molecular weight HSP has been shown to be expressed following cerebral ischemia in astrocytes but not in neurons. HSP27 expression is involved in ischemic tolerance of the brain. This study investigated the effect of Dexmd on HSP27 in rat C6 glioma cells. 12-O-tetradecanoylphorbol-13-actate (TPA), a direct activator of protein kinase C (PKC), stimulated the phosphorylation of HSP27 at Ser82, but not Ser15 in a time-dependent manner. Prostaglandin (PG) E(1) or PGE(2) which activates the adenylyl cyclase-cAMP system as well as forskolin and dibutyryl-cAMP, suppressed the TPA-induced phosphorylation of HSP27. Dexmd reversed the suppression of HSP27 phosphorylation by the adenylyl cyclase-cAMP system. Therefore, these results strongly suggest that Dexmd reverses the suppression of HSP27 phosphorylation by the adenylyl cyclase-cAMP system activation through the inhibition of its system in C6 cells. alpha(2) Adrenoreceptor agonists may therefore show a neuroprotective effect through the modification of HSP27 phosphorylation induced by PKC activation.

(-)-Epigallocatechin gallate reduces transforming growth factor beta-stimulated HSP27 induction through the suppression of stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts.

We previously reported that transforming growth factor-beta (TGF-beta) stimulates heat shock protein 27 (HSP27) induction through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the TGF-beta-stimulated induction of HSP27 in these cells, and its underlying mechanism. EGCG significantly suppressed the HSP27 induction stimulated by TGF-beta in a dose-dependent manner between 10 and 30 microM without affecting the HSP70 levels. TGF-beta with or without EGCG did not affect the advanced oxidation protein products. The TGF-beta-induced phosphorylation of p38 MAP kinase and ERK1/2 was not affected by EGCG. SP600125, a specific inhibitor of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), markedly reduced the HSP27 expression induced by TGF-beta. EGCG significantly suppressed the TGF-beta-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of Smad2. EGCG attenuated the phosphorylation of both MKK4 and TAK1 induced by TGF-beta. These results strongly suggest that EGCG suppresses the TGF-beta-stimulated induction of HSP27 via the attenuation of the SAPK/JNK pathway in osteoblasts, and that this effect is exerted at a point upstream from TAK1.

(-)-epigallocatechin gallate inhibits prostaglandin D2-stimulated HSP27 induction via suppression of the p44/p42 MAP kinase pathway in osteoblasts.

We previously reported that prostaglandin D2 (PGD2) stimulates heat shock protein 27 (HSP27) induction through p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the induction of HSP27 in these cells and the mechanism. EGCG significantly reduced the HSP27 induction stimulated by PGD2 without affecting the levels of HSP70. The PGD2-induced phosphorylation of p38 MAP kinase or SAPK/JNK was not affected by EGCG. On the contrary, EGCG markedly suppressed the PGD2-induced phosphorylation of p44/p42 MAP kinase and MEK1/2. However, the PGD2-induced phosphorylation of Raf-1 was not inhibited by EGCG. These results strongly suggest that EGCG suppresses the PGD2-stimulated induction of HSP27 at the point between Raf-1 and MEK1/2 in osteoblasts.

Expression levels of heat shock protein 20 decrease in parallel with tumor progression in patients with hepatocellular carcinoma.

Heat shock protein (HSP) 20, a low-molecular-weight HSP, is constitutively expressed in various tissues, such as smooth muscle, skeletal muscle, and liver. However, the characteristics and function of HSP20 have not been precisely understood. In the present study, we investigated correlations of expression levels of HSP20 in hepatocellular carcinoma (HCC) tissues and the surrounding tissues with clinical and pathologic characteristics in 53 resected HCC specimens. Although HSP20 was detected in all 53 HCC tissues, the expression levels were reduced compared with those in the adjacent non-tumor tissues. The expression levels of HSP20 were inversely correlated with tumor stage by TNM classification (p<0.01), presence of microvascular invasion (p<0.05), and tumor size (p<0.05). Our findings strongly suggest that HSP20 may play a role against the progression of human HCC.

Expression of smooth muscle cell-specific proteins in neural progenitor cells induced by agonists of G protein-coupled receptors and transforming growth factor-beta.

