Loss of Raf kinase inhibitor protein promotes cell proliferation and migration of human hepatoma cells

BACKGROUND & AIMS: The Raf kinase inhibitor protein (RKIP) has been identified as a suppressor of the mitogen-activated protein kinase (MAPK) pathway. Loss of RKIP function promotes tumor metastasis in prostate cancer and melanoma. The insulin-like growth factor I (IGF-I)-mediated MAPK cascade is often activated in hepatocellular carcinoma (HCC), but the role of RKIP in the molecular pathogenesis of these tumors is unknown. This study was performed to evaluate the role of RKIP in the development of HCC. METHODS: The levels of RKIP expression in HCC tumor and corresponding peritumoral tissues were determined by immunohistochemistry and Western blot analysis. The underlying mechanisms of RKIP were assessed with immunoblot analysis, Raf kinase activity assay, cell proliferation, and migration assays after either overexpression or knockdown of RKIP expression in HCC cell lines. RESULTS: RKIP expression is down-regulated in human HCC compared with adjacent peritumoral tissues. Low RKIP levels were correlated with enhanced extracellular signal-regulated-kinase (ERK)/MAPK pathway activation. Reconstitution experiments antagonized IGF-I-mediated MAPK pathway activation, resulting in reduced nuclear accumulation of phospho-ERK. In contrast, knockdown of RKIP expression using small interfering RNA induced activation of the ERK/MAPK pathway. Ectopic expression of RKIP altered HCC cell proliferation and migration. CONCLUSIONS: Our findings indicate that down-regulation of RKIP expression is a major factor in activation of the IGF-I/ERK/MAPK pathway during human hepatocarcinogenesis.     

胰腺癌细胞系血管紧张素Ⅱ1型受体蛋白表达的研究

目的：探讨血管紧张素Ⅱ1型受体（angiotensin Ⅱ type 1 receptor,AT1）在胰腺癌细胞中的表达。方法：用免疫细胞化学染色检测胰腺癌细胞系SW1990、PaTu8988s和PANC－1中AT1蛋白的表达。结果：胰腺癌细胞系SW1990、PaTu8988s和PANC－1中均有AT1蛋白的表达，结论：AT1在胰腺癌细胞生长中可能发挥重要作用，应用AT1拮抗剂对防治胰腺癌似乎有一定意义。     

Activation of AMP-activated protein kinase enhances angiotensin ii-induced proliferation in cardiac fibroblasts

AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic regulator of cellular enzymes involved in carbohydrate and fat metabolism, which regulate ATP conservation and synthesis. Here, we investigated whether AMPK signaling has a role in the regulation of angiotensin II (Ang II)-induced proliferation in rat cardiac fibroblasts. Aminoimidazole-4-carboxamide-1-beta-ribofuranoside (AICAR) activated AMPK in rat cardiac fibroblasts and increased Ang II-induced extracellular signal-regulated kinase 1/2 phosphorylation and activity. AICAR also increased Ang II-induced c-fos mRNA expression in the cells. [3H]-thymidine and [3H]-proline incorporation by cardiac fibroblasts treated with Ang II was enhanced when the cells were pretreated with AICAR. Inhibition of AMPK by small interfering RNA for AMPKalpha1 suppressed Ang II-induced extracellular signal-regulated kinase activity, c-fos mRNA expression, and cell proliferation. Treatment of rats with AICAR (1 mg/g body weight per day) for 1 week significantly enhanced Ang II-induced hypertrophy of the myocardium. Our findings indicate that AMPK works as a stimulator of the Ang II-induced proliferative pathway in cardiac fibroblasts. Inhibition of AMPK signaling might serve as a new therapeutic target of remodeling of the hypertrophic myocardium.     

Activation of lamina propria T cells induces crypt epithelial proliferation and goblet cell depletion in cultured human fetal colon.

