miR-9-3p plays a tumour-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells.

BACKGROUND: The inactivation of the Hippo pathway lead to TAZ (PDZ-binding motif)/YAP (yes-associated protein) overexpression, and is associated with worse prognostic outcomes in various cancers including hepatocellular carcinoma (HCC). Although there are several reports of microRNA (miR) targeting for YAP, miR targeting for TAZ remains unclear. The aim of this study is to identify the miR targeting TAZ expression in HCC. METHODS: MicroRNA expression was analysed using the Human miFinder 384HC miScript miR PCR array, and was compared between low and high TAZ expression cell lines. Then, we extracted miR-9-3p as a tumour-suppressor miR targeting TAZ. We examined the functional role of miR-9-3p using miR-9-3p mimic and inhibitor in HCC cell lines). RESULTS: In HCC cell lines and HCC clinical samples, there was the inverse correlation between miR-9-3p and TAZ expressions. TAZ expression was induced by treatment of miR-9-3p inhibitor and was downregulated by treatment of miR-9-3p mimic. Treatment of miR-9-3p mimic inhibited cell proliferative ability with downregulated phosphorylations of Erk1/2, AKT, and ß-catenin in HLF. Inversely, treatment of miR-9-3p inhibitor accelerated cell growth compared with control in HuH1. CONCLUSIONS: MicroRNA-9-3p was identified as the tumour-suppressor miR targetting TAZ expression in HCC cells.

Cataract induced by short-term administration of large doses of busulfan: a case report.

Busulfan is a carcinostatic which is used for myelocytic leukemia. A 42-year-old Japanese woman given high doses of busulfan for a short time was referred to us because of decreased visual acuity and blurred vision in both eyes. She received 212 mg/day of busulfan for only 4 days. Ophthalmoscopic examination demonstrated a posterior polar subcapsular opacity of the lens in both eyes. Although the occurrence of cataract due to busulfan has been reported after long-term administration of busulfan, this is the first clinical case of cataract caused by high doses of busulfan for a short period. It is necessary to pay more attention to busulfan cataract.

A new animal model of thrombophilia confirms that high plasma factor VIII levels are thrombogenic.

The thrombotic risk associated with elevated plasma levels of clotting factor VIII (FVIII) was investigated in a mouse model of thrombophilia. After the intravenous injection of recombinant human FVIII and/or of purified FVIII-free human von Willebrand factor (vWF), a controlled mild injury was inflicted on the carotid artery of FVB mice by irradiation with filtered green light in combination with intravenous injection of the dye rose bengal. Formation of a platelet-rich thrombus was continuously monitored for 40 min via transillumination and the thrombus size was measured via image analysis. Administration of recombinant human FVIII at 40 microg/kg led to initial FVIII plasma activities equivalent to 250% of normal human plasma FVIII activity and significantly enhanced thrombus size. Immunohistochemical staining illustrated the accumulation of FVIII within the thrombi. Human vWF, even at 10 mg/kg, had no effect on thrombus formation. The thrombotic tendency induced by FVIII was significantly inhibited by the administration of human vWF in a dose-dependent manner. Separate plasma measurements revealed that human FVIII has comparable affinities for human and murine vWF but that human vWF does not effectively bind murine platelets. The inhibition by human vWF of the thrombotic tendency induced by human FVIII could therefore be explained by a lack of accumulation of FVIII within the developing thrombus because of the reduced affinity of human vWF for murine platelets and the reduced occupancy of murine von Willebrand factor by human FVIII after injection of human vWF. These results show that vWF actively participates in FVIII accumulation in the arterial thrombus and provide experimental evidence for epidemiological findings that elevated plasma FVIII levels are associated with an increased thrombotic risk, also in arteries.

Vasopressin stimulates the induction of heat shock protein 27 and alphaB-crystallin via protein kinase C activation in vascular smooth muscle cells.