Neural progenitor cells isolated from the embryonic cerebral cortex are well known to differentiate into neurons and glial cells, but recent reports have demonstrated differentiation into smooth muscle cells (SMCs) under the influence of fetal bovine serum. In this study, we report that agonists for G protein-coupled receptors (GPCRs), including endothelin, lysophosphatidic acid and carbachol, effectively promote the expression of SMC-specific proteins in the presence of transforming growth factor-beta (TGF-beta). Incubation of neural progenitor cells with agonists for GPCRs or TGF-beta alone induced the expression of an SMC-specific protein, alpha-smooth muscle actin (SMA), and their combination resulted in incremental increase. Stimulation with combinations of each GPCR agonist and TGF-beta increased the numbers of large, flat cells with thick actin fibers and also caused expression of other SMC marker proteins. Endothelin and TGF-beta enhanced SMA promoter-luciferase reporter activity at different times after stimulation. The mutation of TGF-beta control element of SMA promoter constructs decreased TGF-beta-enhanced luciferase activity but not endothelin-stimulated activity. Transfection of active forms of RhoA and its effector, mDia, strongly enhanced SMA promoter activity, and a dominant negative form of RhoA inhibited endothelin-stimulated promoter activity but not TGF-beta-stimulated activity. Whereas endothelin consistently activated RhoA, TGF-beta did not, and a specific inhibitor of TGF-beta type I receptor blocked TGF-beta-enhanced SMA promoter activity, suggesting involvement of Smad phosphorylation. These results suggest that separate signaling pathways of G protein and TGF-beta cooperatively promote the expression of SMC-specific proteins in neural progenitor cells.

TNF-alpha decreases hsp 27 in human blood mononuclear cells: involvement of protein kinase c.

Treatment of PBMCs with TNF-alpha decreased the levels of heat shock protein (HSP) 27, but had little effect on the level of HSP70. Parallel to the decrease of HSP27, TNF-alpha increased the level of HSP27 in the incubation medium of the cells. The decrease of HSP27 induced by TNF-alpha was suppressed by the pretreatment of PBMCs with the specific protein kinase C (PKC) inhibitor, GF109203X. Furthermore, phorbol myristate acetate (PMA), a PKC stimulant, but not dibutyryl cyclic AMP, a protein kinase A stimulant, decreased the levels of HSP27. To investigate the effect of TNF-alpha on the oligomerization state of HSP27 in PBMCs, we performed sucrose density gradient centrifugation with subsequent fractionation and immunoassay. Extract of vehicle-treated PBMCs contained mainly dissociated forms of HSP27. The amounts of dissociated forms of HSP27 in PBMCs was decreased by TNF-alpha, while the amounts of aggregated form of HSP27 was little changed. In intact PBMCs, HSP27 is constitutively phosphorylated at Ser78, but not at Ser15 or at Ser82. The amount of phosphorylated HSP27 at Ser78 was decreased by TNF-alpha. These results indicate that TNF-alpha reduces HSP27 in PBMCs through PKC activation. This decrease may be due to efflux of dissociated form of HSP27, phosphorylated HSP27 at Ser78, from the cells.

Cell signaling pathways to alphaB-crystallin following stresses of the cytoskeleton.

Small heat shock proteins (sHSPs) act as chaperone, but also in protecting the different cytoskeletal components. Recent results suggest that alphaB-crystallin, a member of sHSPs family, might regulate actin filament dynamics, stabilize them in a phosphorylation dependent manner, and protect the integrity of intermediate filaments (IF) against extracellular stress. We demonstrate that vinblastin and cytochalasin D, which respectively disorganize microtubules and actin microfilaments, trigger the activation of the p38/MAPKAP2 kinase pathway and lead to the specific alphaB-crystallin phosphorylation at serine 59. Upstream of p38, we found that RhoK, PKC and PKA are selectively involved in the activation of p38 and phosphorylation of alphaB-crystallin, depending on the cytoskeletal network disorganized. Moreover, we demonstrate that chronic perturbations of IF network result in the same activation of p38 MAPK and alphaB-crystallin phosphorylation, as with severe disorganization of other cytoskeletal networks. Finally, we also show that Ser 59 phosphorylated alphaB-crystallin colocalizes with cytoskeletal components. Thus, disturbance of cytoskeleton leads by converging signaling pathways to the phosphorylation of alphaB-crystallin, which probably acts as a protective effector of the cytoskeleton.

