Inhibitors such as staurosporine,H-7 or polymyxin B cannot be used in skeletal muscle to prove the role of protein kinase C on insulin action

The precise role of protein kinase C in insulin action in skeletal muscle is not well defined. Based on the fact that inhibitors of protein kinase C block some insulin effects, it has been concluded that some of the biological actions of insulin are mediated via protein kinase C. In this study, we present evidence that inhibitors of protein kinase C such as staurosporine, H-7 or polymyxin B cannot be used to ascertain the role of protein kinase C in skeletal muscle. This is based on the following experimental evidences: a) staurosporine, H-7 and polymyxin B markedly block in muscle the effect of insulin on System A transport activity; however, this effect of insulin is not mimicked in muscle by TPA-induced stimulation of protein kinase C, b) H-7 and polymyxin B block insulin action on System A transport activity in an additive manner to the inhibitory effect of phorbol esters, c) staurosporine, H-7 and polymyxin B block the effect of insulin on lactate production, a process that is activated by insulin and TPA in an additive fashion, and d) staurosporine completely blocks the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle.Abbreviations MeAIB a-(methyl)aminoisobutyric acid - TPA 12-O-tetradecanoylphorbol-13-acetate - H-7 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine     

Dissimilar effects of the protein kinase C inhibitors, staurosporine and H-7, on cholecystokinin-induced enzyme secretion from rabbit pancreatic acini.

The effects of two putative inhibitors of protein kinase C activity, staurosporine and H-7, on partially purified protein kinase C and amylase secretion from isolated rabbit pancreatic acini were investigated. Staurosporine dose-dependently inhibited amylase release stimulated by an optimal concentration of cholecystokinin C-terminal octapeptide. At a concentration of 100 nM, the drug inhibited the secretory response to the secretagogue by approximately 50%. At the same concentration, staurosporine inhibited 12-O-tetradecanoylphorbol 13-acetate-stimulated enzyme secretion by 90%. Moreover, the potentiating effect of this phorbol ester on cholecystokinin-induced amylase release was completely abolished in the presence of staurosporine. Interestingly, amylase release was decreased to the level observed with the combination of cholecystokinin and staurosporine. In contrast, H-7, potentiated rather than inhibited cholecystokinin-stimulated enzyme secretion, whereas the secretory response to 12-O-tetradecanoylphorbol 13-acetate was not affected by the drug. Both staurosporine and H-7, however, inhibited protein kinase C purified from exocrine pancreatic tissue. Kinetic analysis revealed that both compounds inhibited protein kinase C competitively with respect to ATP. The Ki value for staurosporine was 0.55 nM and for H-7 13.5 microM. Our results obtained with staurosporine are in line with a stimulatory role of protein kinase C in cholecystokinin-induced enzyme secretion from the exocrine pancreas. The results obtained with H-7 emphasize that care has to be taken in interpreting the biological effects of this drug.     

Protein kinase C is likely to be involved in zoosporogenesis and maintenance of flagellar motility in the peronosporomycete zoospores

The motility of zoospores is critical in the disease cycles of Peronosporomycetes that cause devastating diseases in plants, fishes, vertebrates, and microbes. In the course of screening for secondary metabolites, we found that ethyl acetate extracts of a marine Streptomyces sp. strain B5136 rapidly impaired the motility of zoospores of the grapevine downy mildew pathogen Plasmopara viticola at 0.1 μg/ml. The active principle in the extracts was identified as staurosporine, a known broad-spectrum inhibitor of protein kinases, including protein kinase C (PKC). In the presence of staurosporine (2 nM), zoospores moved very slowly in their axis or spun in tight circles, instead of displaying straight swimming in a helical fashion. Compounds such as K-252a, K-252b, and K-252c structurally related to staurosporine also impaired the motility of zoospores in a similar manner but at varying doses. Among the 22 known kinase inhibitors tested, the PKC inhibitor chelerythrine was the most potent to arrest the motility of zoospores at concentrations starting from 5 nM. Inhibitors that targeted kinase pathways other than PKC pathways did not practically show any activity in impairing zoospore motility. Interestingly, both staurosporine (5 nM) and chelerythrine (10 nM) also inhibited the release of zoospores from the P. viticola sporangia in a dose-dependent manner. In addition, staurosporine completely suppressed downy mildew disease in grapevine leaves at 2 μM, suggesting the potential of small-molecule PKC inhibitors for the control of peronosporomycete phytopathogens. Taken together, these results suggest that PKC is likely to be a key signaling mediator associated with zoosporogenesis and the maintenance of flagellar motility in peronosporomycete zoospores.     

