PKCα Protein Expression Research Articles

Processed product (Pinelliae Rhizoma Praeparatum) of Pinellia ternata (Thunb.) Breit. Alleviates the allergic airway inflammation of cold phlegm via regulation of PKC/EGFR/MAPK/PI3K-AKT signaling pathway

Ethnopharmacological relevancePinelliae Rhizoma Praeparatum (PRP) is a traditional processed product of Pinellia ternata (Thunb.) Berit., which mainly used for treating cold asthma (CA). However, the mechanism of action of PRP for treating CA have not been fully elucidated. Aim of the studyTo investigate the core active constituents and the pharmacological mechanism of PRP against CA. Materials and methodsOvalbumin (OVA) and cold water-induced cold asthma model were established in male mice. The effects of water extract from PRP were evaluated by general morphological observation, expectorant activity, airway hyperresponsiveness, mucus hypersecretion, inflammatory cytokines, etc. Additionally, the mRNA and protein expression of mucin 5AC (MUC5AC) and aquaporin 5 (AQP5) in vivo and in vitro were detected by immunohistochemistry (IHC), qRT-PCR, and western blotting. The mechanisms of action were investigated through network pharmacology and transcriptomic, and validated through western blotting and molecular docking. ResultsPRP exhibited a favorable expectorant activity, and significantly reduced the airway inflammation, mucus secretion, and hyperresponsiveness in cold asthma model. It also reduced the levels of IL-4, IL-5, IL-8, and IL-13 in bronchoalveolar lavage fluid (BALF) and IL-4 and total IgE in serum, while obviously increased the levels of IL-10 and IFN-γ in serum for asthmatic mice. Meanwhile, PRP also attenuated the pathological changes and mucus production in cold asthmatic mice. Moreover, the downregulation of MUC5AC and upregulation of AQP 5 were detected by western blotting and qRT-PCR after administration with PRP both in vivo and in vitro. PRP expectedly inhibited the protein expression of PKC-α, SRC, p-EGFR, p-ERK1/2, p-JNK, p-p38, p-PI3K, and p-Akt levels in vivo. ConclusionsThese combined data showed that PRP suppressed the allergic airway inflammation of CA by regulating the balance of Th1 and Th2 cytokines and the possible involvement of the PKC/EGFR/MAPK/PI3K-Akt signaling pathway. Pentadecanoic acid, licochalcone A, β-sitosterol, etc. were considered as main active ingredients of PRP against CA. This study provides a novel perspective of the classical herbal processed product PRP in the treatment of CA.

Effects of formula composed of active monomers of removing heat and phlegm prescription on malignant phenotypes and growth signaling in esophageal carcinoma cells.

e16051 Background: Esophageal carcinoma (EC) ranks seventh in the incidence of malignant tumors and the sixth mortality worldwide. Removing heat and phlegm prescription(RHPP, composed of Rhizoma Dioscoreae Nipponicae, Rhizoma Paridis, Saponin, Rhizoma Bolbostemmae) was an effective formula screened from 531 Chinese herbs for treating EC in our previous study. The purpose of this study is to further track, isolate, purify and identify the active monomers of RHPP, study the effect of each active monomer on Eca109, EC9706, EC-1, TE-1 four EC cells and the signaling mechanism, then optimize and compose new active monomers formula and study its mechanism on EC cells. Methods: The active monomer of RHPP was extracted, separated and identified by ethanol extractioncolumn, chromatography, recrystallization, HPLC, spectroscopic method,etc. The effects of four kinds of active monomers and their composed formula on the proliferation, migration, clone formation, cell cycle and signal pathway proteins expression of Eca109 cell, EC9706 cell and TE-1 cell were investigated by MTT assay, RTCA assay, soft agar assay, flow cytometry and western blot analysis. Results: Four active monomers were identified from Rhizoma Dioscoreae Nipponicae (CSL), Saponin (ZJ), Rhizoma Bolbostemmae (TBM), Rhizoma Paridis (CL) respectively with molecular weights of 868, 1003, 1365, 854.5, and corresponding molecular formulas of C45H72O16, C50H82O20, C64H100O31 and C44H70O16(China patient No. ZL202010136431.6). Four active monomers significantly affected the cell morphology, proliferation, migration, cloning ability, cell cycle of four EC cells. The IC50 values of the active monomer of CSL, ZJ, TBM and CL for Eca109 were 1.29±0.12, 2.42±0.09, 1.93±0.09, 3.04±0.28μg/ml respectively; for EC9706 were 1.41±0.02, 5.94±0.45, 1.97±0.09, 0.63±0.04μg/ml respectively; for TE-1 were 3.72±0.28, 35.58±0.58, 7.90±0.41, 1.85±0.09μg/ml respectively; for EC-1 were 1.11±0.14, 3.56±0.28, 1.23±0.02, 0.61±0.02μg/ml respectively. Four active monomers could downregulate EGFR, PLC-γ1, and PKCα protein expression. Using the baseline proportional increase and decrease design method to compose new removing heat prescription(RH), removing phlegm prescription(RP) and removing heat and phlegm prescription (RHPP), the ratio of each drug monomer in RH, RP and RHPP were mCSL: mCL = 6: 4, mZJ: mTBM = 3: 7, mCSL: mCL: mZJ: mTBM = 48: 32: 6: 14. RH, RP and RHPP all could inhibit the proliferation, migration, cloning ability, cell cycle of four EC cells. Conclusions: The four active monomers and new composed RHPP all can inhibit the proliferation of EC9706, Eca109, TE-1, EC-1 four EC cells, which have close relationship with EGFR, PLC-γ1, and PKCαproteins expression. This study provides new formula and basis for the development of anti-esophageal carcinoma drugs in traditional Chinese medicine.