In the present study, we examined the effect of vasopressin on the induction of the low-molecular-weight heat shock proteins heat shock protein 27 (HSP27) and alphaB-crystallin in an aortic smooth muscle cell line, A10 cells. Vasopressin induced a time-dependent accumulation of HSP27 and alphaB-crystallin. The stimulatory effects of vasopressin were dose-dependent over the range 0.1 nmol/L to 0.1 micromol/L. The EC50 values for vasopressin were 2 (HSP27) and 4 nmol/L (alphaB-crystallin). Vasopressin induced increases in the levels of the mRNAs for HSP27 and alphaB-crystallin. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, induced an accumulation of HSP27 (EC50, 20 nmol/L) and alphaB-crystallin (EC50, 2 nmol/L). In contrast, 4alpha-phorbol 12,13-didecanoate, a non-PKC-activating phorbol ester, had no such effect. Staurosporine and calphostin C, inhibitors of PKC, significantly reduced the vasopressin-induced accumulation of HSP27 and alphaB-crystallin as well as that induced by TPA. BAPTA/AM and TMB-8, inhibitors of intracellular Ca2+ mobilization, significantly reduced the vasopressin-induced accumulation of HSP27 and alphaB-crystallin. These results strongly suggest that vasopressin stimulates the induction of HSP27 and alphaB-crystallin via PKC activation in vascular smooth muscle cells and that this effect of vasopressin is dependent on intracellular Ca2+ mobilization.

Retinoic acid suppresses interleukin-6 synthesis induced by prostaglandins in osteoblasts.

We previously reported that prostaglandin E1 (PGE1) induces the synthesis of interleukin-6 (IL-6) via cAMP production in osteoblast-like MC3T3-E1 cells, and that, on the other hand, prostaglandin F2alpha (PGF2alpha) stimulates IL-6 synthesis via activation of protein kinase C. In the present study, we examined the effect of retinoic acid on IL-6 synthesis induced by these two prostaglandins in MC3T3-E1 cells. Retinoic acid inhibited the IL-6 synthesis induced by PGF2alpha or PGE1 in a dose-dependent manner in the range between 0.1 and 10 nM. Retinoic acid also suppressed the IL-6 synthesis stimulated by 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C. The IL-6 synthesis induced by cholera toxin, forskolin or dibutyryl cAMP was inhibited by retinoic acid. However, retinoic acid had little effect on the IL-6 synthesis induced by interleukin-1. These results indicate that retinoic acid inhibits IL-6 synthesis induced by prostaglandins in osteoblasts as follows: the inhibitory effect on the PGE1-induced IL-6 synthesis is exerted at a point downstream from cAMP, and the inhibitory effect on the PGF2alpha-induced IL-6 synthesis is exerted at a point downstream from protein kinase C.

Effects of several agents on UVB- and UVA plus systemic fluoroquinolone-induced erythema of guinea pig skin evaluated by reflectance colorimetry.

The aim of this study was to clarify the mechanisms underlying the erythema of guinea pig skin induced by ultraviolet (UV) irradiation alone and in combination with a systemic fluoroquinolone (FQ). The effects of several drugs which may modify the actions of some inflammatory mediators and radicals possibly released in the inflamed site on the erythema were examined and compared in an objective and quantitative way by measuring the change in color of the irradiated skin, determined as the change in chroma (C*) with use of reflectance colorimetry. After confirming that the C* value increased in an irradiation dose-dependent manner and reached a plateau 1-2 h after irradiation of UVB alone or UVA coadministered with an FQ, Y-26611 (10 mg/kg, i.p.), guinea pigs were pretreated with indomethacin, butylated hydroxytoluene (BHT) or beta-carotene before, or treated with H1- or H2-receptor antagonist, superoxide dismutase or N omega-nitro-L- arginine methyl ester after UV irradiation, and their inhibitory effects against erythema were evaluated. It was suggested that there are some substantial differences between UVB- and UVA plus FQ-induced erythemas. Although histamine makes little contribution to both types of erythema, metabolites of arachidonic acid catalyzed by cyclooxygenase contribute more to UVB-induced erythema, whereas superoxides take more part in UVA plus FQ-induced erythema. Furthermore, nitric oxide seems to participate in both types of erythema; however, the pretreatment with BHT or beta-carotene was ineffective against both erythemas. From these results, interventions should be directed to powerfully scavenging radicals for prevention and treatment of UV plus FQ-induced phototoxicity.

Effect of vitamin D3 on interleukin-6 synthesis induced by prostaglandins in osteoblasts.