Prostaglandin D2 induces the phosphorylation of HSP27 in osteoblasts: function of the MAP kinase superfamily.

We previously reported that prostaglandin D(2) (PGD(2)) stimulates the induction of heat shock protein 27 (HSP27) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether PGD(2) stimulates the phosphorylation of HSP27 in MC3T3-E1 cells exposed to heat shock. In the cultured MC3T3-E1 cells, PGD(2) markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85 in a time-dependent manner. Among the mitogen-activated protein (MAP) kinase superfamily, p44/p42 MAP kinase and p38 MAP kinase were phosphorylated by PGD(2) which had little effect on the phosphorylation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). The PGD(2)-induced phosphorylation of HSP27 was attenuated by PD169316, an inhibitor of p38 MAP kinase or PD98059, a MEK inhibitor. SP600125, a SAPK/JNK inhibitor did not affect the HSP27 phosphorylation. In addition, PD169316 suppressed the PGD(2)-induced phosphorylation of MAPKAP kinase 2. These results strongly suggest that PGD(2) stimulates HSP27 phosphorylation via p44/p42 MAP kinase and p38 MAP kinase but not SAPK/JNK in osteoblasts.

Cu/Zn- and Mn-superoxide dismutase distribution and concentration in adrenal tumors.

The tissue distribution of Cu/Zn- and Mn-superoxide dismutases (SOD) in adrenal tumors was studied by an immunohistochemical technique, and the concentrations of both SODs were measured by a sensitive sandwich enzyme immunoassay technique. In the normal adrenal gland, both Cu/Zn- and Mn-SODs were localized predominantly in the reticular zone of the cortex. Cu/Zn-SOD was stained clearly in the inner fascicular zone of the cortex, but not in the medulla, whereas Mn-SOD was stained weakly in the medulla. In different adrenal tumors, the localization of both stained SODs reflected the origin of the tumor cell. Thus, in one section of a pheochromocytoma only Mn-SOD was stained clearly. The concentrations of both SODs in the tissues of medullary tumors were lower than those in the normal adrenal gland and adrenocortical adenomas. The concentration of Cu/Zn-SOD in the tumor tissue of Cushing's syndrome adenoma was higher, and that of Mn-SOD was lower than the concentrations in the normal adrenal gland. The ratio of the tissue concentrations of Mn-SOD to Cu/Zn-SOD was lower in adrenal medullary tumors and Cushing's syndrome adenomas than in the normal adrenal gland and primary aldosteronism adenomas, indicating the predominance of Cu/Zn-SOD in the former, and Mn-SOD in the latter. These data suggest that the localization of Cu/Zn- and Mn-SODs in adrenal tissues reflects the specificity of the adrenal cells that produce the tissue-specific hormones. An investigation of changes in these enzymes in adrenal tumors may also provide useful information on adrenal tumor cell differentiation.

Phosphatidylinositol 3-kinase/Akt plays a role in sphingosine 1-phosphate-stimulated HSP27 induction in osteoblasts.

We previously reported that p38 mitogen-activated protein (MAP) kinase plays a part in sphingosine 1-phosphate-stimulated heat shock protein 27 (HSP27) induction in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is involved in the induction of HSP27 in these cells. Sphingosine 1-phosphate time dependently induced the phosphorylation of Akt. Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, reduced the HSP27 induction stimulated by sphingosine 1-phosphate. The sphingosine 1-phosphate-induced phosphorylation of GSK-3beta was suppressed by Akt inhibitor. The sphingosine 1-phosphate-induced HSP27 levels were attenuated by LY294002 or wortmannin, PI3K inhibitors. Furthermore, LY294002 or Akt inhibitor did not affect the sphingosine 1-phosphate-induced phosphorylation of p38 MAP kinase and SB203580, a p38 MAP kinase inhibitor, had little effect on the phosphorylation of Akt. These results suggest that PI3K/Akt plays a part in the sphingosine 1-phosphate-stimulated induction of HSP27, maybe independently of p38 MAP kinase, in osteoblasts.

Plasminogen/plasmin regulates alpha-enolase expression through the MEK/ERK pathway.