An organ culture model has been used to study the effects of T cell activation in the human colon. Lamina propria T cells in explant cultures of human fetal colon (11 to 23 weeks gestation) were activated in situ using pokeweed mitogen or an anti-CD3 monoclonal antibody, and compared with unstimulated controls. After three days of culture, there was a two to four-fold increase in crypt epithelial cell proliferation in T cell stimulated explants of more than 15 weeks gestation, associated with a fall in crypt goblet cell numbers of up to 20-fold. By three days, the surface epithelium of stimulated explants appeared thin with loss of goblet cells, and by day 7, severe and extensive mucosal damage was observed by light and electron microscopy. These changes did not occur in control cultures and explants deficient in T cells (less than 16 weeks gestation), and were inhibited by cyclosporin A. These experiments indicate that the increase in epithelial cell proliferation and accompanying goblet cell depletion observed in colorectal crypts in chronic inflammatory bowel disease may be mediated by activated T cells.     

Functional antagonism of different angiotensin II type I receptor blockers in human arteries

Objectives. To evaluate and compare the functional type and the degree of antagonism of the selective angiotensin II type 1 receptor blockers (ARB) losartan, EXP 3174 (the active metabolite of losartan), valsartan and candesartan in human internal mammary arteries. Methods. Human internal mammary arteries were obtained as excess graft material during coronary bypass surgery. Vessels were prepared as rings and mounted in an organ bath in which vasoconstriction and -dilation can be measured. Concentration-response curves of angiotensin II-mediated vasoconstriction were measured in absence or presence of different concentrations of one of the ARBs. Results. Losartan showed a rightward shift of the angiotensin II-mediated vasoconstriction, whereas addition of its metabolite EXP 3174 caused a decrease of the maximal effect of angiotensin II. Incubation with valsartan and candesartan also resulted in a decrease of the maximal effect. The inhibiting effects on the angiotensin II-mediated vasoconstriction by the highest concentration of EXP 3174, valsartan and candesartan did not differ significantly. Conclusion. In human internal mammary arteries, losartan acts as a surmountable antagonist. On the other hand, EXP 3174, valsartan and candesartan demonstrate an insurmountable type of antagonism. Furthermore, the inhibiting effects of EXP 3174, valsartan and candesartan in our study are equal in the highest concentrations.     

Raf kinase inhibitor protein inhibits cell proliferation but promotes cell migration in rat hepatic stellate cells

Aim: Hepatic stellate cells (HSCs) play an important role in the pathogenesis of liver fibrosis and cirrhosis. Raf kinase inhibitor protein (RKIP), an inhibitor of extracellular signal‐regulated kinases (ERK)/mitogen‐activated protein kinase (MAPK) signalling pathway, has been proved to suppress tumor metastasis. Interestingly, RKIP promotes cell migration in Madin–Darby canine kidney epithelial cells. However, the effects of RKIP on HSC behaviours are unknown. The purpose of the present study is to investigate the role of RKIP in HSC proliferation, apoptosis and migration. Methods: Two types of cells, freshly isolated HSC and HSC‐T6 cell line, were used in this study. The amount of RKIP, the phosphorylation of RKIP, Raf and ERK (pRKIP, pRaf and pERK) were analysed in quiescent and activated HSCs by Western blots. HSC‐T6 cells were transfected with RKIP‐expressing plasmid or treated with locostatin, a RKIP inhibitor. HSC proliferation, apoptosis and migration were evaluated with 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labelling (TUNEL) staining and Transwell cell migration assay respectively. Results: In activated HSCs, RKIP protein expression was downregulated whereas pRKIP, pRaf and pERK were upregulated. RKIP overexpression significantly mitigated the phosphorylation of RKIP, Raf and ERK. This in turn inhibited HSC proliferation. Locostatin not only inhibited RKIP protein expression but also, to some extent, reversed the RKIP‐inhibited phosphorylation of RKIP, Raf and ERK. RKIP augmented HSC migration and enhanced wound closure. Locostatin reversed the effects of RKIP. Conclusion: Raf kinase inhibitor protein inhibits ERK/MAPK signalling and this inhibition impedes HSC proliferation. RKIP promotes HSC migration and wound closure.     