Normal spontaneous firing of cardiac pacemaker cells is regulated by basal pkc delta activation

Abstract The spontaneous beating rate of rabbit sinoatrial node cells (SANC) is regulated by local subsarcolemmal calcium releases (LCRs) from sarcoplasmic reticulum (SR). LCRs appear during diastolic depolarization (DD) and activate an inward sodium/calcium exchange current which increases DD rate and thus accelerates spontaneous SANC firing. High basal level of protein kinase A and calcium/calmodulin-dependent protein kinase II phosphorylation are required to sustain basal LCRs and normal spontaneous SANC firing. Recently we discovered that basal PKC activation is also obligatory for cardiac pacemaker function: inhibition of PKC activity by broad spectrum PKC inhibitors Bis I or calphostin C markedly suppressed SR calcium cycling and decreased or abolished spontaneous beating of freshly isolated rabbit SANC. Here we studied which PKC isoforms mediate PKC-dependent effects on cardiac pacemaker cell automaticity. The PKC superfamily consists of 3 major subgroups: conventional, novel and atypical. All PKC isoforms were detected at the RNA level (RT-qPCR) in the rabbit SA node and ventricle, and expression levels were comparable in both tissues. Expression of PKCβ, however, was markedly higher in the rabbit SA node, compared to other PKC isoenzymes in either tissue. We verified expression of conventional PKC (α, β) and novel PKC-delta at the protein level in SANC and ventricular myocytes (VM). Western blot confirmed RNA results, showing a 6-fold higher PKCβ protein abundance in SANC compared to VM. Expression of PKCα protein was similar in both cell types, while PKC-delta protein was more abundant in VM. To study whether PKCβ regulates spontaneous beating of SANC we employed selective inhibitor of conventional (α, β, gamma) PKC isoforms Go6976 (10 μmol/L), which had no effects on either LCR characteristics (confocal microscopy, calcium indicator Fluo-3AM) or spontaneous beating of freshly isolated rabbit SANC (perforated patch-clamp technique). Because selective PKC-delta inhibitors are not available, we explored effects of PKC-delta inhibition comparing effects of Go6976 (the inhibitor of conventional PKCs) and Go6983, which inhibits conventional PKCs and PKC-delta. In contrast to Go6976, Go6983 (5 μmol/L) markedly decreased the LCR size (from 7.1±0.4 to 4.5±0.3 μm) and number per each spontaneous cycle (from 1.3±0.1 to 0.8±0.1). It also markedly increased the LCR period (time from the prior AP-induced calcium transient to the subsequent LCR) which was paralleled by an increase in the spontaneous SANC cycle length. Rottlerin, another PKC-delta inhibitor, produced similar effects on LCR characteristics, and markedly and time-dependently decreased DD rate, leading to an increase in the spontaneous cycle length, and finally abrogated the spontaneous SANC firing. Thus, our data indicate that basal activity of PKC-delta, but not that of PKCβ, is essential for generation of LCRs and normal spontaneous firing of cardiac pacemaker cells. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Intramural Research Program, National Institute on Aging, National Institute of Health, USA

Dietary supplementation of Huangshan Maofeng green tea preventing hypertension of older C57BL/6 mice induced by desoxycorticosterone acetate and salt

Senile hypertension affects the life quality of aged population. Dietary intervention plays a pivotal role in the prevention of hypertension. There are few reports concerning the effects and mechanisms of green tea supplementation preventing age related hypertension. The current study investigated the effect and mechanism of dietary supplement of Huangshan Maofeng green tea (HSMF) on prevention of hypertension induced by deoxycorticosterone acetate (DOCA) and salt in old C57BL/6 mice. Our results showed that HSMF dose-dependently prevented the increase of systolic blood pressure and diastolic blood pressure induced by DOCA plus salt (DS) at 51-week-old mice. And HSMF significantly reduced the agonists' stimulated contraction of mesenteric arteries isolated from the old mice. The expression of vasoconstrictor genes and inflammatory cytokines in aorta were suppressed observably by HSMF supplementation compared with DS group. The protein expression of PKCα in the aorta was dose-dependently decreased by HSMF compared to DS group. The phosphorylation level of MYPT1, CPI-17and MLC20 was also restrained by HSMF in the aorta. Furthermore, HSMF protected kidney by maintaining integrity of glomeruli and tubules and remarkably decreased the NGAL level in plasma. HSMF also suppressed the kidney inflammation by decreasing inflammatory cytokines expression and the macrophage infiltration. Our results proved that dietary supplement of HSMF remarkably improved the vascular functions and protected kidney injury, and thus prevented hypertension induced by DS in older C57BL/6 mice. Our data indicated that the dietary supplement of HSMF may potentially be used as a food additive for preventing hypertension for aged people.

