Protein Kinase C Protein Expression Research Articles

Chemopreventive and hepatoprotective effects of genistein via inhibition of oxidative stress and the versican/PDGF/PKC signaling pathway in experimentally induced hepatocellular carcinoma in rats by thioacetamide

ABSTRACT Objective: Genistein is a recognized isoflavone present in soybeans with antioxidant, anti-inflammatory, antiangiogenic and antitumor activities. This study aimed to test ability of genistein in modulating versican/platelet derived growth factor (PDGF) axis in HCC. Methods: HCC was experimentally induced in male Sprague-Dawley rats then treated with 25 or 75 mg/kg genistein. Antioxidant activities of genistein was assessed by measuring the gene expression of Nrf2 and the hepatic levels of malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione. Expression of versican, PDGF, protein kinase C (PKC) and ERK-1 protein was assessed by Western blotting and immunostaining. Results: HCC induced an elevation in oxidative stress, PDGF, versican, PKC and ERK protein expression levels. Genistein significantly reduced an HCC-induced increase in oxidative stress. Moreover, genistein dose-dependently reduced HCC-induced elevation of PDGF, versican, PKC and ERK protein expression levels. Moreover, genistein helped retain a normal hepatocyte structure and reduced fibrous tissue deposition, especially in high dose. Conclusions: Genistein exerted antitumor and antioxidant effects and therefore suppress HCC development via inhibition of the PDGF/versican bidirectional axis, suppressing both ERK1 and PKC as downstream regulators. Therefore, genistein is a potential novel therapeutic candidate for improving the outcome of patients with HCC. Highlights Genistein is an isoflavones present in various soybeans and soy products. Genistein produced antitumor activity not only due to its antioxidant activity. Genistein expression of versican, PDGF, PKC and ERK. Genistein produced hepatoprotective effects.

Diabetes Mellitus Promotes Smooth Muscle Cell Proliferation in Mouse Ureteral Tissue through the P-ERK/P-JNK/VEGF/PKC Signaling Pathway

Background and objectives: The aim of our study was to evaluate the role of diabetes mellitus (DM) as a significant factor affecting spontaneous stone expulsion, as suggested by previous research. Materials and methods: We investigated the influence of DM on the ureter using a murine model. The mouse-model arm of this study used 20 15 -week-old mice, including 10 normal (control) mice and 10 DM mice. We measured the proximal, middle and distal ureteral smooth muscle thickness in each mouse and the differences among ureteral sections were analyzed. Mouse ureteral specimens were also analyzed via western blotting to detect relative protein expression of phosphor–extracellular signal regulated kinases (P–ERK), phosphor–C–Jun N–terminal kinase (P–JNK), vascular endothelial growth factor (VEGF), and protein kinase C (PKC), which are representative factors involved in cell regulation. Results: We observed significant hyperproliferation of ureteral smooth muscle in DM mice compared to normal mice, which may provoke reduced peristalsis. The ureteral smooth muscle of DM mice was significantly thicker than that of normal mice in all ureteral tissues: proximal (p = 0.040), mid (p = 0.010), and distal (p = 0.028). The relative protein expression of P-ERK (p = 0.005) and P–JNK (p = 0.001) was higher in the diabetic group compared to the normal group. Additionally, protein expression of VEGF (p = 0.002) and PKC (p = 0.001) were remarkably up-regulated in DM mice. Conclusions: Hyperproliferation of ureteral smooth muscle was observed in DM mice, but not in normal mice. The pathways mediated by P–ERK, P–JNK, VEGF, and PKC may play an important role in pathological ureteral conditions.

Protein and mRNA expression of protein kinase C (PKC) in the postmortem brain of bipolar and schizophrenic subjects

Abnormalities of protein kinase C (PKC) have been implicated in the pathophysiology of bipolar (BP) illness. This is primarily based on studies of PKC in platelets of BP patients. Whether such abnormalities of PKC activity and isoforms exist in the brain is unclear. We have therefore determined PKC activity, protein and mRNA expression of PKC isoforms in the prefrontal cortex (PFC), cingulate cortex (CING) and temporal cortex (TEMP) from BP (n = 19), schizophrenic (SZ) (n = 20) and normal control (NC) (n = 25) subjects. The brain samples were obtained from the Harvard Brain Bank, and the subjects were diagnosed according to DSM-IV criteria. Protein levels were determined using Western blot technique and mRNA levels were determined using real-time PCR (qPCR) method. We found that there was a significant decrease in the PKC activity in the cytosol and membrane fractions of PFC and TEMP obtained from BP subjects but not from SZ subjects. When we compared the expression of PKC isozymes, we found that the protein and mRNA expression of several isozymes was significantly decreased in the PFC (i.e., PKCα, PKCβI, PKCβII and PKCε) and TEMP (i.e., PKCα, PKCβI, PKCβII, PKCε and PKCγ) of BP subjects, but not in the CING. Overall, there was no difference in the mRNA or protein expression of PKC isozymes between SZ and NC subjects in any of the three brain areas we studied. Our results show that there is a region-specific decrease of certain PKC isozymes in the membrane and cytosol fractions of BP but not SZ subjects.

