Effects of betaine on ethanol-stimulated secretion of IGF-Ⅰ and IGFBP-1 in rat primary hepatocytes: involvement of p42/44 MAPK activation

AIM: To evaluate the effects of betaine on the ethanolinduced secretion of IGF-Ⅰ and IGFBP-1 using radioimmunoassay and Western blotting, respectively, in primary cultured rat hepatocytes.METHODS: Hepatocytes isolated from male Sprague-Dawley rats were incubated with various concentrations of ethanol and PD98059 procedures. The hepatocytes were also treated with different doses of betaine (10-5,10-4, and 10-3 mol/L). We measured IGF-Ⅰ and IGFBP-1 using radioimmunoassay and Western blotting, respectively.RESULTS: The ethanol-induced inhibition of IGF-Ⅰ secretion was attenuated by betaine in a concentration-dependent manner in primary cultured rat hepatocytes. At 10-3 mol/L, betaine significantly increased IGF-Ⅰ secretion but decreased IGFBP-1 secretion. In addition, p42/44 mitogen-activated protein kinase (MAPK) activity was accelerated significantly from 10 min to 5 h after treatment with 10-3 mol/L betaine. Furthermore, the changes in IGF-1 and IGFBP-1 secretion resulting from the increased betaine-induced p42/44 MAPK activity in primary cultured rat hepatocytes was blocked by treatment with the MAPK inhibitor PD98059. Betaine treatment blocked the ethanol-induced inhibition of IGF-Ⅰ secretion and p42/44 MAPK activity, and the ethanol-induced increase in IGFBP-1 secretion.CONCLUSION: Betaine modulates the secretion of IGF-Ⅰ and IGFBP-1 via the activation of p42/44 MAPK in primary cultured rat hepatocytes. Betaine also alters the MAPK activations induced by ethanol.     

Induction of protein synthesis in cardiac fibroblasts by cardiotrophin-1: integration of multiple signaling pathways

OBJECTIVE: Cardiotrophin-1 (CT-1) is a member of the IL-6 family of cytokines and is expressed in various cardiovascular disease states. CT-1 induces cardiomyocyte hypertrophy, and protects myocytes from ischemia reperfusion injury. We sought to elucidate CT-1 signaling in cardiac fibroblasts with respect to initiation of protein synthesis. METHODS: Cardiac fibroblasts were isolated from the ventricles of 200-g Sprague-Dawley rats and stimulated with CT-1 at specified concentrations with or without inhibitors of cell signaling pathways. Activation of intracellular signaling pathways was determined by Western analysis and immunocytochemistry. Protein synthesis was measured by incorporation of [3H]leucine. RESULTS: CT-1 treatment resulted in activation of the Jak/STAT, MAPK, and Akt pathways in addition to protein synthesis regulatory proteins with resultant increase in overall protein synthesis. Analysis with phospho-specific antibodies revealed that AG490 (Jak inhibitor), PD98059 (MEK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), LY294002 (PI3-K inhibitor) and rapamycin (mTOR inhibitor) act at different levels in the signaling cascade to inhibit CT-1 induced protein synthesis. CONCLUSION: Cardiotrophin-1 activates the Jak/STAT, PI3K/Akt, p38 and p42/44 MAPK pathways in cardiac fibroblasts. Use of pharmacologic inhibitors reveals that each of these pathways play a role in CT-1 induced protein synthesis.     

Mediation of basic fibroblast growth factor-induced lactotropic cell proliferation by Src-Ras-mitogen-activated protein kinase p44/42 signaling

Basic fibroblast growth factor (bFGF), which is secreted from folliculostellate cells in the anterior pituitary, is known to be involved in the communication between folliculostellate cells and lactotropes during estradiol-induced lactotropic cell proliferation. We studied the role of MAPK p44/42 in bFGF-regulated cell proliferation using enriched lactotropes and the lactotrope-derived PR1 cell line. In cell cultures, bFGF increased cell proliferation of PR1 cells and enriched lactotropes. In both of these cell populations, bFGF also increased phosphorylation of MAPK p44/42. U0126, an inhibitor of MAPK p44/42, blocked the bFGF-induced activation of MAPK p44/42 as well as the bFGF-induced cell proliferation of enriched lactotropes and PR1 cells. Treatment of PR1 cells with bFGF increased the activity of Ras p21, whereas overexpression of a dominant negative mutant of Ras p21 abrogated the bFGF-induced activation of MAPK p44/42 in these cells. Furthermore, the Src kinase inhibitor PP1 suppressed bFGF-induced activation of MAPK p44/42 in both enriched lactotropes and PR1 cells. The Src kinase inhibitor PP1 also reduced bFGF activation of Ras p21 and cell proliferation in PR1 cells. On the other hand, the bFGF-induced activation of MAPK p44/42 in enriched lactotropes and PR1 cells was not affected by protein kinase C inhibitors. These data suggest that bFGF induction of lactotropic cell proliferation is possibly mediated by activation of Src kinase, Ras p21, and MAPK p44/42.     

Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation

An early component of atherogenesis is abnormal vascular smooth muscle cell (VSMC) proliferation. The presence of Chlamydia pneumoniae in many atherosclerotic lesions raises the possibility that this organism plays a causal role in atherogenesis. In this study, C pneumoniae elementary bodies (EBs) rapidly activated p44/p42 mitogen-activated protein kinases (MAPKs) and stimulated proliferation of VSMCs in vitro. Exposure of VSMCs derived from human saphenous vein to C pneumoniae EBs (3x10(7) inclusion forming units/mL) enhanced bromodeoxyuridine (BrdU) incorporation 12+/-3-fold. UV- and heat-inactivated C pneumoniae EBs also stimulated VSMC proliferation, indicating a role of direct stimulation by chlamydial antigens. However, the mitogenic activity of C pneumoniae was heat-labile, thus excluding a role of lipopolysaccharide. Chlamydial hsp60 (25 microg/mL) replicated the effect of C pneumoniae, stimulating BrdU incorporation 7+/-3-fold. Exposure to C pneumoniae or chlamydial hsp60 rapidly activated p44/p42 MAPK, within 5 to 10 minutes of exposure. In addition, PD98059 and U0126, which are two distinct inhibitors of upstream MAPK kinase 1/2 (MEK1/2), abolished the mitogenic effect of C pneumoniae and chlamydial hsp60. Toll-like receptors (TLRs) act as sensors for microbial antigens and can signal via the p44/p42 MAPK pathway. Human VSMCs were shown to express TLR4 mRNA and protein, and a TLR4 antagonist abolished chlamydial hsp60-induced VSMC proliferation and attenuated C pneumoniae-induced MAPK activation and VSMC proliferation. Together these results indicate that C pneumoniae and chlamydial hsp60 are potent inducers of human VSMC proliferation and that these effects are mediated, at least in part, by rapid TLR4-mediated activation of p44/p42 MAPK.     

p38MAPK inhibition enhances basal and norepinephrine-stimulated p42/44MAPK phosphorylation in rat pinealocytes

Interaction between p38MAPK and p42/44MAPK in rat pinealocytes was examined by determining the effects of p38MAPK inhibitors on the phosphorylation of p42/44MAPK using Western blot analysis. Treatment with SB202190, a specific inhibitor of p38MAPK, increased p42/44MAPK phosphorylation in a concentration-dependent manner. SB202190 also enhanced the magnitude and the duration of norepinephrine-activated p42/44MAPK phosphorylation. The effect of SB202190 on p42/44MAPK phosphorylation was abolished by PD98059 or UO126, inhibitors of MEK, suggesting that SB202190 is acting upstream of MEK in activating p42/44MAPK. The SB202190-induced phosphorylation of p42/44MAPK was not blocked by inhibitors of cGMP-dependent kinase (KT5823), protein kinase C (calphostin C) or Ca2+/calmodulin dependent kinase (KN93) suggesting that these pathways may not be involved in the effect of SB202190. SB202190 further increased p42/44MAPK phosphorylation that was stimulated by 8-bromo-cGMP, 4beta phorbol 12-myristate 13-acetate, or ionomycin. In contrast, inhibition of p42/44MAPK phosphorylation by dibutyryl-cAMP persisted when p42/44MAPK phosphorylation was increased by SB202190. Furthermore, inhibition of p42/44MAPK phosphorylation had no effect on p38MAPK activation. These results suggest that inhibition of p38MAPK causes activation of p42/44MAPK and acts synergistically with norepinephrine in the regulation of p42/44MAPK activation in rat pinealocytes.     