In previous studies, we have shown that prostaglandin F2alpha (PGF2alpha) stimulates interleukin-6 (IL-6) synthesis via activation of protein kinase C in osteoblast-like MC3T3-E1 cells, and that prostaglandin E1 (PGE1) induces the synthesis of IL-6 through protein kinase A activation. In the present study, we investigated the effect of vitamin D3 on IL-6 synthesis in MC3T3-E1 cells. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), an active form of vitamin D3, inhibited the IL-6 synthesis induced by PGF2alpha or PGE1. On the contrary, 24,25-dihydroxyvitamin D3, an inactive form of vitamin D3, had no effect. 1,25-(OH)2D3 did not affect the IL-6 synthesis stimulated by 12-O-tetradecanoyl-phorbol-13-acetate, an activator of protein kinase C. The IL-6 synthesis induced by cholera toxin or forskolin was significantly inhibited by 1,25-(OH)2D3. However, 1,25-(OH)2D3 had little effect on the IL-6 synthesis induced by dibutyryl cAMP. These results strongly suggest that 1,25-(OH)2D3, an active form of vitamin D3, inhibits IL-6 synthesis at both the protein kinase C pathway and the protein kinase A pathway in osteoblasts.

Interleukin-6 synthesis induced by prostaglandin E2: cross-talk regulation by protein kinase C.

We previously showed that prostaglandin E2 (PGE2) stimulates multiple intracellular signaling pathways as follows: by activation of adenylate cyclase; phosphoinositide (PI)-hydrolyzing phospholipase C and phosphatidylcholine (PC)-hydrolyzing phospholipase D; and by induction of Ca2+ influx in osteoblast-like MC3T3-E1 cells. In this study, we investigated the effect of PGE2 on the synthesis of interleukin-6 (IL-6) and its regulatory mechanism in MC3T3-E1 cells. PGE2 significantly stimulated IL-6 secretion in a dose-dependent manner in the range between 1 nmol/L and 10 micromol/L. A23187, a calcium ionophore, or dibutyryl-cAMP significantly induced IL-6 secretion. The effect of a combination of A23187 and dibutyryl-cAMP on IL-6 secretion was additive. The depletion of extracellular Ca2+ by EGTA reduced the PGE2-induced IL-6 secretion. EP1 receptor antagonist inhibited the PGE2-induced IL-6 secretion. H-89, an inhibitor of cAMP-dependent protein kinase, decreased the PGE2-induced IL-6 secretion. EP2 receptor agonist alone stimulated IL-6 secretion. However, EP4 receptor antagonist had little effect on IL-6 secretion. Calphostin C, a specific inhibitor of protein kinase C (PKC), enhanced the secretion of IL-6 induced by PGE2. The stimulative effect of PGE2 on IL-6 secretion was significantly enhanced in PKC downregulated MC3T3-E1 cells. Pertussis toxin enhanced PGE2-induced IL-6 secretion. These results strongly suggest that PGE2 stimulates IL-6 synthesis through both Ca2+ mobilization from extracellular space via EP1 receptor and cAMP production via EP2 receptor in osteoblast-like cells, and that the PKC activation by PGE2 itself regulates oversynthesis of IL-6.

Involvement of protein kinase C activation in endothelin-1-induced secretion of interleukin-6 in osteoblast-like cells.

We previously reported that endothelin-1 (ET)-1 stimulates phospholipase D (PLD) independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of ET-1 on the secretion of interleukin-6 (IL-6) and the involvement of protein kinase C (PKC) activation in the IL-6 secretion in these cells. ET-1 significantly stimulated IL-6 secretion time-dependently up to 72 h. The stimulative effect was dose-dependent in the range between 1 nM and 1 microM. BQ123, a selective antagonist of endothelinA (ETA) receptor, inhibited the ET-1-induced IL-6 secretion. On the contrary, BQ788, a selective antagonist of endothelinB (ETB) receptor, had no effect. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, significantly stimulated IL-6 secretion. However, 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, did not affect IL-6 secretion. The effect of a combination of ET-1 and TPA on IL-6 secretion was not additive. Calphostin C, a specific PKC inhibitor, significantly inhibited the ET-1-induced IL-6 secretion. Both ET-1- and TPA-induced IL-6 secretion were reduced in PKC downregulated MC3T3-E1 cells. These results strongly suggest that ET-1 stimulates IL-6 secretion via ETA receptor in osteoblast-like cells and that PKC activation is involved in the ET-1-induced IL-6 secretion.

Inhibitory effects of propofol on intracellular signaling by endothelin-1 in aortic smooth muscle cells.