Involvement of the protein kinase pathway in melanin synthesis by chick retinal pigment epithelial cells

Protein kinases are involved in a variety of cellular functions and cell proliferation in eyes. We have explored the involvement of protein kinase C (PKC) in cell proliferation and melanin synthesis by chick retinal pigment epithelial (RPE) cells in vitro. This was achieved by incubation of confluent RPE cells with known inhibitors of protein kinase, H-7, W-7, H-8, and staurosporine. Chick RPE cells were cultured in the presence or absence of the protein kinase inhibitors for a 10-day period. Effects of the inhibitors on cell proliferation and melanin synthesis, as an indication of cell differentiation, were assessed by counting the number of surviving cells and by measuring the melanin content in the cells, respectively. H-7, W-7, and staurosporine inhibited cell proliferation and increased melanin synthesis in a concentration-dependent manner during culture; however, H-8 did not produce these cellular effects. These findings indicate that PKC and calcium/calmodulin-dependent kinase pathways are involved in the proliferation and differentiation of chick RPE cells.     

Involvement of protein kinase C in translocation of desmoplakins from cytosol to plasma membrane during desmosome formation in human squamous cell carcinoma cells grown in low to normal calcium concentration

The intracellular signal transduction mechanism leading to desmosome formation in low-calcium-grown keratinocytes after addition of calcium to the medium was studied by immunofluorescence using antibodies to desmoplakins I and II (cytoplasmic desmosomal proteins) and by electron microscopy before and after addition of calcium; protein kinase C (PKC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoylglycerol (DOG); calcium ionophore A23187; selective PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine; and a Ca2+/calmodulin-dependent kinase inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). In previous studies using a low-calcium-grown human epidermal squamous cell carcinoma, we have shown that an increase in extracellular Ca2+ caused a four-fold increase in PKC activity and addition of TPA (10 ng/ml) induced a transient increase in membrane-bound PKC activity in association with cell-cell contact formation. The present study showed that TPA (10 ng/ml). PDBu (10 ng/ml), and DOG (1 mg/ml) induced a rapid cell-cell contact and redistribution of desmoplakins from cytoplasm to the plasma membrane with desmosome formation within 60-120 min, which was similar, although less marked, to the effect of increased Ca2+. The TPA-induced desmosome formation was inhibited by selective PKC inhibitors, H-7 (20 microM) or staurosporine (100 nM). On the other hand, calcium ionophore A23187 induced only a temporary increase in the number of desmoplakin-containing fluorescent spots in the cytoplasm and a temporary cell-cell attachment without desmosome formation. The calcium-induced desmosome formation was partially inhibited by 20-100 microM H-7 or 100 nM staurosporine; however, it was not inhibited by W-7 at a concentration of 25 microM, at which this agent selectively inhibits calmodulin-dependent protein kinase. These results suggest that PKC activation plays an important role in desmoplakin translocation from the cytoplasm to the plasma membrane as one of the processes of calcium-induced desmosome formation.     

Effects of protein phosphatase and kinase inhibitors on the cardiac L- type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase

We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10- 30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel.     

Enhancement of differentiation of rat adipocyte precursor cells by pertussis toxin

To examine whether GTP-binding protein(s) is (are) involved in adipocyte differentiation, the effect of pertussis toxin (PT) was studied in rat adipocyte precursor cell culture. PT potentiated adipose conversion induced by dexamethasone, insulin, and 1-methyl-3-isobutylxanthine in a dose- and time-dependent fashion. Attenuation of an inhibitory control of adenylate cyclase was not the mechanism of action of PT. The dose-dependent inhibition of PT-catalyzed ADP-ribosylation of the Mr 40,000 protein of the cell membrane by preincubation of the toxin was inversely related to the potentiating effect on differentiation. PT-sensitive G protein(s) may be involved in adipocyte differentiation in a negative fashion.     