Effects of apolipoprotein E on proliferation of mouse pulmonary arterial smooth muscle cells induced by hypoxia

To investigate the effects of apolipoprotein E (apoE) on the proliferation of pulmonary arterial smooth muscle cells (PASMCs) induced by hypoxia. Primary culture of mouse PASMCs was prepared from male C57BL/6 mouse pulmonary artery by the method of tissue block anchorage. PASMCs were divided into four groups: normoxia group, normoxia with apoE administration group, hypoxia group and hypoxia with apoE administration group. The proliferation of PASMCs was observed by EdU incorporation. The protein levels of apoE, proliferating cell nuclear antigen (PCNA), protein kinase C (PKC) and phosphorylated protein kinase C (p-PKC) were analyzed by Western blot. The percentage of PASMCs proliferation of hypoxia group was significantly higher than that of normoxia group by 64.7% (P＜0.05), and the protein expression levels of PCNA and p-PKC of hypoxia group were up-regulated than those of normoxia group by 69.0% and 120.0%, while the protein expression of apoE was down-regulated by 51.0% (P＜0.05), respectively. The percentage of PASMCs proliferation of hypoxia with apoE administration group was significantly lower than that of hypoxia group by 19.6% (P＜0.05), and the protein expression levels of PCNA and p-PKC of hypoxia with apoE administration group were down-regulated than those of hypoxia group by 19.8% and 103.2% (P＜0.05), respectively. There was no significant difference among each group in the protein expression of PKC, nor do there any significant difference between normoxia group and hypoxia group in the protein expression of p-PKC (P＞0.05). ApoE can inhibit the proliferation of PASMCs induced by hypoxia, and the mechanism of its effect may be attributed to blocking PKC pathway.

Phosphorylated SNAP25 in the CA1 regulates morphine-associated contextual memory retrieval via increasing GluN2B-NMDAR surface localization.

Although our previous studies have demonstrated both protein kinase C (PKC) and GluN2B-containing N-methyl-d-aspartate receptor (GluN2B-NMDAR) play crucial roles in morphine-associated learning and memory, the relationship between them remains unexplored. In this study, we validated the enhanced PKC and membrane GluN2B protein expression in the hippocampal CA1 after morphine conditioned place preference (CPP) expression in rats. Interestingly, we also found that phosphorylation of SNAP25 at Ser187 (pSer187-SNAP25), a PKC-activated target, was significantly increased following morphine CPP expression. Blocking the pSer187-SNAP25 by intra-CA1 injection of an interfering peptide impaired morphine CPP expression and accompanied by the reduced ratio of GluN2B membrane/total in the CA1. In addition, intra-CA1 blockade of pSer187-SNAP25 did not affect natural learning and memory process as evidenced by intact sucrose-induced CPP expression and normal locomotor activity in rats. Therefore, our results reveal that enhanced pSer187-SNAP25 by PKC recruits GluN2B-NMDAR to the membrane surface in the hippocampal CA1 and mediates context-induced addiction memory retrieval. Our findings in this study fill in the missing link and provide better understanding of the molecular mechanisms involved in morphine-associated contextual memory retrieval.

Protective role of Pollen Typhae total flavone against the palmitic acid-induced impairment of glucose-stimulated insulin secretion involving GPR40 signaling in INS-1 cells.

