抑制细胞外信号调节激酶转导结直肠癌细胞组蛋白磷酸化和基因表达的影响

目的 观察人结直肠癌细胞中细胞外信号调节激酶-丝裂原激活蛋白激酶(ERK-MAPK)信号转导途径与组蛋白磷酸化水平及其相关蛋白表达的关系,及其对细胞增殖和细胞周期的影响.方法 培养结直肠癌细胞系SW1116、HCT116,以终浓度分别为0、10、20及40μmol/L的ERK-MAPK阻断剂U0126干预细胞.细胞计数试剂盒(cell counting kit,CCK-8)检测细胞活力影响,以流式细胞术(flow cytometry,FCM)研究细胞周期变化,Western免疫印迹法分析组蛋白H3激酶核糖体S6丝氨酸-苏氨酸激酶2(ribosomal S6 kinase 2,RSK-2)、丝裂原和应激-激活的激酶1和2(mitogen-and stress-activated kinase 1 and 2,MSK1和MSK2)、组蛋白H3(Ser10)磷酸化水平及c-Fos蛋白表达变化情况.结果 CCK-8法检测发现ERK-MAPK阻断剂能以剂量和时间依赖方式明显抑制2种结直肠癌细胞的增殖,增殖率可下降至对照组的47%;并能明显增加G0/G1期细胞百分比(P<0.01),而S期细胞百分比明显减少(P<0.01).Western印迹结果显示U0126干预后,H3激酶MSK1和RSK2表达明显降低,分别为对照HCT116的28%和40%,而MSK2蛋白水平在2个细胞系中均无明显变化.相应地,组蛋白H3磷酸化(Ser10)水平亦降低,相关蛋白c-Fos的表达亦明显下降.结论 抑制ERK-MAPK信号转导可能通过抑制组蛋白H3激酶MSK1、RSK2活性而降低组蛋白H3磷酸化水平,进而减少c-Fos蛋白表达,从而抑制人结直肠癌细胞的增殖和生长.     

Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis

BACKGROUND & AIMS: Stimulation of canalicular secretion by tauroursodeoxycholate (TUDC) involves dual activation of p38 mitogen-activated protein kinase (p38(MAPK)) and extracellular signal-regulated kinase (ERK). This study investigates the sensing and upstream signaling events of TUDC-induced choleresis. METHODS: TUDC and hypo-osmolarity effects on protein kinase activities and taurocholate excretion were studied in perfused rat liver. RESULTS: TUDC induced a rapid activation of focal adhesion kinase (FAK) and Src, as shown by an increase in Y418 phosphorylation and a decrease in Y529 phosphorylation of Src. Inhibition of Src by PP-2 abolished the TUDC-induced activation of p38(MAPK) but not of FAK and ERKs. An integrin-inhibitory peptide with an RGD motif blocked TUDC-induced FAK, Src, ERK, and p38(MAPK) activation, suggesting that integrin signaling toward FAK/Src is required for TUDC-induced MAPK activation. The RGD peptide and PP-2 also abolished the stimulation of taurocholate excretion in perfused rat liver in response to TUDC. Integrin-dependent Src activation was also identified as an upstream event in hypo-osmotic signaling toward MAPKs and choleresis. CONCLUSIONS: TUDC-induced stimulation of canalicular taurocholate excretion involves integrin sensing, FAK, and Src activation as upstream events for dual MAPK activation. Integrins may also represent one long-searched sensor for cell hydration changes in response to hypo-osmolarity.     

Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode

AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.     

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.     

Acid exposure activates the mitogen-activated protein kinase pathways in Barrett's esophagus

BACKGROUND & AIMS: To explore mechanisms whereby acid reflux might contribute to carcinogenesis in Barrett's esophagus (BE) we studied: (1) the effects of acid on the mitogen-activated protein kinase (MAPK) pathways, cell proliferation, and apoptosis in a Barrett's adenocarcinoma cell line (SEG-1); and (2) the ability of acid to activate the MAPK pathways in vivo in patients with BE. METHODS: SEG-1 cells were exposed to acidic media for 3 minutes, and the activities of 3 MAPKs (ERK, p38, and JNK) were determined. Proliferation was assessed using flow cytometry; cell growth and apoptosis were assessed using cell counts and an apoptosis ELISA assay. MAPK activation was studied in biopsy specimens taken from patients with BE before and after esophageal perfusion for 3 minutes with 0.1N HCl. RESULTS: Acid-exposed SEG-1 cells exhibited a significant increase in proliferation and total cell numbers, and a significant decrease in apoptosis. These effects were preceded by a rapid increase in the activities of ERK and p38, and a delayed increase in JNK activity. PD 98059 abolished the acid-induced increase in G0/G1 and decrease in subG0 phases of the cell cycle. Both SB 203580 and DN-JNK 1/2 inhibited the acid-induced progression from G0/G1 to G2/M. The acid-induced decrease in apoptosis was abolished by inhibition of either ERK or p38. In the patients, acid exposure significantly increased the activity of p38 in the metaplastic epithelium. CONCLUSIONS: Acid increases proliferation and survival, and decreases apoptosis in SEG-1 cells by activating the MAPK pathways. Acid also activates the MAPK pathways in BE in vivo. These findings suggest that acid might contribute to carcinogenesis in BE through activation of MAPK pathways.     