Bone-edge electroacupuncture relieves pain by regulating expression of GRK5, β-arrestin 2 and PKCα proteins in locus coeruleus in bone cancer pain rats with morphine tolerance

To observe the effect of bone-edge electroacupuncture (EA) intervention on mechanical pain threshold (PT) and expression of G protein-coupled receptor kinase (GRK5), β-arrestin 2, total and phosphorylated PKC alpha (p-PKCα) proteins in the locus coeruleus (LC) of rats with bone cancer pain induced morphine tolerance, so as to reveal its partial central mechanisms underlying pain relief. Forty SD rats were randomly divided into 5 groups, namely sham bone cancer, bone cancer pain, morphine tolerance, bone-edge EA, and sham EA (n＝ 8 rats in each group). The bone cancer with morphine tolerance model was established by intramedullary injection of MRMT-1 cells into the tibial cavity, and then intraperitoneal injection of morphine hydrochloride injection. After successful establishment of morphine tolerance model, the bone-edge EA (2 Hz/100 Hz，0.5－1.5 mA) was applied to bilateral "Zusanli" (ST36) and "Kunlun" (BL60) for 30 min, once a day for 7 days, after inserting the needle-tip to the tibial bone surface. The ipsilateral mechanical paw withdrawal thresholds (PWTs) were detected dynamically. The expression levels of GRK5, β-arrestin 2, PKCα and p-PKCα in the LC area were measured by Western blot. The PWTs of bone cancer pain rats were decreased on day 10 after inoculation of cancer cells (P<0.01). After i．p. of morphine for 11 days, no analgesic effect and pain tolerance appeared (P>0.05). The PWTs were significantly increased in the bone-edge EA intervention group (P<0.01), not in the sham EA group (P>0.05). In comparison with the sham bone cancer group, the expression of GRK5 protein in morphine tolerance group was significantly decreased (P<0.01); compared with morphine tolerance group, the expression of GRK5 protein in bone-edge EA group was increased(P<0.01). In comparison with the sham bone cancer group, the expression of β-arrestin 2 and p-PKCα in bone cancer group significantly increased (P<0.01). After the intervention, the increased β-arrestin 2 and p-PKCα expressions were reversed in the bone-edge EA group (P<0.01); compared with morphine tolerance group and sham EA group, the expression of PKCα protein was decreased(P<0.01). Bone-edge EA can effectively relieve morphine tolerance in bone cancer pain rats, which may be related to its functions in up-regulating GRK5 protein and down-regulating β-arrestin 2, PKCα and p-PKCα proteins in LC. .

Changes of myocardial function in diabetic rats and its mechanism

To investigate the role of calcium-sensing receptor (CaSR) in the decrease of cardiac function in type 2 diabetic rats. Wistar rats were randomly divided into 3 groups including control, diabetic-4 week and diabetic-8 week groups. Rats in the diabetes group were fed with high-glucose and high-fat diet, and intraperitoneal injection of streptozocin (STZ,30 mg/kg) was conducted 4 weeks later to establish a type 2 diabetes model. Cardiac morphological changes were observed by HE staining, cardiac function was detected by echocardiography, and CaSR and PKC-αprotein expressions in cardiac tissue were detected by Western blot. Compared with the control group, the myocardium of diabetic rats showed irregular contraction zone, decreased expression of CaSR protein, increased expression of PKC-α protein, decreased systolic and diastolic functions, and gradually worsened with the prolongation of the course of the disease. Hyperglycemia inhibits the expression of CaSR protein in myocardium of diabetic rats by activating PKC-α, which can cause intracellular calcium disorder and lead to decreased cardiac function.

MicroRNA-200b inhibits pituitary tumor cell proliferation and invasion by targeting PKCα.