Proinflammatory cytokines,IL-1β and TNF-α, induce expression of interleukin-34 mRNA via JNK- and p44/42 MAPK-NF-κB pathway but not p38 pathway in osteoblasts

The aim of this study is to investigate the induction of interleukin-34 (IL-34) and macrophage colony-stimulating factor (M-CSF) mRNA by inflammatory cytokines and the involvement of mitogen-activated protein kinases (MAPKs) in this signaling pathway in human osteoblasts as both IL-34 and M-CSF bind to the same receptor c-FMS. Among four inflammatory cytokines [(IL-1β, IL-6, IL-17, and tumor necrosis factor-α (TNF-α)], IL-34 mRNA expression level was dramatically induced by IL-1β (17-fold) and TNF-α (74-fold). IL-1β and TNF-α activated the intracellular mitogen-activated protein kinases (MAPKs): p44/42 MAPK, p38, and c-Jun N-terminal kinase (JNK) as well as nuclear factor-κB (NF-κB) in osteoblasts. IL-1β- and TNF-α-mediated induction of IL-34 mRNA expression was decreased by JNK inhibitor. Interestingly, although treatment of MEK-1/2 inhibitor showed no reduction in the increase of IL-34 mRNA expression by cytokines, combination of MEK-1/2 inhibitor and JNK inhibitor significantly inhibited IL-1β- and TNF-α-mediated IL-34 mRNA expression level compared to those by each inhibitor alone. On the other hand, M-CSF mRNA expression level was significantly induced by both IL-1β and TNF-α by up to 7- and 11-fold, respectively. IL-1β- and TNF-α-mediated induction of M-CSF mRNA was not affected by p38, JNK, and MEK-1/2 inhibitors. However, NF-κB inhibitor completely inhibited the elevation of M-CSF mRNA expression by these cytokines. These results showed that proinflammatory cytokines, IL-1β and TNF-α, induced the expression of IL-34 mRNA via JNK and p44/42 MAPK but not p38 in human osteoblasts while p38, JNK, and p44/42 MAPK were not involved in the induction of M-CSF mRNA expression by these cytokines.     

Role of cyclooxygenase 2, p38 and p42/44 MAPK in the secretion of prostacyclin induced by epidermal growth factor,endothelin-1 and angiotensin II in rat ventricular cardiomyocytes

We studied the respective roles of cyclooxygenases (COX) isoforms as well as the p38 and p42/44 MAP kinase cascades in angiotensin II (AngII)-, endothelin-1 (ET-1)- and epidermal growth factor (EGF)-induced prostacyclin (PGI(2)) secretion in neonatal rat ventricular cardiomyocytes. Exposure of these cells for 1 h to 100 nM AngII, ET-1 or EGF resulted in an increase in prostacyclin formation which was abolished by the COX-2 specific inhibitor NS-398 (1 microM), while the COX-1 inhibitor valeryl salicylate (5 microM) had no effect. Agonist-induced prostacyclin secretion was also abolished in the presence of cycloheximide (10 microg/ml), indicating that newly synthesized proteins are necessary for this response. In this context, the COX-2 protein amount was significantly increased following 1 h incubation of cardiomyocytes, with AngII, ET-1 and EGF. These results indicate that in cardiomyocytes AngII, ET-1 and EGF induce both the synthesis and the activity of COX-2. Investigating the role of MAPK in the stimulation of prostacyclin induced by these three agonists, we found that both the p42/44 MAPK inhibitor PD 98059 (50 microM) and the p38 MAPK blocker SB 203580 (5 microM) prevented agonist-induced PGI(2) secretion without affecting COX-2 activity or synthesis. Our results show that p42/44 and p38 MAPK activation is at the basis of AngII-, ET-1- and EGF-induced prostacyclin secretion in cardiomyocytes. They further suggest that these MAPK act on a target(s) located upstream of COX-2.     

Effects of betaine on ethanol-stimulated secretion of IGF-I and IGFBP-1 in rat primary hepatocytes： involvement of p42/44 MAPK activation

AIM:To evaluate the effects of betaine on the ethanol-induced secretion of IGF-I and IGFBP-1 using radioimmunoassay and Western blotting, respectively, in primary cultured rat hepatocytes. METHODS: Hepatocytes isolated from male Sprague-Dawley rats were incubated with various concentrations of ethanol and PD98059 procedures. The hepatocytes were also treated with different doses of betaine (10(-5), 10(-4), and 10(-3) mol/L). We measured IGF-I and IGFBP-1 using radioimmunoassay and Western blotting, respectively. RESULTS:The ethanol-induced inhibition of IGF-I secretion was attenuated by betaine in a concentration-dependent manner in primary cultured rat hepatocytes. At 10(-3) mol/L, betaine significantly increased IGF-I secretion but decreased IGFBP-1 secretion. In addition, p42/44 mitogen-activated protein kinase (MAPK) activity was accelerated significantly from 10 min to 5 h after treatment with 10(-3) mol/L betaine. Furthermore, the changes in IGF-1 and IGFBP-1 secretion resulting from the increased betaine-induced p42/44 MAPK activity in primary cultured rat hepatocytes was blocked by treatment with the MAPK inhibitor PD98059. Betaine treatment blocked the ethanol-induced inhibition of IGF-I secretion and p42/44 MAPK activity, and the ethanol-induced increase in IGFBP-1 secretion. CONCLUSION: Betaine modulates the secretion of IGF-I and IGFBP-1 via the activation of p42/44 MAPK in primary cultured rat hepatocytes. Betaine also alters the MAPK activations induced by ethanol.     