Prolonged elevated levels of free fatty acids (FFAs) contribute to the impairment of insulin secretion function of pancreatic βcells, a hallmark of type2 diabetes, which is partly attributed to the dysfunction of G-protein-coupled receptor40 (GPR40) signaling. Pollen Typhae total flavone(PTF), an extract from a Chinese herbal medicine named Pollen Typhae, has been reported to effectively treat type2 diabetes, but the underlying mechanisms remain to be fully elucidated. In the present study, palmitic acid(PA), a saturated fatty acid, severely impaired glucose-stimulated insulin secretion (GSIS) in a time-dependent manner in INS-1 cells, and PTF treatment prevented the impairment in a dose-dependent manner. Moreover, PTF improved insulin secretion function in rats presenting with type2 diabetes induced by a high-fat diet and low-dose streptozotocin. Furthermore, PA exposure for 24h decreased the protein expression of GPR40, phospholipaseC(PLC)β1, PLCβ3, and protein kinaseC(PKC), and inhibited the activity of PLC and PKC stimulated by GW9508, a GPR40 agonist. In addition, PTF enhanced the protein expression of GPR40 and to a certain extent strengthened the protein expression of PKC, increased cellular levels of triphosphoinositide (IP3) and diacylglycerol (DAG), and promoted GW9508-stimulated activity of PLC and PKC reduced by PA in INS-1 cells, which were blocked by PLC inhibitor U-73122 and PKC inhibitor staurosporine, respectively. Additionally, the improvement in PA-induced impairment of GSIS by PTF in INS-1 cells was restrained by U-73122, staurosporine, and calcium channel inhibitor nifedipine, respectively. The results indicate that PTF exerts a protective role against PA-induced impairment of GSIS involving GPR40 signaling in INS-1 cells.

Cyclic stretch induces human bladder smooth muscle cell proliferation in vitro through muscarinic receptors.

The present study aimed to investigate whether the cyclic stretch‑induced proliferation of human bladder smooth muscle cells (HBSMCs) is mediated by muscarinic (M) receptors, together with the signal transduction mechanisms involved in this process. HBSMCs seeded onto silicone membranes were subjected to different cyclic stretches (5, 10, 15 and 20%) for 6 and 12 h. As the effect of cyclic stretch on M2 and M3 mRNA expression levels was maximal at 6 h 10% stretch, all subsequent experiments were performed at this stretch. Western blot analysis was used to quantify M2, M3, protein kinase C (PKC) and phosphorylated (p)‑PKC protein expression levels, flow cytometry was employed to examine cell cycle distribution and a 5-bromo‑2-deoxyuridine (BrdU) incorporation assay was used to assess cell proliferation at this stretch. Subsequently, HBSMCs were exposed to different acetylcholine concentrations and/or cyclic stretch, M receptor antagonists [AF-DX16, an M2 receptor antagonist; 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), an M3 receptor antagonist and atropine, a non‑selective antagonist] and GF 109203X, a PKC antagonist, to assess the possible underlying signaling mechanisms. Cyclic stretch was found to increase the proliferation of HBSMCs and the expression levels of M2, M3, PKC and p‑PKC proteins. M receptor and PKC antagonists exerted no apparent effect on nonstretched cells, but reduced the incorporation of BrdU into stretched cells; the most pronounced effects were observed when non‑selective M receptor and PKC antagonists were applied. Notably, 4‑DAMP did not inhibit stretch‑induced PKC activation. These results indicate that the activation of the M3 receptor signaling pathway in stretch‑induced HBSMC proliferation occurs via PKC-independent mechanisms.

Protein kinase C-nuclear factor-erythroid 2-related factor 2 regulating the expression of heme oxygenase-1 in rat airway epithelial cells

To observe the effect of the signaling pathway of protein kinase C (PKC)-nuclear factor-erythroid 2-related factor 2 (Nrf2) on the expression of heme oxygenase -1 (HO-1) induced by cigarette smoke extract in rat airway epithelial cells. The airway epithelial cells of 25 male SD rats were randomly divided into a control group, a CSE3h group, a RO318220 group (PKC inhibitor), a Nrf2 siRNA group and a Nrf2 siRNA+RO318220 group, 5 rats in each group. The control group was incubated with DMEM/F12 alone. The CSE3h group was treated with 10% CSE for 3 h. The RO318220 group was pretreated with 3 μmol/L RO318220 for 0.5 h and subsequently treated with 10% CSE for 3 h. The Nrf2 siRNA group was pretreated with Nrf2 siRNA, and then treated with 10% CSE for 3 h. The Nrf2 siRNA+RO318220 group was pretreated with Nrf2 siRNA and 3 μmol/L RO318220 for 0.5 h, and then treated with 10% CSE for 3 h. The protein levels of Nrf2 in the nucleus and cytoplasm, and HO-1 and PKC in the whole cells were semi-quantified by Western blot. The protein expression of HO-1 was measured by immunocytochemistry. HO-1 mRNA expression was detected by RTPCR. Immunofluorescence staining was used to observe the nuclear translocatin of Nrf2. CSE markedly induced Nrf2 nuclear translocation in the rat airway epithelial cells, and RO318220 pretreatment blocked the CSE induced Nrf2 nuclear translocation. Immunocytochemistry showed that HO-1 protein expression was strongly positive in the CSE3h group, weakly positive in the other 4 groups. The expression of PKC protein, HO-1 mRNA and protein significantly increased in the CSE3h group, and HO-1 activity markedly improved in the CSE group (P<0.05). The level of PKC protein expression was not significantly different in the Nrf2 siRNA group compared with that in the CSE3h group (P>0.05). CSE induces the nuclear translocation of Nrf2 by PKC signaling pathway, thus upregulating the HO-1 expression in the rat airway epithelial cells.