The present study aimed to investigate the expression of miR-200b and protein kinase Cα (PKCα) in pituitary tumors and to determine whether miR-200b may inhibit proliferation and invasion of pituitary tumor cells. The regulation of PKCα expression was targeted in order to find novel targets for the treatment of pituitary tumors. In total, 53 pituitary tumor tissue samples were collected; these included 28 cases of invasive pituitary tumors and 25 cases of non-invasive tumors, in addition to 5 normal pituitaries. The expression level of miR-200b in the pituitary tumor tissue was detected by quantitative polymerase chain reaction (qPCR) and the expression of PKCα protein was detected by immunohistochemistry. A PKCα 3'untranslated region (UTR) luciferase vector was constructed and a dual luciferase reporter gene assay was employed in order to examine the effect of miR-200b on the PKCα 3'UTR luciferase activity. AtT-20 cells were transfected with miR-200b mimics, PKCα siRNA and miR-200b mimics + PKCα, and the changes in cellular proliferation, invasion and apoptosis were observed via MTT, Transwell assay and flow cytometric analysis. Furthermore, PKCα mRNA expression was determined by qPCR, and Western blotting was performed to detect the expression of PKCα protein. miR-200b revealed downregulation in invasive pituitary tumor tissue, and the expression level was significantly down-regulated compared with normal and non-invasive pituitary tumor tissue (P<0.01). In addition, the positive rate of PKCα protein expression in invasive pituitary tumor tissues was significantly higher than in normal and non-invasive tissues (P<0.01). PKCα protein levels are inversely correlated with miR-200b levels in invasive pituitary tumor tissues (r=-0.436, P=0.021). The dual luciferase reporter gene assay revealed that miR-200b could specifically bind to the 3'UTR of PKCα and significantly inhibit the luciferase activity by 39% (P<0.01). Upregulation of miR-200b or downregulation of PKCα could suppress cell proliferation and invasion, and increase apoptosis of AtT-20 cells. It was revealed that PKCα siRNA could suppress both proliferation and invasion of AtT-20 cells and partially simulate the function of miR-200b. Expression of PKCα mRNA and protein decreased significantly in AtT-20 cells overexpressing miR-200b. Additionally, miR-200b was significantly down-regulated in invasive pituitary tumor tissue and inversely correlated with PKCα protein levels. In conclusion, miR-200b inhibited proliferation and invasiveness and promoted the apoptosis of pituitary tumor cells by targeting PKCα. The observations of the present study indicate that miR-200b and PKCα may serve as promising therapeutic targets for invasive pituitary tumors.

Role of Small Interfering RNA Silencing Protein Kinase C-α Gene on the Occurrence of Ultrafiltration Failure in Peritoneal Dialysis Rats.

This study aims to explore the effect of PKC-α gene silencing on the occurrence of ultrafiltration failure (UFF) in peritoneal dialysis (PD) rats. Forty-eight male SD rats were collected to establish 5/6 renal resection uremic and uremic PD rats models. Rats were assigned into control, sham operation, uremia, PD-2 W (peritoneal dialysis for 2 weeks), PD-4 W (peritoneal dialysis for 4 weeks), negative control (NC) (peritoneal dialysis for 4 weeks, and injected 0.1 mg/kg blank plasmid into abdominal cavity) and PKC-α siRNA (peritoneal dialysis for 4 weeks, and injected 0.1 mg/kg PKC-α siRNA into abdominal cavity) groups. CD34 staining was performed to determine microvessel density (MVD) for peritoneal tissues. The mRNA and protein expression of PKC-α in peritoneal tissue were detected by qRT-PCR and Western blot. Compared with the control group, MVD, the mRNA and protein expression of PKC-α were significantly increased in rats of the uremia, PD-2 W, PD-4 W, NC, and PKC-α siRNA groups. Compared with the uremia group, MVD, the mRNA and protein expression of PKC-α were increased, the changes observed in the PD-4 W and NC groups were better obvious than in the PD-2 W group. In comparison with the PD-4 W and NC groups, MVD, the mRNA and protein expression of PKC-α in rats were decreased in the PKC-α siRNA group. PKC-α gene has a high expression in uremic PD rats, and PKC-α gene silencing is able to increase UF while decrease MVD and glucose transport in peritoneal tissues thus reversing UFF in PD rats. J. Cell. Biochem. 118: 4607-4616, 2017. © 2017 Wiley Periodicals, Inc.

Low expression of PKCα and high expression of KRAS predict poor prognosis in patients with colorectal cancer.

The current study aimed to determine the association between protein kinase Cα (PKCα) and Kirsten rat sarcoma viral oncogene homolog (KRAS) expression and the response to folinic acid, 5-fluorouracil and oxaliplatin (FOLFOX regimen) in patients with colorectal cancer (CRC). The protein levels of PKCα and KRAS were analyzed by immunohistochemistry in tissue samples from patients with CRC and in non-cancerous tissues, including 152 cases of colorectal adenocarcinoma, 30 cases of colorectal adenoma and 20 normal colonic mucosa samples. The association between PKCα and KRAS expression and clinicopathological features was analyzed. The rates of positive PKCα protein expression in patients with poorly, moderately and well-differentiated adenocarcinoma were 16.7% (6/36), 40.0% (24/60), and 57.1% (32/56), respectively (P<0.013). The rate of positive KRAS expression in CRC patients was significantly higher than in patients with colon adenoma and normal colon mucosa (P<0.001). Expression levels of KRAS were associated with the degree of differentiation of CRC (P<0.001). Expression of PKCα was negatively correlated with KRAS expression in CRC tissues. The mean progression-free survival (PFS) times in patients with high and low expression of PKCα were 43.9 and 38.8 months, respectively (P<0.001). The mean PFS times were 38.5 and 45.5 months in patients with high and low expression of KRAS, respectively (P=0.001). In conclusion, low PKCα and high KRAS expression predicted relatively poor prognosis in patients with CRC.

