Protein kinase C activation inhibits stress-induced synthesis of heat shock protein 27 in osteoblast-like cells: Function of arachidonic acid

Exposure of osteoblast-like MC3T3-E1 cells to sodium arsenite (arsenite) increased the level of heat shock protein 27 (hsp27). The effect of arsenite was dose-dependent in the range of 50 to 200 μM. Arsenite also stimulated arachidonic acid release dose-dependently in the range between 50 and 200 μM in these cells. Both indomethacin, an inhibitor of cyclooxygenase, and nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly enhanced the arsenite-induced accumulation of hsp27. Melittin, an activator of phospholipase A₂, significantly enhanced the arsenite-induced accumulation of hsp27. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, inhibited the arsenite-induced accumulation of hsp27. In contrast, 4α-phorbol 12, 13-didecanoate (4α-PDD), a PKC-nonactivating phorbol ester, had little effect. TPA suppressed the arsenite-induced arachidonic acid release, but 4α-PDD had little effect. Arsenite no longer affected cAMP accumulation, inositol phosphates formation nor the formation of choline and phosphocholine in these cells. These results suggest that the response to stress of hsp27 is coupled with the metabolic activity of the arachidonic acid cascade, and the activation of PKC inhibits the induction of hsp27 through the suppression of arachidonic acid release in osteoblast-like cells. © 1996 Wiley-Liss, Inc.     

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.     

Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma

We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.     

Activation of protein kinase B (Akt/RAC-protein kinase) by cellular stress and its association with heat shock protein Hsp27

Protein kinase B (PKB, also named as Akt or RAC-protein kinase), that is activated by cellular stress such as heat shock and hyperosmotic treatment, was revealed to be activated by oxidative stress and by chemical stressors of CdCl₂ and NaAsO₂ by measuring the activity of the enzyme immunoprecipitated from the transfected COS-7 cells. Upon stress treatment, a 30-kDa phosphoprotein was co-immunoprecipitated with PKB from the cells metabolic labeled with [³²P]orthophosphate. The phosphoprotein was identified as Hsp27, a small heat shock protein, by immunoblot analysis and co-immunoprecipitation. The association of Hsp27 was specific to PKB as the heat shock protein was not co-immunoprecipitated with other protein kinases such as protein kinase C and PKN. When the cells were treated with H₂O₂, PKB was activated gradually and the association of Hsp27 with PKB increased concurrently with the enhancement of PKB activity. In heat-shocked cells, activation of PKB and the association of Hsp27 were detected immediately after the treatment, and the association of the heat shock protein decreased while PKB kept stimulated activity when the cells were further incubated at 37°C. These results suggest that Hsp27 is involved in the activation process of PKB in the signal transduction pathway of various forms of stress.     

Specific induction of heat shock protein 27 by glucocorticoid in osteoblasts

It is generally recognized that osteoporosis is a common complication of patients with glucocorticoid excess and that glucocorticoid receptor is associated with heat shock protein (HSP) 70 and HSP90 in a heterocomplex. In the present study, we investigated whether glucocorticoid induces HSP27, HSP70, and HSP90 in osteoblast-like MC3T3-E1 cells. Dexamethasone time-dependently increased the levels of HSP27, while having no effect on the levels of HSP70 or HSP90. The effect of dexamethasone was dose-dependent in the range between 0.1 nM and 0.1 microM. Dexamethasone induced an increase of the levels of mRNA for HSP27. Dexamethasone induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the HSP27 accumulation by dexamethasone. In addition, SB203580 reduced the dexamethasone-stimulated increase of the mRNA levels for HSP27. The dexamethasone-induced phosphorylation of p38 MAP kinase was reduced by SB203580. These results strongly suggest that glucocorticoid stimulates the induction of neither HSP70 nor HSP90, but HSP27 in osteoblasts, and that p38 MAP kinase is involved in the induction of HSP27.     

Constitutive phosphorylation by protein kinase C regulates D1 dopamine receptor signaling

The D(1) dopamine receptor (D(1) DAR) is robustly phosphorylated by multiple protein kinases, yet the phosphorylation sites and functional consequences of these modifications are not fully understood. Here, we report that the D(1) DAR is phosphorylated by protein kinase C (PKC) in the absence of agonist stimulation. Phosphorylation of the D(1) DAR by PKC is constitutive in nature, can be induced by phorbol ester treatment or through activation of Gq-mediated signal transduction pathways, and is abolished by PKC inhibitors. We demonstrate that most, but not all, isoforms of PKC are capable of phosphorylating the receptor. To directly assess the functional role of PKC phosphorylation of the D(1) DAR, a site-directed mutagenesis approach was used to identify the PKC sites within the receptor. Five serine residues were found to mediate the PKC phosphorylation. Replacement of these residues had no effect on D(1) DAR expression or agonist-induced desensitization; however, G protein coupling and cAMP accumulation were significantly enhanced in PKC-null D(1) DAR. Thus, constitutive or heterologous PKC phosphorylation of the D(1) DAR dampens dopamine activation of the receptor, most likely occurring in a context-specific manner, mediated by the repertoire of PKC isozymes within the cell.     