BACKGROUND: Blood pressure decreases when propofol is administered. However, the exact mechanism underlying the vascular effects of propofol has not yet been elucidated. Endothelin produced by vascular endothelial cells is a potent vasoactive peptide that elicits prolonged contraction of vascular smooth muscle cells. The effects of propofol on endothelin-1-induced intracellular signaling in an aortic smooth muscle cell line, A10 cells, were examined. METHODS: Cultured A10 cells were pretreated with propofol for 20 min and then stimulated with endothelin-1. The effect of propofol on the endothelin-1-induced Ca2+ influx into A10 cells was evaluated by measuring intracellular 45Ca2+. The effects of propofol on the endothelin-1-induced activation of phosphatidylinositol-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D were evaluated by measuring the formation of inositol phosphates and choline, respectively. The effect of propofol on endothelin-1 binding to its receptor was determined by an [125I] endothelin-1-binding assay. RESULTS: Propofol inhibited the endothelin-1-induced Ca2+ influx, but this was significant only at supuraclinical concentrations. The endothelin-1-stimulated formation of inositol phosphates was significantly suppressed by propofol. However, propofol had no effect on the formation of inositol phosphates induced by NaF, an activator of heterotrimeric guanosine triphosphate (GTP)-binding proteins. Propofol inhibited the endothelin-1-induced formation of choline. Propofol had no effect on the binding of endothelin-1 to its receptor. CONCLUSIONS: These results suggest that propofol inhibits endothelin-1-induced intracellular signaling in vascular smooth muscle cells. The inhibitory effect of propofol might be exerted at a point between the endothelin-1 receptor and its GTP-binding protein. However, because all significant effects are observed at high concentrations, clinical relevance is unclear.

Protein kinase C activation by interleukin (IL)-1 limits IL-1-induced IL-6 synthesis in osteoblast-like cells: involvement of phosphatidylcholine-specific phospholipase C.

We investigated the regulatory mechanism of interleukin-6 (IL-6) synthesis induced by interleukin-1 (IL-1) in osteoblast-like MC3T3-E1 cells. IL-1 stimulated the secretion of IL-6 in a dose-dependent manner in the range between 0.1 and 100 ng/ml. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), significantly enhanced the IL-1-induced secretion of IL-6. The stimulative effect of IL-1 was markedly amplified in PKC down-regulated MC3T3-E1 cells. IL-1 produced diacylglycerol in MC3T3-E1 cells. IL-1 had little effect on the formation of inositol phosphates and choline. On the contrary, IL-1 significantly stimulated the formation of phosphocholine dose-dependently. D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed the IL-1-induced diacylglycerol production. The IL-1-induced IL-6 secretion was significantly enhanced by D-609. These results indicate that IL-1 activates PKC via phosphatidylcholine-specific phospholipase C in osteoblast-like cells, and the PKC activation then limits IL-6 synthesis induced by IL-1 itself.

Tumor necrosis factor-alpha autoregulates interleukin-6 synthesis via activation of protein kinase C. Function of sphingosine 1-phosphate and phosphatidylcholine-specific phospholipase C.

We investigated the mechanism of interleukin-6 (IL-6) synthesis induced by tumor necrosis factor-alpha (TNF) in osteoblast-like MC3T3-E1 cells. TNF stimulated the synthesis of IL-6 dose dependently in the range between 1 and 30 ng/ml. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), significantly enhanced the TNF-induced synthesis of IL-6. 1-Oleoyl-2-acetylglycerol, a specific activator of PKC, inhibited the TNF-induced IL-6 synthesis. The stimulative effect of TNF was markedly increased in the PKC down-regulated cells. TNF produced diacylglycerol. TNF had little effect on the formation of inositol phosphates and choline. On the contrary, TNF significantly stimulated the formation of phosphocholine dose dependently. D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed the TNF-induced diacylglycerol production. The TNF-induced IL-6 synthesis was significantly enhanced by D-609. TNF induced sphingomyelin hydrolysis. Neither C2-ceramide nor sphingosine but sphingosine 1-phosphate significantly stimulated the synthesis of IL-6. PKC down-regulation amplified the IL-6 synthesis by sphingosine 1-phosphate. These results strongly suggest that sphingosine 1-phosphate may act as a second messenger for TNF-induced IL-6 synthesis and that TNF autoregulates IL-6 synthesis due to PKC activation via phosphatidylcholine-specific phospholipase C in osteoblast-like cells.

Thrombin regulates interleukin-6 synthesis through phosphatidylcholine hydrolysis by phospholipase D in osteoblasts.