Enhanced expression of angiotensin II type 2 receptor, inositol 1,4, 5-trisphosphate receptor, and protein kinase cepsilon during cardioprotection induced by angiotensin II type 2 receptor blockade

We hypothesized that the cardioprotective effect of angiotensin II type 2 receptor (AT(2)R) blockade with PD 123,319 (PD) on the recovery of left ventricular (LV) mechanical function after ischemia/reperfusion (IR) in the isolated working rat heart is associated with the enhanced expression of AT(2)R protein and mRNA as well as an increase in inositol 1,4,5-trisphosphate type 2 receptor (IP(3)R) and protein kinase Cepsilon (PKCepsilon) proteins. We assessed AT(2)R, angiotensin II type 1 receptor (AT(1)R), IP(3)R, and PKCepsilon protein expression (Western blots) and AT(2)R mRNA levels (Northern blots) in myocardium from isolated working rat hearts that were subjected to global ischemia (30 minutes) followed by reperfusion (30 minutes). Groups of adult rat hearts (n=6) were exposed to no IR, no IR+PD (0.3 micromol/L), IR, and IR+PD. Compared with no IR and no IR+PD, IR decreased (P<0.05) functional recovery and AT(2)R mRNA and protein, as well as AT(1)R mRNA (not protein) and IP(3)R and PKCepsilon proteins. Compared with IR, PD+IR improved LV functional recovery (P<0.05) and markedly increased AT(2)R mRNA and protein (P<0.001). However, PD did not change AT(1)R mRNA or protein. More importantly, PD+IR markedly increased IP(3)R and PKCepsilon proteins. The downregulation of AT(2)R mRNA and protein with IR and their upregulation with PD indicate that the effects of PD are AT(2)R specific. The overall results suggest that the cardioprotective effect of acute PD treatment on LV functional recovery after IR in the isolated working rat heart is specifically due to AT(2)R blockade and is associated with enhanced downstream IP(3)R and PKCepsilon signaling.     

Selective activation of protein kinase C isoforms by angiotensin II in neuroblastoma X glioma cells

Differential activation of PKC isoforms by angiotensin II (AII) has been found in a variety of tissues in which this important octapeptide mediates its multitude of effects. To date, the PKC isoforms involved in mediating brain-specific effects are yet to be defined. In the present study, the identity of PKC isoforms coupled to AII stimulation was examined in the neuroblastoma X glioma hybrid cell line, NG108-15, by Western blot analysis. This cell line expresses both the AT1 and AT2 receptor subtypes, with the AT1 subtype predominating, and expression levels highly-upregulated when cells are in the differentiated state. Six PKC isoforms were examined in the present study, including three Ca(2+) dependent (alpha, beta, and gamma), and three Ca(2+) independent (delta, and zeta) isoforms. NG108-15 cells were found to express PKC alpha, delta, and zeta isoforms but not beta or gamma isoforms. Differential sensitivity of the PKC isoforms to AII stimulation was demonstrated, with AII causing a rapid and transient activation of the PKC alpha only in undifferentiated cells, whereas both PKC alpha and isoforms were responsive in differentiated cells. PKC activation was found to be both dose- and time-dependent. The data demonstrate the differential activation of PKC isoforms to AII stimulation in NG108-15 cells, with evidence supporting the involvement of the PKC alpha and isoforms in AII-mediated effects in the brain.     

Putative involvement of protein kinase C in proliferation of human myeloid progenitor cells

The effects of two different potent inhibitors of protein kinase C, 1- (5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine on human myeloid (CFU-C) and late erythroid progenitor cells (CFU-E) were studied using an in vitro clonal assay. Our objective was to determine whether protein kinase C has a role in signal transduction related to proliferation of these committed progenitor cells. The presence of H-7 or staurosporine led to an inhibition of colony formation stimulated by crude colony-stimulating factor (CSF), interleukin-3 (IL-3), granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), or macrophage CSF (M-CSF) in a dose-dependent manner. N-(2-guanidinoethyl)- 5-isoquinolinesulfonamide (HA-1004), a weaker analog of H-7, did not inhibit proliferation of CFU-C. Neither H-7 nor staurosporine had any effect on CFU-E formation. H-7 and staurosporine dose-dependently inhibited the protein kinase C from K562 cells. The potential of these compounds to inhibit proliferation of CFU-C correlated well with the magnitude of their inhibition of protein kinase C from K562 cells. The inhibition of proliferation of CFU-C appears to relate to the potential of these compounds to inhibit protein kinase C. Thus, activation of protein kinase C is presumably involved in the proliferation of CFU-C, and the regulatory system of CFU-E appears to differ from that of CFU-C.     