Adenylyl cyclase-cAMP system inhibits thrombin-induced HSP27 in vascular smooth muscle cells

We previously reported that thrombin stimulates the induction of heat shock protein (HSP) 27 via p38 mitogen-activated protein (MAP) kinase activation in aortic smooth muscle A10 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on the thrombin-stimulated induction of HSP27 in A10 cells. Forskolin, a direct activator of adenylyl cyclase, reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the HSP27 accumulation. Furthermore, dibutyryl-cAMP (DBcAMP), a permeable analog of cAMP, significantly suppressed the accumulation of HSP27. On the other hand, calphostin C, an inhibitor of protein kinase C (PKC), reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. Moreover, forskolin reduced the p38 MAP kinase phosphorylation induced by the 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, and significantly suppressed the TPA-stimulated accumulation of HSP27. These results indicate that adenylyl cyclase-cAMP system has an inhibitory role in thrombin-stimulated HSP27 induction in aortic smooth muscle cells, and the effect seems to be exerted on the thrombin-induced PKC- p38 MAP kinase signaling pathway.     

Effect of methylglyoxal modification and phosphorylation on the chaperone and anti-apoptotic properties of heat shock protein 27

Heat shock protein 27 (Hsp27) is a stress-inducible protein in cells that functions as a molecular chaperone and also as an anti-apoptotic protein. Methylglyoxal (MGO) is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine. We found considerable amount of Hsp27 in phosphorylated form (pHsp27) in human cataractous lenses. pHsp27 was the major argpyrimidine-modified protein in brunescent cataractous lenses. Modification by MGO enhanced the chaperone function of both pHsp27 and native Hsp27, but the effect on Hsp27 was at least three-times greater than on pHsp27. Phosphorylation of Hsp27 abolished its chaperone function. Transfer of Hsp27 using a cationic lipid inhibited staurosporine (SP)-induced apoptotic cell death by 53% in a human lens epithelial cell line (HLE B-3). MGO-modified Hsp27 had an even greater effect (62% inhibition). SP-induced reactive oxygen species in HLE-B3 cells was significantly lower in cells transferred with MGO-modified Hsp27 when compared to native Hsp27. In vitro incubation experiments showed that MGO-modified Hsp27 reduced the activity of caspase-9, and MGO-modified pHsp27 reduced activities of both caspase-9 and caspase-3. Based on these results, we propose that Hsp27 becomes a better anti-apoptotic protein after modification by MGO, which may be due to multiple mechanisms that include enhancement of chaperone function, reduction in oxidative stress, and inhibition of activity of caspases. Our results suggest that MGO modification and phosphorylation of Hsp27 may have important consequences for lens transparency and cataract development.     

热休克蛋白27在肿瘤中的研究进展

热休克蛋白27（HSP27）作为热应激蛋白表达于各种生理或是环境损伤之后,保护细胞生存,其具有多种功能包括：分子伴侣,抗凋亡,参与细胞运动等。近年来发现HSP27在多种肿瘤中过度表达,参与肿瘤的发生发展,分化,耐药以及转移等方面,因而抑制HSP27成为一种新的肿瘤治疗策略。本文就相关研究进展进行综述。     

Serum heat shock protein 27 levels in patients with hepatocellular carcinoma

Levels of serum heat shock protein 27 (sHsp27) have been studied in numerous cancer types, but their potential relevance in patients with hepatocellular carcinoma (HCC) is undetermined. Our aim was to compare sHsp27 levels in patients with HCC and HCC-free controls. Specifically, we recruited 71 patients with HCC (80 % with early tumour), 80 patients with chronic liver disease (59 with liver cirrhosis and 21 with chronic active hepatitis) and 42 healthy subjects. sHsp27 was measured by immunoenzymatic assay. Results showed that sHsp27 levels were significantly (p < 0.001) higher in patients with HCC than in the other groups, particularly in those with hepatitis C virus (HCV)-related disease. In HCC patients, sHsp27 levels were not associated with prognostic risk factors, such as size/multiplicity of nodules and stage. In logistic regression analysis, performed in patients with liver disease, log-sHsp27 was associated with a significant age-adjusted 2.5-fold increased odds ratio of HCC and with a significant 4.4-fold higher odds ratio of HCC in the subgroup with HCV-related liver disease. In receiver operating characteristic curve analysis, sensitivity and specificity of the best sHsp27 cut-off value (456.5 pg/ml) for differentiating patients with HCC from those with HCC-free chronic liver disease were 70 and 73 %, respectively. In conclusion, sHsp27 levels are enhanced in patients with HCC and may represent a candidate biomarker of HCC.     