Previously we have demonstrated that both plasminogen (Plg) and plasmin (Pla) regulate the expression of the transcription factors c-FOS and EGR-1 [L.P. De Sousa, B.S. Brasil, B.M. Silva, M.H. Freitas, S.V. Nogueira, P.C. Ferreira, E.G. Kroon, C.A. Bonjardim, Plasminogen/plasmin regulates c-fos and egr-1 expression via the MEK/ERK pathway, Biochem. Biophys. Res. Commun. 329 (2005) 237-245]. Here we show that Plg activates the mitogen-activated protein kinases MEK and ERK which leads to alpha-enolase (alpha-ENO) gene expression not only in fibroblasts, but also in peripheral blood mononuclear cells. The alpha-ENO mRNA accumulation was apparent three hours post-Plg treatment and remained elevated out to 28h, a process that seems to require both de novo protein synthesis and active gene transcription. Pla mimics Plg-stimulated alpha-ENO expression through its serine protease activity, suggesting that conversion of Plg to active Pla is required. Pharmacological and genetic blockade of MEK caused inhibition of alpha-ENO mRNA accumulation, implicating MEK/ERK as the transduction pathway that leads to alpha-ENO expression upon Plg stimulation. Furthermore, Plg stimulated DNA binding activity of the transcription factors activator-protein 1 and early growth response gene-1 to their cognate regulatory sequences at alpha-ENO promoter. Altogether, our data show that Plg/Pla regulates alpha-ENO expression through the MEK/ERK pathway.

Attenuated phosphorylation of heat shock protein 27 correlates with tumor progression in patients with hepatocellular carcinoma.

Heat shock protein 27 (HSP27) is expressed at high levels in human hepatocellular carcinoma (HCC). We examined correlations of total HSP27 and serine phosphorylated (Ser-15, Ser-78, and Ser-82) HSP27 levels in HCC tissues with clinical and pathologic characteristics in 48 resected HCC specimens. The levels of total and Ser-phosphorylated HSP27 were evaluated by Western blot analysis. Immunohistochemical analysis of HSP27 expression was also performed on some samples. Phosphorylation of HSP27 was detected in all 48 HCC tissues. Levels of phosphorylated HSP27 were correlated inversely with tumor size, microvascular invasion of HCC, and tumor stage by TNM classification. In contrast, only microvascular invasion showed an inverse correlation with total HSP27 levels. The decrease in phosphorylated HSP27 in progressed HCC was also observed by immunohistochemistry. Levels of phosphorylated HSP27 gradually decreased in parallel with HCC progression. Our findings suggest that phosphorylated HSP27 may have a suppressive role in progression of human HCC.

Nuclear import of {alpha}B-crystallin is phosphorylation-dependent and hampered by hyperphosphorylation of the myopathy-related mutant R120G.

Phosphorylation modulates the functioning of alphaB-crystallin as a molecular chaperone. We here explore the role of phosphorylation in the nuclear import and cellular localization of alphaB-crystallin in HeLa cells. Inhibition of nuclear export demonstrated that phosphorylation of alphaB-crystallin is required for import into the nucleus. As revealed by mutant analysis, phosphorylation at Ser-59 is crucial for nuclear import, and phosphorylation at Ser-45 is required for speckle localization. Co-immunoprecipitation experiments suggested that the import of alphaB-crystallin is possibly regulated by its phosphorylation-dependent interaction with the survival motor neuron (SMN) protein, an important factor in small nuclear ribonucleoprotein nuclear import and assembly. This interaction was supported by co-localization of endogenous phosphorylated alphaB-crystallin with SMN in nuclear structures. The cardiomyopathy-causing alphaB-crystallin mutant R120G was found to be excessively phosphorylated, which disturbed SMN interaction and nuclear import, and resulted in the formation of cytoplasmic inclusions. Like for other protein aggregation disorders, hyperphosphorylation appears as an important aspect of the pathogenicity of alphaB-crystallin R120G.

Midazolam suppresses thrombin-induced heat shock protein 27 phosphorylation through inhibition of p38 mitogen-activated protein kinase in cardiac myocytes.