Shifts in temperature within the physiologic range modify strand-specific expression of select human microRNAs

Previous studies have revealed that clinically relevant changes in temperature modify clinically relevant gene expression profiles through transcriptional regulation. Temperature dependence of post-transcriptional regulation, specifically, through expression of miRNAs has been less studied. We comprehensively analyzed the effect of 24 h exposure to 32°C or 39.5°C on miRNA expression profile in primary cultured human small airway epithelial cells (hSAECs) and its impact on expression of a targeted protein, protein kinase C α (PKCα). Using microarray, and solution hybridization-based nCounter assays, with confirmation by quantitative RT-PCR, we found significant temperature-dependent changes in expression level of only five mature human miRNAs, representing only 1% of detected miRNAs. Four of these five miRNAs are the less abundant passenger (star) strands. They exhibited a similar pattern of increased expression at 32°C and reduced expression at 39.5°C relative to 37°C. As PKCα mRNA has multiple potential binding sites for three of these miRNAs, we analyzed PKCα protein expression in HEK 293T cells and hSAECs. PKCα protein levels were lowest at 32°C and highest at 39.5°C and specific miRNA inhibitors reduced these effects. Finally, we analyzed cell-cycle progression in hSAECs and found 32°C cells exhibited the greatest G1 to S transition, a process known to be inhibited by PKCα, and the effect was mitigated by specific miRNA inhibitors. These results demonstrate that exposure to clinically relevant hypothermia or hyperthermia modifies expression of a narrow subset of miRNAs and impacts expression of at least one signaling protein involved in multiple important cellular processes.

Platelet derived growth factor receptor α-phospholipase Cγ1-protein kinase Cα signal transductionin in human lung cancer cell GLC-82 for combination of the drugs tumor necrosis factor-related apoptosis-inducing ligand and proteasome inhibitor MG132

Objective To tumor necrosis factor- related apoptosis- inducing ligand(TRAIL)combined with a protease inhibitor MG132 observed in human lung adenocarcinoma cell line GLC- 82 inhibition of proliferation and platelet derived growth factor receptor α(PDGFRα)- phospholipase Cγ1(PLCγ1)-protein kinase Cα(PKCα)signal transduction pathway. Methods Methyl thiazol tetrazolium(MTT)assay inhibition of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell growth detection; using flow cytometry to detect the effects of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell apoptosis, using SP immunohistochemical detection of protease inhibitors MG132 joint expression of TRAIL- associated protein PDGFRα, PLCγ1,PKCα's, Western blotting to determine the effect of protease inhibitors MG132 TRAIL combined for GLC- 82 cells PDGFRα, PLCγ1, PKCαprotein expression. Results MTT assay inhibition of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell growth detection. The inhibitory rates were(34.92±3.95)%,(33.17±1.78)% and(76.96±1.28)%, respectively; using flow cytometry to detect the effects of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell apoptosis. Apoptosis rates of the four group were 6.78%, 38.72%, 45.60% and 91.74%, respectively. Using SPimmunohistochemical detection of protease inhibitors MG132 joint expression of TRAIL- associated protein PDGFRα, PLCγ1, PKCα's, Western blotting to determine the effect of protease inhibitors MG132 TRAIL combined for GLC- 82 cells PDGFRα, PLCγ1, PKCα protein expression. Conclusion TRAIL combined protease inhibitor MG132 on human lung adenocarcinoma GLC- 82 cells significantly inhibited the growth, can reduce the level of GLC- 82 cells PDGFRα- PLCγ1- PKCα each protein signaling pathway, the incidence of lung cancer and PDGFRα- PLCγ1- PKCα enhanced signal transduction pathways are closely related. Key words: Tumor necrosis factor-related apoptosis-inducing ligand; MG132; Lung adenocarcinoma; Signal transduction