The effect of blue light on Ca2+-protein kinase C signaling pathway in human retinal pigment epithelial cells in vitro

Objective To investigate the effect of blue light on Ca2+-protein kinase C (PKC)signaling pathway in human retinal pigment epithelial (RPE) cells in vitro.Methods Primary human RPE cells were cultured in vitro and characterized.The experiments were carried out using the 4th generation of human RPE cells.The PKC protein level was measured by Western blot to determine the most appropriate concentration of phorbol ester (PMA) and calcium phosphate binding protein (calphostin C) on PKC expression.Non-radioactive isotope method was used to determine the effect of blue light on PKC expression of cultured cells.Blue-light damage model of human RPE cells was established by 6 hour irradiation of medical blue-light lamp [20 W,450-500 nm wavelength,(2000± 500) Lux],and 24 hours prolongation of post-exposure culture.The human RPE cells were randomly divided into 5 groups.Group A did not receive light irradiation,group B only received blue light irradiation,group C was blue light irradiation and 0.1 mmol/L nifedipine treatment,group D was blue light irradiation and 100.0 nmol/L calphostin C treatment,group E was blue light irradiation and 100.0 nmol/L PMA treatment.Intracellular Ca2-concentration was measured by acetoxymethyl ester (Fluo 3-AM) labelling and confocal microscope imaging.Results The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L PMA-treated groups was higher than 0.1,1.0,10.0,and 50.0 nmol/L PMA-treated groups,the difference was statistically significant (F=217.537,P＜0.05),but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L PMA-treated groups (P =0.072).The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L calphostin C-treated groups was lower than 5.0,25.0,50.0,and 75.0 nmol/L calphostin C-treated groups,the difference was statistically significant (F=164.543,P＜0.05),but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L calphostin C treated groups (P=0.385).PKC level in blue light group was higher than non-light group,the difference was statistically significant (t =-9.869,P＜0.05).The Ca2+ fluorescence intensity values in group B,C,D and E was higher than group A,the difference was statistically significant (F=26 764.92,P＜0.05).The Ca2+ fluorescence intensity values in group E was higher than group B,C and D (P＜0.05),and that in group B was higher than group C and D (P＜0.05).Conclusions The PKC activity and intracellular Ca2+ concentration in human RPE cells increase after blue light irradiation.Both calcium channel inhibitor nifedipine and PKC inhibitor calphostin C can reduce intracellular Ca2+ concentration in human RPE cells.PMA can induce intracellular Ca2+ concentration in human RPE cells after blue light irradiation. Key words: Eye injuries/physiopathology; Retinal pigment epithelium; Apoptosis; Photic stimulation

Role of PKC and CaV1.2 in detrusor overactivity in a model of obesity associated with insulin resistance in mice.

Obesity/metabolic syndrome are common risk factors for overactive bladder. This study aimed to investigate the functional and molecular changes of detrusor smooth muscle (DSM) in high-fat insulin resistant obese mice, focusing on the role of protein kinase C (PKC) and Cav1.2 in causing bladder dysfunction. Male C57BL/6 mice were fed with high-fat diet for 10 weeks. In vitro functional responses and cystometry, as well as PKC and Cav1.2 expression in bladder were evaluated. Obese mice exhibited higher body weight, epididymal fat mass, fasting glucose and insulin resistance. Carbachol (0.001–100 µM), α,β-methylene ATP (1–10 µM), KCl (1–300 mM), extracellular Ca2+ (0.01–100 mM) and phorbol-12,13-dibutyrate (PDBu; 0.001–3 µM) all produced greater DSM contractions in obese mice, which were fully reversed by the Cav1.2 blocker amlodipine. Cystometry evidenced augmented frequency, non-void contractions and post-void pressure in obese mice that were also prevented by amlodipine. Metformin treatment improved the insulin sensitivity, and normalized the in vitro bladder hypercontractility and cystometric dysfunction in obese mice. The PKC inhibitor GF109203X (1 µM) also reduced the carbachol induced contractions. PKC protein expression was markedly higher in bladder tissues from obese mice, which was normalized by metformin treatment. The Cav1.2 channel protein expression was not modified in any experimental group. Our findings show that Cav1.2 blockade and improvement of insulin sensitization restores the enhanced PKC protein expression in bladder tissues and normalizes the overactive detrusor. It is likely that insulin resistance importantly contributes for the pathophysiology of this urological disorder in obese mice.