热休克蛋白27 在肿瘤中的研究进展

热休克蛋白27(HSP27)作为热应激蛋白表达于各种生理或是环境损伤之后,保护细胞生存,其具有多种功能包括:分子伴侣,抗凋亡,参与细胞运动等。近年来发现HSP27在多种肿瘤中过度表达,参与肿瘤的发生发展,分化,耐药以及转移等方面,因而抑制HSP27成为一种新的肿瘤治疗策略。本文就相关研究进展进行综述。     

Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

Mitogenic signaling via endogenous kappa-opioid receptors in C6 glioma cells: evidence for the involvement of protein kinase C and the mitogen-activated protein kinase signaling cascade

As reports on G protein-coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous kappa-opioid receptors in C6 glioma cells, an astrocytic model system. We find that the kappa-opioid receptor-selective agonist U69,593 stimulates phospholipase C activity, extracellular signal-regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593-stimulated extracellular signal-regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin-sensitive G proteins, L-type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen-activated protein or extracellular signal-regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant-negative or sequestering mutants, we provide evidence that extracellular signal-regulated kinase 1/2 phosphorylation is Ras-dependent and transduced by Gbetagamma subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous kappa-opioid receptor in C6 glioma cells.     

Modulation of protein phosphorylation and stress protein expression by okadaic acid on heat shock cells

We have demonstrated that pretreatment but not post-treatment with okadaic acid (OA) can aggravate cytotoxicity as well as alter the kinetics of stress protein expression and protein phosphorylation in heat shocked cells. Compared to heat shock, cells recovering from 1 hr pretreatment of OA at 200 nM and cotreated with heat shock at 45°C for the last 15 min of incubation (OA→HS treatment) exhibited enhanced induction of heat shock proteins (HSPs) 70 and 110. In addition to enhanced expression, the attenuation of HSC70 and HSP90 after the induction peaks was also delayed in OA→HS-treated cells. The above treatment also resulted in the rapid induction of the 78 kDa glucose-regulated protein (GRP78), which expression remained constant in cells recovering from treatment with 200 nM OA for 1 hr, heat shocked at 45°C for 15 min, or in combined treatment in reversed order (HS→OA treatment). Enhanced phosphorylation of vimentin and proteins with molecular weights of 65, 40, and 33 kDa and decreased phosphorylation of a protein with a molecular weight of 29 kDa were also observed in cells recovering from OA→HS treatment. Again, protein phosphorylation in cells recovering from HS→OA treatment did not differ from those in cells treated only with heat shock. Since the alteration in the kinetics of stress protein expression and protein phosphorylation was tightly correlated, we concluded that there is a critical link between induction of the stress proteins and phosphorylation of specific proteins. Furthermore, the rapid induction of GRP78 under the experimental condition offered a novel avenue for studying the regulation of its expression. © 1996 Wiley-Liss, Inc.     

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.     

Increase in anti-apoptotic molecules,nucleolin, and heat shock protein 70, against upregulated LRRK2 kinase activity

Leucine-rich repeat kinase 2 (LRRK2) is involved in Parkinson’s disease (PD) pathology. A previous study showed that rotenone treatment induced apoptosis, mitochondrial damage, and nucleolar disruption via up-regulated LRRK2 kinase activity, and these effects were rescued by an LRRK2 kinase inhibitor. Heat-shock protein 70 (Hsp70) is an anti-oxidative stress chaperone, and overexpression of Hsp70 enhanced tolerance to rotenone. Nucleolin (NCL) is a component of the nucleolus; overexpression of NCL reduced cellular vulnerability to rotenone. Thus, we hypothesized that rotenone-induced LRRK2 activity would promote changes in neuronal Hsp70 and NCL expressions. Moreover, LRRK2 G2019S, the most prevalent LRRK2 pathogenic mutant with increased kinase activity, could induce changes in Hsp70 and NCL expression. Rotenone treatment of differentiated SH-SY5Y (dSY5Y) cells increased LRKK2 levels and kinase activity, including phospho-S935-LRRK2, phospho-S1292-LRRK2, and the phospho-moesin/moesin ratio, in a dose-dependent manner. Neuronal toxicity and the elevation of cleaved poly (ADP-ribose) polymerase, NCL, and Hsp70 were increased by rotenone. To validate the induction of NCL and Hsp70 expression in response to rotenone, cycloheximide (CHX), a protein synthesis blocker, was administered with rotenone. Post-rotenone increased NCL and Hsp70 expression was repressed by CHX; whereas, rotenone-induced kinase activity and apoptotic toxicity remained unchanged. Transient expression of G2019S in dSY5Y increased the NCL and Hsp70 levels, while administration of a kinase inhibitor diminished these changes. Similar results were observed in rat primary neurons after rotenone treatment or G2019S transfection. Brains from G2019S-transgenic mice also showed increased NCL and Hsp70 levels. Accordingly, LRRK2 kinase inhibition might prevent oxidative stress-mediated PD progression.

Abbreviations: 6-OHDA: 6-hydroxydopamine; CHX: cycloheximide; dSY5Y: differentiated SH-SY5Y; g2019S tg: g2019S transgenic mouse; GSK/A-KI: GSK2578215A kinase inhibitor; HSP70: heat shock protein 70; LDH: lactose dehydrogenase; LRRK2: leucine rich-repeat kinase 2; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; myc-GS LRRK2: myc-tagged g2019S LRRK2; NCL: nucleolin; PARP: poly(ADP-ribose) polymerase; PD: Parkinson’s disease; PINK1: PTEN-induced putative kinase 1; pmoesin: phosphorylated moesin at t558; ROS: reactive oxygen species