Abstract 4211: Protein kinase C alpha (PKCα) signaling in endometrial cancer

Abstract Endometrial cancer is the most common gynecological malignancy in the US and the fourth most common cancer in women. Despite its high incidence, there is limited knowledge of the molecular etiology of this disease. With obesity being a major risk factor, the incidence of endometrial cancer has been on a steady incline and is expected to become an increasing cause of cancer mortality in future years. Thus, improved understanding of endometrial tumorigenesis is of critical importance. Our analysis of human endometrial tumors identified loss of the signaling molecule PKCα in approximately 60% of endometrioid (Type I) tumors. Loss of PKCα trends with increasing tumor grade and is associated with aggressive disease features such as lymphovascular involvement and myometrial invasion, pointing to an important role for this molecule in regulating endometrial cancer progression. Inactivation of the tumor suppressor PTEN and deregulation of the PI3K/AKT pathway are key drivers of Type I endometrial cancer. Thus, mice with allele-specific knock-in of cancer-related Pten mutants (i.e., PtenΔ4-5, PtenC124R, and PtenG120E) offer unique models for the disease. Precancerous endometrial hyperplasias arising in these mice show loss of PTEN and increased AKT activity. While PKCα is expressed in the normal murine endometrial epithelium at all phases of the estrus cycle, the enzyme is uniformly lost in these precancerous lesions. Thus, disruption of PKCα signaling can occur early in endometrial tumorigenesis. In human endometrial cancer cell lines, low PKCα levels also correlate with PTEN loss; however, shRNA-mediated knockdown of PTEN in PTEN expressing cells did not affect PKCα expression indicating that downregulation of the kinase is not the direct result of changes in PTEN activity. Notably, PKC agonists suppress AKT activity in cells expressing high levels PKCα (HEC-1-A, HEC-50) but not in PKCα-low cells (Ishikawa, RL95-2). Collectively, these data suggest that, in addition to PTEN inactivation, loss of PKCα is required for full activation of the PI3K/AKT signaling pathway during endometrial carcinogenesis. Analysis of the molecular basis for loss of PKCα expression in endometrial tumors identified multiple regulatory mechanisms. PKCα protein expression in tumors generally parallels that of its mRNA and promoter activity assays point to transcriptional mechanisms for PKCα downregulation. Differential mRNA and protein stability were identified as additional levels of regulation of PKCα expression in endometrial cancer. Taken together, our findings indicate that 1) PKCα signaling regulates the PI3K/AKT pathway in the endometrial epithelium; 2) concomitant loss of PKCα and PTEN may act in a co-operative manner in regulation of endometrial turmorigenesis; and 3) loss of PKCα can occur by multiple mechanisms, as often seen with tumor suppressive molecules during neoplastic progression. Supported by NIH grants CA036727, CA016056, and DK60632. Citation Format: Alice H. Hsu, Kathryn J. Curry, Kang-Sup Shim, Peter Frederick, Carl D. Morrison, Baojing Chen, Subodh M. Lele, Gustavo Leone, Adrian R. Black, Jennifer D. Black. Protein kinase C alpha (PKCα) signaling in endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4211. doi:10.1158/1538-7445.AM2014-4211

Tonic and Phasic Smooth Muscle Contraction Is Not Regulated by the PKCα - CPI-17 Pathway in Swine Stomach Antrum and Fundus

Regulation of myosin light chain phosphatase (MLCP) via protein kinase C (PKC) and the 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) has been reported as a Ca2+ sensitization signaling pathway in smooth muscle (SM), and thus may be involved in tonic vs. phasic contractions. This study examined the protein expression and spatial-temporal distribution of PKCα and CPI-17 in intact SM tissues. KCl or carbachol (CCh) stimulation of tonic stomach fundus SM generates a sustained contraction while the phasic stomach antrum generates a transient contraction. In addition, the tonic fundus generates greater relative force than phasic antrum with 1 µM phorbol 12, 13-dibutyrate (PDBu) stimulation which is reported to activate the PKCα – CPI-17 pathway. Western blot analyses demonstrated that this contractile difference was not caused by a difference in the protein expression of PKCα or CPI-17 between these two tissues. Immunohistochemical results show that the distribution of PKCα in the longitudinal and circular layers of the fundus and antrum do not differ, being predominantly localized near the SM cell plasma membrane. Stimulation of either tissue with 1 µM PDBu or 1 µM CCh does not alter this peripheral PKCα distribution. There are no differences between these two tissues for the CPI-17 distribution, but unlike the PKCα distribution, CPI-17 appears to be diffusely distributed throughout the cytoplasm under relaxed tissue conditions but shifts to a primarily peripheral distribution at the plasma membrane with stimulation of the tissues with 1 µM PDBu or 1 µM CCh. Results from double labeling show that neither PKCα nor CPI-17 co-localize at the adherens junction (vinculin/talin) at the membrane but they do co-localize with each other and with caveoli (caveolin) at the membrane. This lack of difference suggests that the PKCα - CPI-17 pathway is not responsible for the tonic vs. phasic contractions observed in stomach fundus and antrum.

GW24-e3077 Inhibition of PICK1 has the Protective Effect on Adriamycin-induced Toxic Myocarditis

ObjectivesAdriamycin (ADR) has broad-spectrum effect such as anti-tumour and antibiotic, but it is limited the application for its dose-dependent cardiac toxicity. PICK1 is the interaction protein of PKC, which has...

Cyclosporine Attenuates Arginine Transport, in Human Endothelial Cells, through Modulation of Cationic Amino Acid Transporter-1