Effect of Huannao Yicong Prescription (还脑益聪方) extract on β-Amyloid precursor protein metabolic signal transduction-related protein in brain tissue of dementia model transgenic mouse

To observe the effect of Huannao Yicong Prescription (, HNYC, a Chinese medical compound) extract on β-amyloid precursor protein (APP) metabolic signal transduction related protein kinase C (PKC), tyrosine amyloid protein kinase (TrKA), and glycogen synthase kinase-3 (GSK-3) in brain tissue of transgenic mouse dementia model induced by APP. Sixty dementia model transgenic 3-month-old mice induced by APP695V717I were randomly allocated in four groups: the model group (A), the Donepezil (0.65×10(-3) g·kg(-1)·(-1))-treated group (B), and the two HNYC-treated groups (C and D) with high dosage (2.8 g·kg(-1)·(-1)) and low dosage (1.4 g·kg(-1)·(-1)) of HNYC extract, respectively, 15 mice in each group. Besides, a normal control group was set up with 15 C57BL/6J mice with the same age and genetic background as the model mice. The drugs for treatment were administered once a day by dissolving in equal-volume distilled water through gastric infusion, continued for 6 months, to mice in group A and to normal control group equal-volume distilled water was administered instead. Spatial learning and memory capacity of mice were observed by Morris water maze; their one-time escape response memory capacity was tested by diving platform; and changes of PKC, TrkA, and GSK-3 levels in hippocampus and cortex of brain were detected by Western blotting. HNYC extract showed significant effects on increasing the time of model mice for swimming through the flat roof and the swimming time and path in the fourth quadrant P<0.05 or P<0.01). Diving platform test showed that the latent times in Groups B and C were longer than that in Group A significantly (P <0.05 and P<0.01). Compared with the normal control group, PKC and TrkA protein expression levels in hippocampus and cortex of model mice's brain lowered significantly (P<0.01), while GSK-3 protein expression increased significantly (P<0.01); compared with Group A (the model group), hippocampal and cortical levels of PKC protein expression in the intervened groups (B-D) as well as those of TrkA in Group C were higher (P<0.01 or P<0.05), while hippocampal levels of GSK-3 in intervened groups were lower (P<0.01). HNYC extract could obviously increase the protein expressions of PKC and TrkA and decrease the expression of GSK-3 protein in brain tissue of transgenetic mice model of dementia, and regulate APP metabolic signal transduction path, and thus to suppress the production of Aβ, which is one of the dominant mechanisms for improving learning/memory capacity of dementia model animals.

779 Androgen Receptor Phosphorylation is Associated With Clinical Outcome Measures in Hormone Naive Prostate Cancer

Introduction: Although prostate cancer (Pca) can be readily diagnosed, estimation of likely outcome remains problematical as many will have a benign course and will not die of their disease, while some patients progress rapidly to castrate resistant disease and die within 18−24 months. Ideally, there would be a factor or factors which would evidently set apart those patients who will, in due course succumb to the disease from those who will not. In Pca activation of signal transduction cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the candidate kinases mediating phosphorylation is not conclusive. Recent evidence suggests that Protein kinase C (PKC) phosphorylates AR at Serine 578 (pAR). Materials and Methods: The aim of the current study was to investigate if the relationship between the AR and PKC is upheld in clinical prostate cancer specimens. Scansite 2.0 predicted PKC as a strong candidate mediating phosphorylation of AR at serine residue 578. Immunohistochemical analysis was performed to assess protein expression levels of PKC and pAR in a cohort of 90 patients with hormone naive prostate adenocarcinoma. Results and Discussion: High cytoplasmic pAR expression (> median) was significantly associated with time to biochemical relapse (p = 0.034) and with decreased time to death from relapse (p = 0.034). Additionally, high expression of cytoplasmic pAR was significantly associated with decreased disease-specific survival (p = 8.6×10−5), as was high nuclear pAR expression (p = 0.036). Protein expression of PKC was not significantly associated with survival. However, nuclear PKC correlated strongly with pAR expression, in both the cytoplasm (c.c 0.426, p = 0.002) and nucleus (c.c. 0.469, p = 0.001). Conclusion: These results suggest that the phosphorylation of the AR by PKC may be of functional importance in the clinical setting. Consistent with predictions by Scansite expression of PKC significantly correlated with phosphorylation of pAR. Phosphorylation pAR is implicated in Pca progression and survival. Further work investigating the functional consequences of inhibition of AR phosphorylation at serine 578 is warranted.