Activation of D3 dopamine receptor decreases angiotensin II type 1 receptor expression in rat renal proximal tubule cells

The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D(3) dopamine receptor gene in mice produces renin-dependent hypertension. In rats, D(2)-like receptors reduce angiotensin II binding sites in renal proximal tubules (RPTs). Because the major D(2)-like receptor in RPTs is the D(3) receptor, we examined whether D(3) receptors regulate angiotensin II type 1 (AT(1)) receptors in rat RPT cells. The effect of D(3) receptors on AT(1) receptors was studied in vitro and in vivo. The D(3) receptor agonist PD128907 decreased AT(1) receptor protein and mRNA in WKY RPT cells and increased it in SHR cells. PD128907 increased D(3) receptors in WKY cells but had no effect in SHR cells. D(3)/AT(1) receptors colocalized in RPT cells; D(3) receptor stimulation decreased the percent amount of D(3) receptors that coimmunoprecipitated with AT(1) receptors to a greater extent in WKY than in SHR cells. However, D(3) receptor stimulation did not change the percent amount of AT(1) receptors that coimmunoprecipitated with D(3) receptors in WKY cells and markedly decreased the coimmunoprecipitation in SHR cells. The D(3) receptor also regulated the AT(1) receptor in vivo because AT(1) receptor expression was increased in kidneys of D(3) receptor-null mice compared with wild type littermates. D(3) receptors may regulate AT(1) receptor function by direct interaction with and regulation of AT(1) receptor expression. One mechanism of hypertension may be related to increased renal expression of AT(1) receptors due decreased D(3) receptor regulation.     

胰腺癌细胞系血管紧张素Ⅱ 1型受体蛋白表达的研究

目的探讨血管紧张素Ⅱ 1型受体(angiotensin Ⅱ type 1 receptor,AT1)在胰腺癌细胞中的表达.方法用免疫细胞化学染色检测胰腺癌细胞系SW1990、PaTu 8988s和PANC-1中AT1蛋白的表达.结果胰腺癌细胞系SW1990、PaTu 8988s和PANC-1中均有AT1蛋白的表达.结论 AT1在胰腺癌细胞生长中可能发挥重要作用,应用AT1拮抗剂对防治胰腺癌似乎有一定意义.     

Endothelin stimulates angiotensin I to angiotensin II conversion in cultured pulmonary artery endothelial cells

We studied the effects of endothelin on the conversion of angiotensin I to angiotensin II in pulmonary artery endothelial cells. Endothelin had a novel effect on angiotensin I conversion. When endothelin was added to pulmonary artery endothelial cells, the conversion of angiotensin I to angiotensin II was enhanced about two-fold. The maximum stimulation was achieved at 10(-8) M of endothelin. This stimulatory effect was suppressed by angiotensin converting enzyme inhibitors such an enalapril. When the calcium antagonist, nifedipine, was incubated with 10(-8) M of endothelin, the conversion of angiotensin I to angiotensin II stimulated with endothelin was slightly suppressed by nifedipine. Enalapril (10(-6) M) completely inhibited the conversion of angiotensin I to angiotensin II in the presence of endothelin. These results suggest that endothelin may play an important role in regulating vascular tone by modulating the conversion of angiotensin I to angiotensin II.     

Activation of AMP-activated protein kinase by human EGF receptor 2/EGF receptor tyrosine kinase inhibitor protects cardiac cells

The human EGF receptor (HER) 2 receptor tyrosine kinase is a survival factor for human cardiomyocytes, and its inhibition may explain the increased incidence of cardiomyopathy associated with the anti-HER2 monoclonal antibody trastuzumab (Genentech, South San Francisco, CA), particularly in patients with prior exposure to cardiotoxic chemotherapies e.g., anthracyclines. Here, we show that GW2974 (HER2/EGF receptor tyrosine kinase inhibitor), but not trastuzumab, activates AMP-activated protein kinase (AMPK), initiating a metabolic stress response in human cardiomyocytes that protects against TNFalpha-induced cell death. GW2974 stimulates calcium dependent fatty acid oxidation in vitro and in the myocardium of GW2974-treated rodents. Calcium chelation or siRNA-targeted AMPK knockdown blocks GW2974 induced fatty acid oxidation. In addition, inhibition of AMPK by a specific inhibitor resulted in increased killing of cardiomyocytes. Elucidating the effects of HER2-targeted therapies on AMPK may predict for risk of cardiomyopathy and provide a novel HER2-targeted strategy designed to protect myocardium from the pro-apoptotic effects of pro-inflammatory cytokines released in response to cardiac injury by chemotherapy or acute ischemia.     