Background: The spectrum of cardiovascular toxicity by cyclosporine (CsA) includes hypertension, accelerated atherosclerosis, and thrombotic microangiopathy, all of which are the result of endothelial cell dysfunction. Endothelial cell dysfunction is characterized by decreased endothelial nitric oxide synthase (eNOS) activity. Cationic amino acid transporter-1 (CAT-1) is the specific arginine transporter for eNOS. CsA has been shown to attenuate nitric oxide (NO) generation. However, the mechanism remains elusive. We hypothesize that CsA inhibits eNOS activity through modulation of its selective arginine supplier CAT-1. Methods: We studied the effect of CsA on arginine uptake, NO₂/NO₃ generation, and CAT-1, protein kinase Cα (PKCα), and phosphorylated PKCα protein expression in human umbilical vein endothelial cell cultures (HUVEC) in the absence and presence of <smlcap>L</smlcap>-arginine. Results: CsA (0.5-2 μg/ml) significantly attenuated arginine transport in a dose- and time-dependent manner, a phenomenon which was prevented by co-incubation with <smlcap>L</smlcap>-arginine (1 m<smlcap>M</smlcap>). The aforementioned findings were accompanied by increased protein nitration, a measure for peroxynitrite accumulation. In contrast, no changes were observed in NO₂/NO₃ generation. CsA significantly decreased the abundance of CAT-1 protein, an effect that was attenuated by <smlcap>L</smlcap>-arginine. PKCα and phosphorylated PKCα (CAT-1 inhibitors) protein contents were not affected by CsA. Conclusion: CsA inhibits arginine transport and induces protein nitration in HUVEC through modulation of CAT-1.

Clinicopathological correlation and prognostic significance of protein kinase cα overexpression in human gastric carcinoma.

ObjectivesThis study investigated the PKCα protein expression in gastric carcinoma, and correlated it with clinicopathological parameters. The prognostic significance of PKCα protein expression in gastric carcinoma was analyzed.MethodsQuantitative real-time PCR test was applied to compare the PKCα mRNA expression in tumorous and nontumorous tissues of gastric carcinoma in ten randomly selected cases. Then PKCα protein expression was evaluated in 215 cases of gastric carcinoma using immunohistochemical method. The immunoreactivity was scored semiquantitatively as: 0 = absent; 1 = weak; 2 = moderate; and 3 = strong. All cases were further classified into two groups, namely PKCα overexpression group with score 2 or 3, and non-overexpression group with score 0 or 1. The PKCα protein expression was correlated with clinicopathological parameters. Survival analysis was performed to determine the prognostic significance of PKCα protein expression in patients with gastric carcinoma.ResultsPKCα mRNA expression was upregulated in all ten cases of gastric carcinoma via quantitative real-time PCR test. In immunohistochemical study, eighty-eight out of 215 cases (41%) of gastric carcinoma revealed PKCα protein overexpression, which was statistically correlated with age (P = 0.0073), histologic type (P<0.0001), tumor differentiation (P = 0.0110), depth of invasion (P = 0.0003), angiolymphatic invasion (P = 0.0373), pathologic stage (P = 0.0047), and distant metastasis (P = 0.0048). We found no significant difference in overall and disease free survival rates between PKCα overexpression and non-overexpression groups (P = 0.0680 and 0.0587). However, PKCα protein overexpression emerged as a significant independent prognostic factor in multivariate Cox regression analysis (hazard ratio 0.632, P = 0.0415).ConclusionsPKCα protein is upregulated in gastric carcinoma. PKCα protein expression is statistically correlated with age, histologic type, tumor differentiation, depth of invasion, angiolymphatic invasion, pathologic stage, and distant metastasis. The PKCα protein overexpression in patients with gastric carcinoma is a significant independent prognostic factor in multivariate Cox regression analysis.

Gene Set Enrichment Analysis Identifies miR-185 as a Negative Regulator of Cardiac Hypertrophy through Targeting the PKC Signaling Pathway

Growing evidence indicates that various microRNAs (miRNAs) are involved in pathogenesis of cardiac hypertrophy, but the precise functions of those miRNAs remain unclear due mainly to their multiple targets. In the present study, we have used a gene set enrichment analysis (GSEA) to identify miRNAs that may regulate multiple gene transcripts in cardiac hypertrophy signaling pathways. Among 23 miRNAs of which targets are preferentially enriched in cardiac hypertrophy signaling pathways, miR-185 was chosen for validation as an essential regulator of cardiac hypertrophy. The results showed that transfection of miR-185 mimic markedly reduced 10 nM endothelin-1 (ET-1)-induced cardiac hypertrophy and the expression of hypertrophic markers such as ANF and BNP. Western blot analysis showed that the increased protein expression of PKC-α, and phosphorylation of ERK1/2 in response to ET-1 treatment were significantly attenuated by treatment with miR-185 mimic. Taken together, our findings suggest that miR-185 acts as a critical regulator of cardiac hypertrophy through controlling the PKC signaling pathway. (Supported by “GIST Systems Biology Infrastructure Establishment Grant (2012)” and “KISTI-KREONET”)

SMB myosin heavy chain knockout enhances tonic contraction and reduces the rate of force generation in ileum and stomach antrum

The role of SMA and SMB smooth muscle myosin heavy chain (MHC) isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in upregulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB((-/-)) ileum and antrum with high potassium (KPSS) but not with carbachol (CCh) stimulation. The increased tonic contraction under the depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC(20) phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKCδ, and CPI-17) did not change significantly in tissues tested except for PKCα protein expression being significantly decreased in the SMB((-/-)) antrum. However, specifically activating PKCα with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild-type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB((-/-)) ileum and antrum. Taken together, these data show removing the SMB MHC protein expression with a compensatory increase in the SMA MHC protein results in enhanced sustained KPSS-induced tonic contraction with a reduced rate of force generation in these phasic tissues.