Involvement of kappa opioid receptors in the formalin-induced inhibition of analgesic tolerance to morphine via suppression of conventional protein kinase C activation

Repeated morphine treatment results in a decreased analgesic effect or the development of analgesic tolerance. However, we reported that some inflammatory chronic pain may inhibit morphine tolerance via kappa opioid receptor (KOR) activation. In this study, we further investigated the role of KOR in the inhibition of morphine tolerance in a chronic pain condition with a focus on the regulation of protein kinase C (PKC) activity. Chronic pain was induced by formalin treatment into the dorsal part of the left hind paws of mice. The analgesic effect of morphine was measured by the tail flick method. We analysed the protein expression of PKC and its activity, and G-protein activity of mu opioid receptor (MOR) under repeated morphine treatment with or without formalin treatment. We found that conventional subtypes of PKC (cPKC) were up-regulated by repeated morphine treatment. Also, antisense oligonucleotide (AS-ODN) targeting cPKC completely suppressed the development of morphine tolerance. The disappearance of the repeated morphine-induced up-regulation of cPKC was completely reversed by treatment with AS-ODN targeting KOR. In addition, AS-ODN targeting KOR significantly reversed the chronic pain-induced down-regulation of PKC activity or up-regulation of MOR [(35)S]GTPgammaS binding activity after repeated morphine treatment. These results indicate that KOR plays an important role in the inhibition of repeated morphine-induced cPKC up-regulation under chronic pain condition. Furthermore, this may result in the increase of MOR activity and in the inhibition of morphine tolerance under chronic pain condition.

Protein kinase C is inhibited by bisphosphonates in prostate cancer PC-3 cells

Bisphosphonates are expected to be effective at preventing tumor metastasis to bone tissue. Since protein kinase C (PKC) plays a crucial role in cancer progression, we examined the effect of bisphosphonates on PKC expression to clarify the mechanism behind the inhibition of the bone metastasis of prostate cancer by bisphosphonates. We found that pamidronate inhibits PKC protein expression and PKC activity in prostate cancer PC-3 cells. PKC protein expression was markedly reduced by treatment with 100 microM of pamidronate. The inhibitory effect of PKC expression by pamidronate was specific for PKCalpha and PKCzeta. Nitrogen-containing bisphosphonates are known to inhibit the mevalonate pathway, but the effect of pamidronate on PKC expression was not due to the inhibition of this pathway. Urokinase-type plasminogen activator (uPA) is one of the critical proteins in tumor metastasis and decreased in bisphosphonate-treated PC-3 cells. We also showed that uPA expression was suppressed by PKC inhibitors (calphostin C and staurosporine) and induced by a PKC activator (PMA) in PC-3 cells, suggesting that the inhibition of uPA by bisphosphonates is involved in PKC inhibition. This is the first finding that bisphosphonates suppress PKC expression in cancer cells. These results strongly suggest that one of the mechanisms behind the inhibitory effect of bisphosphonates on tumor bone metastasis is mediated by PKC inhibition.

C-FLIPL regulates PKC via AP-2 to inhibit Bax-mediated apoptosis induced by HIV-1 gp120 in Jurkat cells

c-FLIPL, an inhibitor of caspase 8, is known to inhibit the Fas/caspase 8 apoptotic pathway; however, its involvement of Bax/mitochondrial apoptosis is not well understood. Using human cells, Jurkat cell line, induced with HIV-1 gp120, we studied the effects of c-FLIPL on Bax/mitochondrial apoptosis. We found that the induction of apoptosis by HIV-1 envelope protein, gp120, involved the activation of both Bax-dependent and death receptor-mediated pathways, and HIV-1 infection deceased c-FLIPL expression. Interestingly, c-FLIPL expression downregulated protein kinase C (PKC) expression at the transcript level involving activated protein-2 (AP-2). c-FLIPL expression reduced AP-2 protein levels required to promote PKC protein expression and PKC-associated inactive form of Bax, and inhibited Bax activation, suggesting that c-FLIPL inhibits Bax activation via modulating PKC expression at the transcriptional level involving AP-2 during gp120 treatment. Collectively, these findings further corroborate the concept that gp120 plays an important role, via involvement of molecules such as c-FLIPL, in apoptotic cell death due to HIV-1 infection.