We previously reported that thrombin stimulates Ca2+ influx and activates phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells. In this study, we investigated the effect of thrombin on interleukin-6 (IL-6) synthesis in these cells. Thrombin stimulated IL-6 synthesis dose-dependently in the range between 0.01 and 1 U/ml. The depletion of extracellular Ca2+ by EGTA suppressed the thrombin-induced IL-6 synthesis. TMB-8, an inhibitor of intracellular Ca2+ mobilization, also inhibited the IL-6 synthesis by thrombin. Propranolol, a phosphatidic acid phosphohydrolase inhibitor, enhanced the IL-6 synthesis by thrombin. Calphostin C, a highly potent and specific inhibitor for protein kinase C, significantly amplified the IL-6 synthesis by thrombin. The thrombin-induced IL-6 synthesis was enhanced in PKC down-regulated MC3T3-E1 cells. These results strongly suggest that thrombin stimulates IL-6 synthesis, which depends on intracellular Ca2+ mobilization mainly from extracellular space in osteoblasts, and that the IL-6 synthesis by thrombin is regulated due to thrombin-activated protein kinase C through phosphatidylcholine-hydrolyzing phospholipase D.

Antiplatelet effect of FK633, a platelet glycoprotein IIb/IIIa antagonist, on thrombus formation and vascular patency after thrombolysis in the injured hamster carotid artery.

The antithrombotic and restenosis-preventing effects of FK633, an inhibitor of platelet aggregation via binding to the glycoprotein (GP) IIb/IIIa receptor, were studied, IC50 value of FK633 against platelet aggregation ex vivo induced by 2.5 microM adenosine diphosphate (ADP) was 5.4 x 10(-7) M as determined using hamster platelet rich plasma. The inhibitory effect was also investigated in vivo on thrombus formation at the carotid arterial wall injured by a modified catheter. As a control, the left carotid artery was injured and the time required to develop a thrombotic occlusion (3.9 +/- 1.1 min, mean +/- S.E.M., n = 18) was determined. Then, the right carotid artery of the same animal was injured while a continuous intravenous (i.v.) infusion of FK633 was administered at doses of 0 (saline), 0.1, 0.3 or 1.0 mg/kg/h. The time to occlusion was dose-dependently prolonged. In a separate experiment, 10% of the total tPA dose (0.52 mg/kg) was injected into the injured artery as a bolus and the remaining was infused i.v. at a constant rate for 30 min. When FK633 (0.3 or 1.0 mg/kg/h) was infused together with tPA, late patency of the reperfused artery was much improved as compared with that of treatment with tPA alone. Bleeding time, measured at the end of the tPA infusion, was markedly prolonged when the higher dose of FK633 (1.0 mg/kg/h) was coadministered, however coadministration of the lower dose of FK633 (0.3 mg/kg/h) was almost without prolongation on the bleeding time, despite a significant effect on the vascular patency after thrombolysis. Next, neointima formation was evaluated 2 weeks after the vascular injury. When FK633 (0.3 mg/kg/h) was continuously infused i.v. by an implanted osmotic pump for 3, 7 or 14 days after the vascular injury, the neointimal area formation was significantly suppressed in the treatment groups for 7 or 14 days. These findings suggest that FK633 inhibits platelet activation in the injured artery and improves vascular patency after thrombolysis with tPA with a concomitant suppression of neointima formation.

GR144053, a fibrinogen receptor antagonist, enhances the suppression of neointima formation by losartan, an angiotensin II receptor antagonist, in the injured carotid artery of hamster.