Effects of betaine on etnanol-stimulated secretion of IGF-I and IGFBP-1 in rat primary hepatocytes: involvement of p42/44 MAPK activation

AIM: To evaluate the effects of betaine on the ethanol-induced secretion of IGF-I and IGFBP-1 using radioim-munoassay and Western blotting, respectively, in primary cultured rat hepatocytes. METHODS: Hepatocytes isolated from male Sprague-Dawley rats were incubated with various concentrations of ethanol and PD98059 procedures. The hepatocytes were also treated with different doses of betaine (10-5, 10-4, and 10-3 mol/L). We measured IGF-I and IGFBP-1 using radioimmunoassay and Western blotting, respectively. RESULTS: The ethanol-induced inhibition of IGF-I secretion was attenuated by betaine in a concentration-dependent manner in primary cultured rat hepatocytes. At 10?mol/L, betaine significantly increased IGF-I secretion but decreased IGFBP-1 secretion. In addition, p42/44 mitogen-activated protein kinase (MAPK) activity was accelerated significantly from 10 min to 5 h after treatment with 10-3 mol/L betaine. Furthermore, the changes in IGF-1 and IGFBP-1 secretion resulting from the increased betaine-induced p42/44 MAPK activity in primary cultured rat hepatocytes was blocked by treatment with the MAPK inhibitor PD98059. Betaine treatment blocked the ethanol-induced inhibition of IGF-I secretion and p42/44 MAPK activity, and the ethanol-induced increase in IGFBP-1 secretion. CONCLUSION: Betaine modulates the secretion of IGF-I and IGFBP-1 via the activation of p42/44 MAPK in primary cultured rat hepatocytes. Betaine also alters the MAPK activations induced by ethanol.     

Aldosterone increases NHE-1 expression and induces NHE-1-dependent hypertrophy in neonatal rat ventricular myocytes

We determined the effect of 24-hour aldosterone (100 nmol/L) treatment on hypertrophic responses in rat neonatal ventricular myocytes and the possible role of Na+-H+ exchange isoform 1 (NHE-1). Aldosterone significantly increased cell size by 61% and expression of atrial natriuretic peptide by 2-fold. NHE-1 mRNA expression and protein abundance were significantly increased, and intracellular Na+ levels were elevated. Both hypertrophy and elevated Na+ levels were prevented by the NHE-1-specific inhibitor EMD87580 as well as the aldosterone antagonist spironolactone, although the increased NHE-1 levels were prevented only by spironolactone. Aldosterone transiently (within 5 minutes) stimulated p44/42 phosphorylation, which decreased thereafter for the remaining 24 hours, whereas p38 phosphorylation was reduced. Neither a p38 nor a p44/42 inhibitor had any effect on aldosterone-induced hypertrophy or NHE-1 regulation. Our results therefore demonstrate a direct hypertrophic effect of aldosterone on cultured myocytes, which is dependent on NHE-1 activity.     

丝裂原激活蛋白激酶信号通路在肝脏缺血预处理细胞保护效应中的作用

目的 研究p44/42丝裂原激活蛋白激酶(MAPK)信号通路在肝脏缺血预处理细胞保护效应中的作用。方法 建立体内和体外肝脏缺血预处理模型,应用蛋白激酶C(PKC)抑制剂和MAPK抑制剂,通过检测p44/42MAPK磷酸化水平,细胞活力,血清天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)的活性变化,同时观察光镜细胞形态学损害,对相关数据进行统计学处理。 结果 体内和体外的缺血预处理两部分实验都观察到相似的结果。和缺血再灌注组比较,预处理组的p44/42 MAPK磷酸化水平显著增高,肝细胞结构损伤改变较小,血清ALT、AST水平显著降低,缺血再灌注组的ALT、AST水平分别为(762.8±130.5)U/L和(820.9±111.3)U/L,预处理组的ALT、AST水平分别为(281.0±35.6)U/L和(407.7±73.7)U/L;和缺血预处理组相比,PKC抑制剂组和丝裂原蛋白激酶(MEK)抑制剂组相应的观察指标呈相反的变化,p44/42 MAPK磷酸化激活显著减少,肝组织细胞结构出现较明显的损伤改变,血清ALT、AST水平显著升高,PKC抑制剂组和MEK抑制剂组的ALT、AST水平分别为(645.61±90.4)U/L、(678.6±136.5)U/L和(466.2±82.8)U/L、(732.9±91.1)U/L。 结论 肝脏缺血预处理细胞保护作用中,p44/42 MAPK通路起到至关重要的作用。     