Differentiation-Associated Expression of Conventional Protein Kinase C Isoforms in Primary Cultures of Bone Marrow Cells Induced by M-CSF and G-CSF

The Protein kinase C (PKC) -associated signal pathway plays crucial roles in regulation of cell growth, differentiation and apoptosis. The present study focuses on conventional PKC (cPKC) expression and its regulation in primary cultures of bone marrow cells induced to undergo macrophage/granulocyte differentiation by macrophage colony-stimulating factor (M-CSF) or granular colony-stimulating factor (G-CSF). By performing western blot analysis with pan anti-PKC antibodies, we found that PKC is transiently induced by M-CSF, reaching a maximum level by day 2, and then declines and diminishes by day 9 in primary culture of bone marrow cells. In contrast, the expression of PKC along G-CSF induced granulocytic differentiation of bone marrow stem cells is low and increases gradually. Reverse transcription-PCR (RT-PCR) assay was utilized to investigate the expression of PKC isoforms. PKC-alpha is constitutively expressed in bone marrow cells independently of hematopoietic growth factors in cultures. PKC-gamma mRNA is undetectable. Similarly, the expression of PKC-beta is transiently induced by M-CSF, yet steadily increased by G-CSF, in agreement with results obtained from PKC protein expression. Furthermore, gel-shift assay showed that the activation of NF-kappaB is transiently induced by M-CSF but not by G-CSF. These data suggest that PKC expression is involved in both macrophage and granulocyte differentiation by bone marrow committed stem cells. Yet, NF-kappaB activation is only detected in macrophage and not granulocyte differentiation. Thus, we conclude that the PKC-mediated signaling pathway is distinctly involved in bone-marrow cell differentiation induced by M-CSF and G-CSF.

Comparison of the involvement of protein kinase C in agonist-induced contractions in mouse aorta and corpus cavernosum

Protein kinase C (PKC) is involved in the regulation of vascular smooth muscle contraction. However, the role of PKC in erectile function is poorly understood. This study investigated whether PKC mediates agonist-induced contractions in mouse penile tissue (corpora cavernosa). We also compared the effects of PKC activators and inhibitors on contractile responses in mouse corpus cavernosum with those in mouse aorta. Aortic rings and corpus cavernosal strips from C57BL/6J mice were mounted in the organ bath for isometric tension recording. Our data showed that a PKC α/β selective inhibitor, G ö6976 (10 µM), inhibited phenylephrine and 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F 2α (U46619, a thromboxane mimetic)-induced contractions in mouse aorta, reducing the maximum contraction by 94% and 17%, respectively. A non-selective PKC inhibitor, chelerythrine (30 µM), also significantly reduced phenylephrine- and U46619-induced maximum contractions in mouse aorta. However, G ö6976 and chelerythrine had no significant effects on phenylephrine- and U46619-induced contractions in corpus cavernosum. Furthermore, a PKC activator, phorbol-12,13-dibutyrate (0.1 µM), significantly increased contractions in aorta (208 ± 14% of KCl-induced maximum contraction) but failed to cause contractions in corpus cavernosum at 1 and 10 µM. Western blot analysis data suggested that protein expression of PKC was similar in aorta and corpus cavernosum. Taken together, our data indicate that PKC does not have a significant role in agonist-induced contractions in mouse corpus cavernosum, whereas it mediates the contractile response to agonists in the aorta.

Altered Penile Vascular Reactivity and Erection in the Zucker Obese-Diabetic Rat

The combination of the independent risk factors for erectile dysfunction, obesity, hypertension, and diabetes are manifested collectively in a condition known as metabolic syndrome X. However, the exact mechanism(s) by which the combination of these factors contributes to erectile dysfunction have yet to be elucidated. We hypothesized that protein kinase C (PKC) and Rho-kinase enhanced vascular tone and thus contributed to erectile dysfunction in this condition. Erectile function was evaluated by recording voltage-dependent increases in intracavernosal pressure following stimulation of the cavernosal nerve in 16- to 20-week-old lean and obese-diabetic Zucker rats. Cavernosal tissue contractile and relaxation responses were evaluated in vitro when contracted with phenylephrine, endothelin-1 and relaxed by Rho-kinase, PKC inhibitors or sodium nitroprusside. Additionally, cavernosal tissue Rho-kinase, protein kinase, and nitric oxide synthase isoform expression were evaluated by Western blot. The voltage-dependent erectile responses were suppressed by >30% in the obese-diabetic Zucker rat. The maximal stress generated by cavernosal tissue from the obese-diabetic was significantly greater than the lean response by greater than 0.8 mN/mm(2) for both phenylephrine and endothelin stimulation. The PKC inhibitor, chelerythrine, inhibited more than 30% of the phenylephrine-induced and 70% of the endothelin-1-induced contractions. Rho-kinase inhibition, with either Y-27632 or HA-1077, revealed impaired relaxations of nearly 30% in tissue from obese-diabetic animals. Western blot analysis revealed increased protein expression of PKC alpha and delta and Rho-kinase alpha and beta but no loss for endothelial or neuronal nitric oxide synthase. In this rodent model both PKC and Rho-kinase signaling elements may contribute to an enhanced vasoconstriction state of the penile smooth muscle that was differentially dependent on the agonist used. The enhanced vasoconstrictive state of the tissue could contribute to the reduced voltage-dependent erectile response in the obese-diabetic Zucker rat.