Arachidonic acid release by basophilic leukemia cells and macrophages stimulated by Ca2+ ionophores, antigen and diacylglycerol: essential role for protein kinase C and prevention by glucocorticosteroids

The role of protein kinase C in phospholipase A2 (PLA2) activation in rat basophilic leukemia cells (RBL-2H3) and macrophages was investigated. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) doubled ionomycin-induced PLA2 activity, assessed by [3H]arachidonate release. Protein kinase C inhibitors, staurosporine and K252a (100 nM) or H-7 (15 micrograms/ml) inhibited ionomycin-stimulation of PLA2 activity by 62, 75 and 80%, respectively. Down-regulation of protein kinase C by prolonged treatment with TPA inhibited Ca2(+)-ionophore A23187 or antigen-stimulation of [3H]arachidonate release by 80%. We examined whether the inhibitory effect of dexamethasone (DEX) on PLA2 activity is related to modulation of protein kinase C activity. The 50% inhibition by DEX of ionomycin elevation of [3H]arachidonate release was almost overcome by addition of TPA. The Ca2+ ionophore and antigen-induced increase in [3H]TPA binding to intact RBL cells was not impaired by DEX. However, DEX markedly reduced phosphorylation of several proteins. 1-Oleoyl-2-acetyl-glycerol (OAG) had a sustained stimulatory effect on PLA2 activity in isolated plasma membranes derived from treated bone-marrow intact mouse macrophages, while both DEX and staurosporine reduced elevated PLA2 activity by 68 and 84%, respectively. The results support an essential role for protein kinase C in regulation of PLA2 activity.     

Prostaglandin F2 alpha inhibits the differentiation of adipocyte precursors in primary culture.

Influence of arachidonate metabolite pathway on adipose differentiation was investigated using primary culture of adipocyte precursors in defined medium. Treatment of the cells with cyclooxygenase inhibitors stimulates adipose differentiation by at least 2-fold. Among the various arachidonate metabolites tested, only prostaglandin F2 alpha (PGF2 alpha) was found to inhibit the differentiation of adipocyte precursors in a dose dependent fashion. Other eicosanoids tested did not have any effect. A 50% inhibition of adipose differentiation was observed with a dose of PGF2 alpha of 3 x 10(-9)M to 7 x 10(-9)M according to the strain of rats used. Maximal inhibition occurred at PGF2 alpha concentrations equal or higher than 10(-8)M. PGF2 alpha inhibited not only the expression of late markers of adipose differentiation such as G3PDH and triglycerides accumulation but also the mRNA expression of early markers of adipose differentiation such as clone 154, lipoprotein lipase and ap2 gene. These results indicate that PGF2 alpha represents a physiological negative modulator of adipose differentiation.     

Effects of Protein Kinase Inhibitors on Morphology and Function of Cultured Bovine Adrenal Chromaffin Cells: KN-62 Inhibits Secretory Function by Blocking Stimulated Ca2+ Entry

Abstract: In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and ⁴⁵Ca²⁺ uptake. Treatment of chromaffin cells with 10 µ M K-252a, 50 µ M H-7, or 50 µ M H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca²⁺/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC₅₀ value of 0.32 µ M ), but had no effect on cell morphology. The reduction of secretory function induced by 1 µ M KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated ⁴⁵Ca²⁺ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated ⁴⁵Ca²⁺ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca²⁺ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.     

Topological regulation of cell-membrane phosphoinositidase C.

Although the translocation of protein kinase C and phospholipase A2 are well documented, no information is available about the possible down-modulation of transmembrane phospholipase C. We found that TPA induced a dose-dependent (10-200 nM) and time-dependent (15 min-6 h) down-modulation of transmembrane phosphoinositidase C (PLC-PI) on lymphoid cells (CEM-CM3 and WIL2-NS) and epitheloid carcinoma cells (HeLa S3) but not on human fibroblasts (MRC-5). Cell-surface expression of PLC-PI on intact cells was assayed by flow cytometry using saturating concentrations of polyclonal anti-PLC-PI antibodies and phycoerythrin-conjugate. A control phorbol-ester which does not activate protein kinase C (PKC) had no internalization effect on PLC-PI. PKC inhibitors staurosporine (2.5 nM) and H-7 (10 microM) partially inhibited the TPA effect. Cytochalasin B (40 micrograms/ml) did not modify the TPA-induced PLC-PI down-modulation. The effect of TPA on PLC-PI seems quite specific since no internalization was induced by TPA on transmembrane phosphatidylcholine-preferring PLC expression. These results show that TPA can translocate the membrane-bound PLC-PI, probably by PKC activation.     