It has been shown that anesthetics have effects of cardiac preconditioning. Heat shock proteins (HSPs) function as molecular chaperone. Among them, HSP27, a low-molecular-weight HSP, abundantly exist in heart. However, the relationship between anesthetics and HSP27 in heart is not yet clarified. We investigated whether thrombin induces or phosphorylates HSP27 in primary cultured mouse myocytes and the effect of midazolam on the thrombin-stimulated HSP27 phosphorylation and the mechanism behind it. Thrombin time dependently phosphorylated HSP27 at Ser-15 and Ser-85 while having no effect on the levels of HSP27. Midazolam markedly suppressed the thrombin-induced phosphorylation of HSP27 at both Ser-15 and Ser-85. Thrombin induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase without affecting stress-activated protein kinase/c-Jun N-terminal kinase. In addition, midazolam attenuated the phosphorylation of thrombin-induced p38 MAP kinase but not that of p44/p42 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the thrombin-induced phosphorylation of HSP27 at both Ser-15 and Ser-85. These results strongly suggest that thrombin induces the HSP27 phosphorylation at least through the p38 MAP kinase activation in cardiac myocytes and that midazolam inhibits the thrombin-induced HSP27 phosphorylation via suppression of p38 MAP kinase activation.

Characterization of alpha-enolase as an interferon-alpha 2 alpha 1 regulated gene.

Interferons (IFNs) are multifunctional cytokines that after binding to the cell surface receptor induce the expression of a large number of genes, which in turn, mediate many biological processes including host defense, cell growth control, signaling, and metabolism. Here we show that IFN-alpha activates the mitogen-activated protein kinases (MAPK) ERK1/2 and the transcription factor CREB/ATF-1, which lead to the alpha-enolase (alpha-ENO) gene expression in fibroblasts. Alpha-ENO mRNA accumulation was apparent 6 h post-IFN stimulation and required both de novo protein synthesis and active gene transcription, which is typical of a secondary response gene. Alpha-ENO expression does not appear to be restricted to fibroblasts, since it was equally verified in peripheral blood mononuclear cells (PBMC). Furthermore, IFN-alpha stimulates the expression of the primary response genes c-fos and egr-1, which was followed by an increase in DNA binding activity of c-FOS and EGR-1 proteins, as verified by shift assays using the cis-acting elements AP-1 and EGR-1 localized at the alpha-ENO promoter. Finally, we also demonstrated that IFN treatment of PBMC cause an increase in both, alpha-ENO expression on the cell surface and plasmin generation followed addition of exogenous plasminogen.

Hsp27-2D-gel electrophoresis is a diagnostic tool to differentiate primary desminopathies from myofibrillar myopathies.

Small heat shock proteins prevent abnormal protein folding and accumulation. We analyzed the expression of hsp27 and alphaB-crystallin in skeletal muscle specimens of patients with desminopathies, plectinopathies, myotilinopathy, and other myofibrillar myopathies by means of differential centrifugation, 2D-gel electrophoresis, Western blotting, and mass spectrometry. Hsp27-P82 and -P15 as well as alphaB-crystallin-P59 and -P45 are the major serine phosphorylation isoforms in normal and diseased human skeletal muscle. 2D-gel-electrophoresis revealed spots of hsp27 in a range of pH 5.3-6.4 in samples of all skeletal muscle specimens, except for the seven desminopathies. They indicated a shift of the main hsp27-spot to alkaline pH degrees, which may help to differentiate primary desminopathies from other myopathies with structural pathology of the desmin cytoskeleton.

alphaB-crystallin is phosphorylated during myocardial infarction: involvement of platelet-derived growth factor-BB.

alphaB-crystallin is the most abundant low-molecular-weight heat shock protein in heart and recent studies have demonstrated that it plays a cardioprotective role during myocardial infarction both in vivo and in vitro. On the other hand, platelet-derived growth factor (PDGF), a potent serum mitogen, has been reported to improve cardiac function after myocardial infarction. In the present study, using a mouse myocardial infarction model, we investigated whether alphaB-crystallin is phosphorylated during myocardial infarction and the implication of PDGF-BB. Phosphorylation of alphaB-crystallin at Ser-59 was time dependently induced and plasma PDGF-BB levels were concomitantly increased. Moreover, PDGF-BB-stimulated phosphorylation of alphaB-crystallin was suppressed by SB203580, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, in primary cultured cardiac myocytes. Our results indicate that PDGF-BB induces phosphorylation of alphaB-crystallin via p38 MAP kinase during myocardial infarction.