Effects of mitochondrial ATP-sensitive K+ channel on protein kinase C pathway and airway smooth muscle cell proliferation in asthma

The effects of ATP-sensitive mitochondrial K(+) channel (mitoK(ATP)) on mitochondrial membrane potential (Δψm), cell proliferation and protein kinase C alpha (PKCα) expression in airway smooth muscle cells (ASMCs) were investigated. Thirty-six Sprague-Dawley (SD) rats were immunized with saline (controls) or ovalbumin (OVA) with alum (asthma models). ASMCs were cultured from the lung of control and asthma rats. ASMCs were treated with diazoxide (the potent activator of mitoK(ATP)) or 5-hydroxydencanote (5-HD, the inhibitor of mitoK(ATP)). Rhodamine-123 (R-123) was used to detect Δψm. The expression of PKCα protein was examined by using Western blotting, while PKCα mRNA expression was detected by using real-time PCR. The proliferation of ASMCs was measured by MTT assay and cell cycle analysis. In diazoxide-treated normal ASMCs, the R-123 fluorescence intensity, protein and mRNA levels of PKCα, MTT A values and percentage of cells in S phase were markedly increased as compared with untreated controls. The ratio of G(0)/G(1) cells was decreased (P<0.05) in diazoxide-treated ASMCs from normal rats. However, there were no significant differences between the ASMCs from healthy rats treated with 5-HD and the normal control group. In untreated and diazoxide-treated ASMCs of asthmatic rats, the R-123 fluorescence intensity, protein and mRNA levels of PKCα, MTT A values and the percentage of cells in S phase were increased in comparison to the normal control group. Furthermore, in comparison to ASMCs from asthmatic rats, these values were considerably increased in asthmatic group treated with diazoxide (P<0.05). After exposure to 5-HD for 24 h, these values were decreased as compared with asthma control group (P<0.05). In ASMCs of asthma, the signal transduction pathway of PKCα may be involved in cell proliferation, which is induced by the opening of mitoK(ATP) and the depolarization of Δψm.

Role of ω-hydroxylase in adenosine-mediated aortic response through MAP kinase using A2A-receptor knockout mice.

Previously, we have shown that A(2A) adenosine receptor (A(2A)AR) knockout mice (KO) have increased contraction to adenosine. The signaling mechanism(s) for A(2A)AR is still not fully understood. In this study, we hypothesize that, in the absence of A(2A)AR, ω-hydroxylase (Cyp4a) induces vasoconstriction through mitogen-activated protein kinase (MAPK) via upregulation of adenosine A(1) receptor (A(1)AR) and protein kinase C (PKC). Organ bath and Western blot experiments were done using isolated aorta from A(2A)KO and corresponding wild-type (WT) mice. Isolated aortic rings from WT and A(2A)KO mice were precontracted with submaximal dose of phenylephrine (10(-6) M), and concentration responses for selective A(1)AR, A(2A)AR agonists, angiotensin II and cytochrome P-450-epoxygenase, 20-hydroxyeicosatrienoic acid (20-HETE) PKC, PKC-α, and ERK1/2 inhibitors were obtained. 2-p-(2-Carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680, A(2A)AR agonist) induced concentration-dependent relaxation in WT, which was blocked by methylsulfonyl-propargyloxyphenylhexanamide (cytochrome P-450-epoxygenase inhibitor; 10(-5) M) and also with removal of endothelium. A(1) agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) produced higher contraction in A(2A)KO aorta than WT (49.2 ± 8.5 vs. 27 ± 5.9% at 10(-6) M, P < 0.05). 20-HETE produced higher contraction in A(2A)KO than WT (50.6 ± 8.8 vs. 21.1 ± 3.3% at 10(-7) M, P < 0.05). Contraction to CCPA in WT and A(2A)KO aorta was inhibited by PD-98059 (p42/p44 MAPK inhibitor; 10(-6) M), chelerythrine chloride (nonselective PKC blocker; 10(-6) M), Gö-6976 (selective PKC-α inhibitor; 10(-7) M), and HET0016 (20-HETE inhibitor; 10(-5) M). Also, contraction to 20-HETE in WT and A(2A)KO aorta was inhibited by PD-98059 and Gö-6976. Western blot analysis indicated the upregulation of A(1)AR, Cyp4a, PKC-α, and phosphorylated-ERK1/2 in A(2A)KO compared with WT (P < 0.05), while expression of Cyp2c29 was significantly higher in WT. CCPA (10(-6) M) increased the protein expression of PKC-α and phosphorylated-ERK1/2, while HET0016 significantly reduced the CCPA-induced increase in expression of these proteins. These data suggest that, in the absence of A(2A)AR, Cyp4a induces vasoconstriction through MAPK via upregulation of A(1)AR and PKC-α.