IL-5-induced IgA synthesis by LPS-stimulated mouse B cells is prevented by protein kinase C inhibitors.

We have previously demonstrated that activation of protein kinase C (PKC) by phorbol esters induces selectively IgA synthesis by mouse B cells. In this study, we investigated the effects of a number of protein kinase inhibitors on IgA secretion induced by a recombinant murine IL-5 in LPS-stimulated mouse B cells. The results show that PKC inhibitors, such as sphingosine (SPH), staurosporine (STP) and H-7, blocked IL-5-induced IgA synthesis; the protein kinase A inhibitor HA-1004 and the inhibitor of calcium/calmodulin dependent protein kinase W-7 had no effect on IgA secretion induced by IL-5. The proliferation of the IL-5 sensitive B13 cell line in response to IL-5 was also inhibited by addition of SPH or STP or H-7. The data suggest an involvement of the PKC pathway in IL-5-induced B cell differentiation into IgA secreting cells.     

Characterization of specific binding sites for [3H]-staurosporine on various protein kinases

Binding of [3H]-staurosporine to different protein kinases was time-dependent, reversible and saturable. Scatchard analysis of saturation isotherms indicated one class of binding sites for [3H]-staurosporine with dissociation constants (KD) of 9.6, 2.0, 3.0 and 7.4 nM for protein kinase C, cAMP-dependent protein kinase, tyrosine protein kinase and calcium/calmodulin-dependent protein kinase respectively. [3H]-staurosporine binding was fully displaced by unlabelled staurosporine or the related compound K-252a whereas other protein kinase inhibitors (H-7, H-8 and W-7) did not compete with [3H]-staurosporine. These data confirm that sataurosporine shows no selectivity for different protein kinases and suggest the putative existence of distinct, specific binding sites for [3H]-staurosporine on these enzymes.     

Genistein, a plant-derived isoflavone, counteracts the antilipolytic action of insulin in isolated rat adipocytes

Genistein is a phytoestrogen exerting numerous biological effects. Its direct influence on adipocyte metabolism and leptin secretion was previously demonstrated. This study aimed to determine whether genistein antagonizes the antilipolytic action of insulin in rat adipocytes. Freshly isolated adipose cells were incubated for 90 min with epinephrine, epinephrine with insulin and epinephrine with a specific inhibitor of protein kinase A (H-89) at different concentrations of genistein (0, 6.25, 12.5, 25, 50 and 100 μM). Genistein failed to affect epinephrine-induced glycerol release, however, the inhibitory action of insulin on epinephrine-induced lipolysis was significantly abrogated in cells exposed to the phytoestrogen (12.5–100 μM). The increase in insulin concentration did not suppress the genistein effect. Its inhibitory influence on the antilipolytic action of insulin was accompanied by a substantial rise in cAMP in adipocytes. This rise appeared despite the presence of 10 nM insulin in the incubation medium. Further experiments, in which insulin was replaced by H-89, revealed that the antilipolytic action of protein kinase A inhibitor on epinephrine-induced lipolysis was not affected by genistein. This means that genistein counteracted the antilipolytic action of insulin due to the increase in cAMP levels and activation of protein kinase A in adipocytes. The observed attenuation of the inhibitory effect of insulin on triglyceride breakdown evoked by genistein was not related to its estrogenic activities, as evidenced in experiments employing the intracellular estrogen receptor blocker, ICI 182,780. Moreover, it was found that genistein-induced impairment of the antilipolytic action of insulin was not accompanied by changes in the proportion between fatty acids and glycerol released from adipocytes. The ability of genistein to counteract the antilipolytic action of insulin may contribute to the decreased triglyceride accumulation in adipose tissue.     