Activation of endothelial nitric oxide synthase by the angiotensin II type 1 receptor

Enhanced angiotensin II (AngII) action has been implicated in endothelial dysfunction that is characterized as decreased nitric oxide availability. Although endothelial cells have been reported to express AngII type 1 (AT1) receptors, the exact role of AT1 in regulating endothelial NO synthase (eNOS) activity remains unclear. We investigated the possible regulation of eNOS through AT1 in bovine aortic endothelial cells (BAECs) and its functional significance in rat aortic vascular smooth muscle cells (VSMCs). In BAECs infected with adenovirus encoding AT1 and in VSMCs infected with adenovirus encoding eNOS, AngII rapidly stimulated phosphorylation of eNOS at Ser1179. This was accompanied with increased cGMP production. These effects were blocked by an AT1 antagonist. The cGMP production was abolished by a NOS inhibitor as well. To explore the importance of eNOS phosphorylation, VSMCs were also infected with adenovirus encoding S1179A-eNOS. AngII did not stimulate cGMP production in VSMCs expressing S1179A. However, S1179A was able to enhance basal NO production as confirmed with cGMP production and enhanced vasodilator-stimulated phosphoprotein phosphorylation. Interestingly, S1179A prevented the hypertrophic response similar to wild type in VSMCs. From these data, we conclude that the AngII/AT1 system positively couples to eNOS via Ser1179 phosphorylation in ECs and VSMCs if eNOS and AT1 coexist. However, basal level NO production may be sufficient for prevention of AngII-induced hypertrophy by eNOS expression. These data demonstrate a novel molecular mechanism of eNOS regulation and function and thus provide useful information for eNOS gene therapy under endothelial dysfunction.     

Activation of resting human T cells requires prolonged stimulation of protein kinase C.

Purified resting human T cells can be induced to express the alpha subunit of the interleukin 2 receptor and to proliferate by treatment with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin but not with 1,2-dioctanoylglycerol plus ionomycin. Determination of the translocation of protein kinase C showed that 12-O-tetradecanoylphorbol-13-acetate plus ionomycin caused a prolonged membrane association of the enzyme for more than 4 hr, whereas 1,2-dioctanoylglycerol plus ionomycin induced a transient membrane association, which was maximal at 20 min. Delivery of multiple additions of 1,2-dioctanoylglycerol plus ionomycin to the T cells resulted in progressively increased expression of the alpha subunit of the interleukin 2 receptor and proliferation commensurate with the number of multiple additions delivered, suggesting that prolonged protein kinase C activity is required for T-cell activation.     

Impaired translocation of protein kinase C activity in human non-insulin-dependent diabetes mellitus

Calcium- and phospholipid-dependent protein kinase (protein kinase C; PKC) may be an important mediator in transduction of some of the cellular actions of insulin. We studied PKC activity in freshly isolated circulating mononuclear cells obtained from healthy subjects and patients with non-insulin-dependent (type II) diabetes mellitus (NIDDM). The kinase activity was measured using a specific nonapeptide substrate, Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide. There was negligible calcium- and phospholipid-independent kinase activity in cytosolic and particulate fractions of cells from both control and diabetic subjects. Total (cytosolic and particulate) PKC activity of mononuclear cells from poorly controlled diabetic patients was significantly reduced compared with controls; this reduction was mainly due to a decrease in the cytosolic kinase activity. Tumor-promoting phorbol ester (TPA, 0.1 mumol/L) induced translocation of PKC activity in control cells; in contrast, this subcellular redistribution was not observed in cells from a majority of poorly controlled diabetic subjects. Increased calcium influx into the cells caused by the calcium ionophore A23187-triggered translocation of PKC activity in control cells, while it was ineffective in cells from poorly controlled diabetic patients. Cells from well-controlled diabetic patients demonstrated TPA-induced translocation of the PKC activity approaching that of control cells. The total PKC activity in cells from patients with good glycemic control was normal. Impaired activation of PKC is thus associated with the insulin resistance found in patients with poorly controlled NIDDM.(ABSTRACT TRUNCATED AT 250 WORDS)