p38丝裂原活化蛋白激酶对急性坏死性胰腺炎大鼠低钙血症的影响

目的 探讨p38丝裂原活化蛋白激酶(p38MAPK)信号转导通路对急性坏死性胰腺炎(ANP)大鼠低钙血症和甲状旁腺激素受体1(PTHR1)表达的影响.方法 将雄性SD大鼠72只按完全随机法分为ANP组、SB203580干预(SB)组和假手术(SO)组,每组分3、6、12 h 3个时间点,每个时间点8只.以5%牛磺脱氧胆酸钠逆行胰胆管注射建立ANP模型,SB组在造模前30 min腹腔注射p38MAPK特异抑制剂SB203580 10 mg/kg体重.观察各组血清钙浓度,蛋白质印迹法(Western blotting)分析骨组织磷酸化p38MAPK(P-p38 MAPK)和TNF-α变化,实时RT-PCR检测骨组织PTHR1 mRNA表达.结果 制模后6 h,SO组、ANP组和SB组血清钙浓度分别为(2.50±0.08)mmoL/L、(2.11±0.06)mmol/L和(2.35±0.10)mmol/L;骨组织P-p38 MAPK表达量分别为0.14±0.04、0.80±0.06和0.33±0.05;骨组织TNF-α表达量分别为0、0.91±0.04和0.44±0.03;骨组织PTHR1 mRNA表达量分别为1.00±0.12、0.23±0.04和0.44±0.06.SB组骨组织P-p38 MAPK及TNF-α表达较ANP组显著降低(P＜0.01);骨组织PTHR1 mRNA表达量及血清钙浓度较ANP组显著增加(P＜0.01).结论 p38MAPK信号转导通路可介导ANP低钙血症的发生,抑制该通路可改善ANP低钙血症.     

A farnesyltransferase inhibitor attenuates cardiac myocyte hypertrophy and gene expression

The overexpression of either oncogenic ras or calmodulin in cardiac myocytes can elicit a hypertrophic response, albeit their recruitment by physiologically relevant stimuli remains unresolved. The present study utilized a pharmacological approach to examine the role of ras and calmodulin in norepinephrine- and endothelin-1-stimulated hypertrophy of neonatal rat cardiac myocytes. The pretreatment of cardiac myocytes with the farnesyltransferase inhibitor BMS-191563 (25 microM) increased the level of unfarnesylated ras in the cytosolic fraction, and caused a concomitant 42 +/- 2% decrease in immunodetectable farnesylated ras in the particulate fraction. In parallel, BMS-191563 pretreatment inhibited norepinephrine-mediated 3H-leucine uptake (80 +/- 10% decrease: n = 6; P<0.01), whereas a significant but less pronounced effect on the endothelin-1 response (46 +/- 6% decrease: n = 6; P<0.05) was observed. The calmodulin inhibitor W7 caused a 50 +/- 10% decrease (n = 8; P<0.05) of norepinephrine stimulated protein synthesis, whereas the endothelin-1 response was unaffected. Consistent with the recruitment of ras, BMS-191563 pretreatment attenuated norepinephrine and endothelin-1-stimulated extracellular signal-regulated kinase (ERK) activity. However, PD098059-mediated inhibition of MEK-dependent stimulation of ERK did not alter the hypertrophic response of either agonist. At the molecular level, the pretreatment with either BMS-191563 or W7 attenuated the norepinephrine-mediated increase of prepro-ANP and -BNP mRNA. Likewise, BMS-191563 caused a significant decrease of endothelin-1-mediated expression of the natriuretic peptide mRNAs, but to a lesser extent, as compared to norepinephrine. Thus, the present study has shown the treatment of neonatal rat cardiac myocytes with a farnesyltransferase inhibitor can attenuate the hypertrophic phenotype in response to physiologically relevant stimuli, thereby supporting a role of the small GTP-binding protein ras. Moreover, these data further suggest alternative ras-independent signaling pathways are also implicated in the hypertrophic response, albeit, there appears to exist a stimulus-specific heterogeneity in their recruitment.