Modulation of the Na–;H antiport by insulin: Interplay between protein kinase C,tyrosine kinase,and protein phosphatases

The insulin modulation of Na-H antiport in rat hepatocytes was studied using the fluorescent, pH-sensitive intracellular probe, 2′,7′ bis (carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Our data show that insulin stimulates the Na? H antiport. The dose-response of insulin effect shows a behavior typical of other insulin responses: a maximum in the physiological range (1 nM) and smaller effects at higher and lower hormone concentrations. The time-course of activation is very fast at high hormone concentrations and slow, but reaching a higher value, for the physiological concentrations (0.26± 0.05 and 0.18 ± 0.022 pH units for 1 nM and 1 μM insulin respectively). The use of phorbol, 12-myristate, 13-acetate (PMA), a potent activator of protein kinase C and its inhibitor staurosporine, and the inhibitor of tyrosine kinase erbstatin analog, suggests that both protein kinase C and tyrosine kinase could be involved in the mechanism leading to Na? H antiport activation by insulin. We suggest that the activation of the antiport involves the two pathways depending on the hormone concentration. In particular, protein kinase C would mediate the effects of high hormone concentrations, acting as a growth factor, since staurosporine fully inhibited insulin 1 μM, but only partially 1 nM effects, and tyrosine kinase would mediate the effect of insulin 1 nM and only partially 1 μM. Okadaic acid 1 μM, a potent inhibitor of protein phosphatases, mimicked the hormone effects on the antiport and abolished the different time-course due to hormone concentration, suggesting a role of kinases and phosphatases in the signal transduction. The effect of all activators was abolished by amiloride analog, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), confirming the specificity of these effects. © 1994 wiley-Liss, Inc.     

Inhibitory effect of calcium-binding protein regucalcin on protein kinase activity in the nuclei of regenerating rat liver

The effect of Ca²⁺-binding protein regucalcin on protein kinase activity in the nuclei of normal and regenerating rat livers was investigated. Protein kinase activity in the nuclei isolated from normal rat liver was significantly increased by addition of Ca²⁺ (500 μM) and calmodulin (10 μg/ml) in the enzyme reaction mixture. Nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), trifluoperazine (TFP; 20 μM), dibucaine (10⁻⁴ M), or staurosporine (10⁻⁷ M), indicating that Ca²⁺-dependent protein kinases are present in the nuclei. Protein kinase activity was significantly elevated in the liver nuclei obtained at 6 to 48 h after a partial hepatectomy. Hepatectomy-increased nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), TFP (20 μM), or staurosporine (10⁻⁷ M) in the enzyme reaction mixture. The presence of regucalcin (0.1–0.5 μM) caused a significant decrease in protein kinase activity in the nuclei obtained from normal and regenerating rat livers. Meanwhile, the nuclear protein kinase activity from normal and regenerating livers was significantly elevated in the presence of anti-regucalcin monoclonal antibody (50–200 ng/ml). The present study suggests that regucalcin plays a role in the regulation of protein kinase activity in the nuclei of proliferative liver cells. J. Cell. Biochem. 71:569–576, 1998. © 1998 Wiley-Liss, Inc.     

Insulin-like growth factor-I mediates osteoclast-like cell formation stimulated by parathyroid hormone

There have been several lines of evidence that parathyroid hormone (PTH) stimulates production of insulinlike growth factor I (IGF-I) in bone and that IGF-I stimulates osteoclast formation. Thus, the present study was performed to clarify the possible role of IGF-I in PTH-stimulated osteoclastlike cell formation and the role of PTH-responsive dual signal transduction systems (cyclic [c] AMP-dependent protein kinase [PKA] and calcium/protein kinase C [PKC]) in its mechanism. Treatment with anti-IGF-I antibody (1–10 μg/ml) partially but significantly blocked hPTH-(1-34)-stimulated osteoclastlike cell formation in unfractionated mouse bone cell cultures, although it did not affect osteoclastlike cell formation stimulated by 1,25-dihydroxyvitamin D₃. Rp-cAMPS (10^-4 M), a direct PKA inhibitor, as well as two types of PKC inhibitors, H-7 (10 μM) and staurosporine (3 nM), and dantrolene (10^-5 M), an inhibitor of calcium mobilization from intracellular calcium stores, all significantly blocked PTH-stimulated osteoclastlike cell formation. Anti-IGF-I antibody (3 μg/ml) significantly blocked osteoclastlike cell formation stimulated by 10^-4 M dbcAMP, 10^-4 M Sp-cAMPS, a direct PKA activator, and 10^-5 M forskolin in mouse bone cell cultures. Dibutyryl cAMP, forskolin, and hPTH-(1-34) significantly stimulated mRNA expression of both IGF-I and IGF-binding protein 5 (IGFBP-5) in these cultures, but neither 10^-7 M PMA, a PKC activator, nor 10^-7 M A23187 did. Moreover, anti-IGF-I antibody significantly blocked osteoclastlike cell formation stimulated by the conditioned medium from MC3T3-E1 cells pretreated with 10^-8 PTH-(1-34), which induced IGF-I and IGFBP-5 mRNA expression in these cells. In conclusion, the present study indicates that IGF-I mediates osteoclastlike cell formation stimulated by PTH and that the PKA pathway is involved in its mechanism. However, IGF-I does not seem to be the sole effector molecule to be active in this system. J. Cell. Physiol. 172:55–62, 1997. © 1997 Wiley-Liss, Inc.     