Elevated Endothelin-1 Levels Impair Nitric Oxide Homeostasis Through a PKC-Dependent Pathway

Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone and pathological states such as ischemia/reperfusion (I/R) injury, coronary vasospasm, and cardiac allograft vasculopathy. We assessed the effects of elevated ET-1 levels as seen after I/R to determine if ET-1 modulates nitric oxide (NO) production via the translocation of specific protein kinase C (PKC) isoforms. Human saphenous vein endothelial cells (HSVECs) (n=8) were incubated with ET-1 or phosphate-buffered saline (PBS) for 24 hours. NO production was determined in the supernatant by measuring nitrate/nitrite levels. Protein expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), caveolin-1 and PKC were determined. Lastly, PKC translocation and activity were assessed after exposure to the drug of interest. HSVECs exposed to ET-1 displayed decreased NO production. PKC inhibition reduced NO production, whereas PKC activation increased production. NO production was maintained when HSVECs exposed to ET-1 were treated with the PKC agonist, PMA. eNOS protein expression was reduced after ET-1 treatment. PKC inhibition also downregulated eNOS protein expression, whereas PMA upregulated expression. ET-1 exposure led to a significant increase in PKCdelta and PKCalpha translocation compared with control, whereas translocation of PKClambda was inhibited. ET-1 exposure significantly reduced overall PKC activity compared with control. Our study demonstrates that high levels of ET-1 impair endothelial NO production via an isoform-specific PKC-mediated inhibition of eNOS expression. ET-1 antagonism with bosentan stimulates translocation of PKClambda and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.

Protein kinase C-α regulation of gallbladder Na + transport becomes progressively more dysfunctional during gallstone formation

Gallbladder Na+ absorption and biliary Ca2+ are both increased during gallstone formation and may promote cholesterol nucleation. Na+/H+ exchange (NHE) is a major pathway for gallbladder Na+ transport. Ca2+-dependent second messengers, including protein kinase C (PKC), inhibit basal gallbladder Na+ transport. Multiple PKC isoforms with species- and tissue-specific expression have been reported. In this study we sought to characterize Ca2+-dependent PKC isoforms in gallbladder and to examine their roles in Na+ transport during gallstone formation. Gallbladders were harvested from prairie dogs fed either nonlithogenic chow or 1.2% cholesterol-enriched diet for varying periods to induce various stages of gallstone formation. PKC was activated with the use of phorboldibutyrate, and we assessed gallbladder NHE regulation by measuring unidirectional Na+ flux and dimethylamiloride-inhibitable 22Na+ uptake. We measured gallbladder PKC activity with the use of histone III-S phosphorylation and used Gö 6976 to determine PKC-alpha contributions. Gallbladder PKC isoform messenger RNA and protein expression were examined with the use of Northern- and Western-blot analysis, respectively. Prairie dog and human gallbladder expresses PKC-alpha, betaII, and delta isoforms. The PKC activation significantly decreased gallbladder J(Na)(ms) and reduced baseline 22Na+ uptake by inhibiting NHE. PKC-alpha mediated roughly 42% of total PKC activity under basal conditions. PKC-alpha regulates basal gallbladder Na+ transport by way of stimulation of NHE isoform NHE-2 and inhibition of isoform NHE-3. PKC-alpha blockade reversed PKC-induced inhibition of J(Na)(ms) and 22Na+ uptake by about 45% in controls but was progressively less effective during gallstone formation. PKC-alpha contribution to total PKC activity is progressively reduced, whereas expression of PKC-alpha mRNA, and protein increases significantly during gallstone formation. We conclude that PKC-alpha regulation of gallbladder NHE becomes progressively more dysfunctional and may in part account for the increased Na+ absorption observed during gallstone formation.