1. The present study investigated the inhibitory effect of losartan, a type 1 angiotensin II (AT1) antagonist, and of combined treatment with losartan and GR144053, a fibrinogen receptor (GPIIb/IIIa) antagonist, on neointima formation subsequent to vascular injury in the hamster carotid artery. Vascular injury was achieved by a roughened-tip 2F catheter and the neointimal area was measured up to 2 weeks inducing the injury. 2. Compared to non-treated hamsters (intimal area (IA/internal elastic laminal area (IELA) ratio = 60.3 +/- 5.9%, n = 12), losartan dissolved in drinking water (1, 3 and 10 mg kg-1 per day, n = 8 each) reduced neointimal area dose-dependently, a significant decrease (IA/IELA = 39.7 +/- 5.6%) being attained with the highest dose when it was administered from 1 day before injury. However, neointima formation was not prevented even with the highest dose of losartan when the administration was started after injury. 3. When the administration of GR144053 (1.0 mg kg-1 per hour) via an implanted osmotic pump was started 30 min before the injury and continued for the next 2 weeks, no suppression of neointima formation was observed, although platelet aggregation evoked ex vivo by adenosine diphosphate (ADP) at the end of treatment period was efficiently inhibited. 4. In separate experiments in which 5-bromo-2-deoxy-Uridine (BrdU) was used to test smooth muscle cell (SMC) proliferation 1 and 7 days after injury, the ratio of SMC proliferation in the injured area was only slightly decreased by losartan when its administration was started after the injury, despite the marked reduction of SMC proliferation when treatment was started before the injury. Treatment with GR144053 as indicated above also significantly decreased the SMC proliferating index 1 day after the injury. 5. To examine the potential benefit of the coadministration of the GPIIb/IIIa antagonist with the AT1 receptor antagonist, GR144053 (1.0 mg kg-1 per hour) was combined with post-injury treatment with losartan (10 mg kg-1 per day). This markedly reduced the proliferation of SMCs and significantly decreased the neointimal area (IA/IELA = 31.2 +/- 4.6%) measured 2 weeks following the catheterization. 6. According to the results of a time-dependent study in which GR144053 was given in combination with post injury treatment with losartan for 1, 3, 7 or 14 days, neointima formation could be reduced by treatment with GR144053 for just 7 days. 7. In conclusion, GR144053, a fibrinogen receptor antagonist, enhanced the inhibitory effect of losartan, an AT1 receptor antagonist, on neointima formation in the damaged carotid artery of hamsters.

Mechanical loading activates mitogen-activated protein kinase and S6 peptide kinase in cultured rat cardiac myocytes.

The molecular mechanisms by which overloaded cardiac myocytes increase the cell size (hypertrophy) remain unknown. We have previously shown that mechanical loading increased the protein synthesis and the expression of proto-oncogene c-fos mRNA (Komuro, I., Kaida, T., Shibazaki, Y., Kurabayashi, M., Katoh, Y. Hoh, E., Takaku, F., and Yazaki, Y. (1990) J. Biol. Chem. 265, 3595-3598; Komuro, I., Katoh, Y., Kaida, T., Shibazaki, Y., Kurabayashi, M., Hoh, E., Takaku, F., and Yazaki, Y. (1991) J. Biol. Chem. 266, 1265-1268). It has been known that both mitogen-activated protein (MAP) kinase and S6 kinase can be activated by many kinds of growth factors. To clarify whether MAP kinase(s) and S6 kinase(s) are associated with the intracellular signaling of cardiac hypertrophy induced by mechanical loading, we cultured neonatal rat cardiac myocytes in deformable dishes and imposed an in vitro mechanical loading by stretching the adherent myocytes. In this study, we demonstrated that 1) myocyte stretching maximally activated a kinase activity toward myelin basic protein (MBP) at 10 min after stretching, and the kinase activity returned to the control level at 30 min after stretching; 2) kinase assays in MBP-containing gel, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that stretch-induced MBP kinase activity mainly migrated at 42 kDa in the immunoprecipitated fraction of anti-MAP kinase antibody, suggesting that the stretching mainly increased the 42-kDa MAP kinase activity in cardiac myocytes; 3) phosphorylation of MAP kinase was induced after stretching cardiac myocytes; 4) when protein kinase C was depleted by preincubating myocytes with 100 nM 12-O-tetradecanoyl-phorbol-13-acetate for 24 h or 2 nM staurosporine for 30 min, stretch-induced MBP kinase activity was decreased by approximately 60-70% as compared with the kinase activity in myocytes without protein kinase C depletion; 5) although the receptor tyrosine kinases were depleted by preincubating myocytes with 50 microM tyrphostin or 20 microM genistein for 30 min, there was no change in the stretch-induced MBP kinase activity; 6) stretch-induced MBP kinase activity was partially dependent on transsarcolemmal influx of Ca2+; 7) myocyte stretching also increased S6 peptide (RRLSSLRA) kinase activity in the anti-S6 kinase II antibody immunoprecipitates. From these results, we conclude that myocyte stretching increases the activities of MAP kinase and S6 peptide kinase, which may play an important role in the induction of the specific genes and the increase in the protein synthesis.