H-89 potentiates adipogenesis in 3T3-L1 cells by activating insulin signaling independently of protein kinase A

Among four kinds of protein kinase A (PKA) inhibitors tested, H-89 exhibited a unique action to remarkably enhance adipocyte differentiation of 3T3-L1 cells, whereas the other three PKA inhibitors, PKA inhibitor Fragment 14-22 (PKI), Rp-cAMP, and KT 5720, did not enhance adipocyte differentiation. H-85, which is an inactive form of H-89, exhibited a similar enhancing effect on adipocyte differentiation. H-89 also potentiated the phosphorylation of Akt and extracellular signal-regulated kinase (ERK) 1/2 in 3T3-L1 cells, which function as downstream signaling of insulin. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and mitogen-activated protein kinase kinase (MEK) inhibitor PD 98059 suppressed both the H-89-induced promotion of adipocyte differentiation and the H-89-induced potentiation of phosphorylation of Akt and ERK1/2. Rho kinase inhibitor Y-27632 also promoted the phosphorylation of both Akt and ERK1/2 and enhanced adipocyte differentiation, although its effect was somewhat less than that of H-89. Even when cells were treated with a mixture of Y-27632 and H-89, the additive enhancing effects on both the insulin signaling and adipocyte differentiation were not detected. Therefore, it is suggested that the major possible mechanism whereby H-89 potentiates adipocyte differentiation of 3T3-L1 cells is activation of insulin signaling that is elicited mostly by inhibiting Rho/Rho kinase pathway.     

Interleukin-1β induction of tissue inhibitor of metalloproteinase (TIMP-1) is functionally antagonized by prostaglandin E2 in human synovial fibroblasts

Elevated levels of tissue inhibitor of metalloproteases-1 (TIMP-1) have been demonstrated in inflamed synovial membranes, and it is believed that the inhibitor may play a critical role in the regulation of connective tissue degradation. The present study was undertaken to define the cellular mechanism of action of the inflammatory mediators, interleukin-1β (IL-1β) and prostaglandin E₂ (PGE₂), in the control of TIMP-1 synthesis and expression in human synovial fibroblasts. Recombinant human IL-1β induced a time- and dose-dependent saturable response in terms of TIMP-1 mRNA expression (effective concentration for 50% maximal response, EC₅₀ = 31.5 ± 3.3 pg/ml) and protein synthesis (EC₅₀ = 30 ± 3.3 pg/ml). The protein kinase C (PKC) inhibitors, H-7, staurosporine, and calphostin C, reversed the rhIL-1β induction of TIMP-1 mRNA. PGE₂ also inhibited rhIL-1β-stimulated TIMP-1 mRNA expression and protein secretion in a dose-dependent fashion. The concentration of PGE₂ necessary to block 50% of rhIL-1β-stimulated TIMP-1 secretion, IC₅₀, was 1.93 ng/ml (4.89 nM). Forskolin, and other stable derivatives of cAMP, mimicked, to a large extent, the effects of PGE₂. The phorbol ester, PMA, up-regulated considerably the mRNA expression of TIMP-1 but had no effect on protein production. Calphostin C substantially reduced PMA-activated TIMP-1 expression. Staurosporine, calphostin C, H-7, and substances that elevate cellular levels of cAMP, like PGE₂, also reduced basal expression and synthesis of TIMP-1. Taken together, the data suggest that PKA and C may mediate opposing effects in terms of TIMP-1 expression and secretion in human synovial fibroblasts.