Aberrant p38 mitogen-activated protein kinase signalling in skeletal muscle from Type 2 diabetic patients

Aims/hypothesis p38 mitogen activated protein kinase (MAPK) is generally thought to facilitate signal transduction to genomic, rather than metabolic responses. However, recent evidence implicates a role for p38 MAPK in the regulation of glucose transport; a site of insulin resistance in Type 2 diabetes. Thus we determined p38 MAPK protein expression and phosphorylation in skeletal muscle from Type 2 diabetic patients and non-diabetic subjects.Methods In vitro effects of insulin (120 nmol/l) or AICAR (1 mmol/l) on p38 MAPK expression and phosphorylation were determined in skeletal muscle from non-diabetic (n=6) and Type 2 diabetic (n=9) subjects.Results p38 MAPK protein expression was similar between Type 2 diabetic patients and non-diabetic subjects. Insulin exposure increased p38 MAPK phosphorylation in non-diabetic, but not in Type 2 diabetic patients. In contrast, basal phosphorylation of p38 MAPK was increased in skeletal muscle from Type 2 diabetic patients.Conclusion/interpretation Insulin increases p38 MAPK phosphorylation in skeletal muscle from non-diabetic subjects, but not in Type 2 diabetic patients. However, basal p38 MAPK phosphorylation is increased in skeletal muscle from Type 2 diabetic patients. Thus, aberrant p38 MAPK signalling might contribute to the pathogenesis of insulin resistance.Abbreviations AICAR 5-aminoimidazole-4-carboxamide ribonucleoside - AMPK 5-AMP activated protein kinase - ERK 1/2 extracellular regulated kinase - GIR glucose infusion rate - IRS-1 insulin receptor substrate 1 - MAPK mitogen-activated protein kinase - PI phosphatidylinositol - VO_2max maximal oxygen uptake     

5-ASA affects cell cycle progression in colorectal cells by reversibly activating a replication checkpoint

BACKGROUND & AIMS: Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. METHODS: CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116(p53-/-), HCT116+chr3, and LoVo were treated with 5-ASA for 2-96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. RESULTS: We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. CONCLUSIONS: Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis.     

Insulin induces PKC-dependent proliferation of mesenteric vascular smooth muscle cells from hypertensive patients

Background and objectives Proliferation of human vascular smooth muscle cells (VSMCs) induced by hyperinsulinemia is a very common clinical pathology. Extensive research has focused on PKC (Protein kinase C)-MAPK (mitogen-activated protein kinase) intracellular signal transduction and the phenotypic modulation accompanied by reorganization of intracellular F-actins in VSMCs. Methods DNA synthesis, signaling of ERK1/2 MAPKs, and changes in a-smooth muscle (SM) actin and F-actin were studied in hypertensive and normotensive human arterial VSMCs exposed to insulin and PMA with and without the PKC inhibitor, GF109203X. Results Differences among cell types in MAPK signaling, a-SM actin, and F-actin isoforms in VSMCs harvested from the arteries of patients with essential hypertension (EH) and normotension (NT) were identified in response to insulin treatment. Proliferation and activation of MAPK were more pronounced in EH VSMCs than in NEH VSMCs. Insulin exposure decreased expression of a-SM actin and was accompanied by rearrangement of intracellular F-actins in VSMCs, especially in the EH group. These effects were reversed by treatment with the PKC inhibitor. Conclusions Human mesenteric VSMCs of EH and NT patients differed in proliferation, MAPK signaling, and degree of changes in a-SM actin and F-actin isoforms immediately following insulin exposure in vitro.     

Helicobacter pylori strain-selective induction of matrix metalloproteinase-7 in vitro and within gastric mucosa

BACKGROUND AND AIMS: Helicobacter pylori strains that possess the cag pathogenicity island (cag(+)) augment the risk for distal gastric cancer. Matrix metalloproteinase (MMP)-7, an epithelial cell-derived MMP that is induced by bacterial contact, is overexpressed within human gastric adenocarcinoma specimens and enhances tumor formation in rodents. We determined whether H. pylori alters MMP-7 expression and investigated the molecular pathways required for these events. METHODS: MMP-7 was detected in human gastric mucosa by immunohistochemistry and in H. pylori/AGS gastric epithelial cell coculture supernatants by Western analysis. AGS cells were cocultured with wild-type H. pylori, or isogenic cagA(-), cagE(-), or vacA(-) mutants, in the absence or presence of inhibitors of nuclear factor kappaB activation, p38, or extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. RESULTS: H. pylori cag(+) strains increased MMP-7 expression in AGS cells 5-7-fold, whereas cag(-) isolates had no effect. Inactivation of cagE, but not cagA or vacA, completely attenuated induction of MMP-7, and inhibition of ERK 1/2 decreased MMP-7 production. In vivo, MMP-7 was expressed in gastric epithelial cells in specimens from 80% of cag(+)-colonized persons but in none of the cag(-) or uninfected subjects. CONCLUSIONS: H. pylori cag(+) strains enhance levels of MMP-7 within inflamed mucosa. In vitro, cag(+) isolates selectively induce MMP-7, and this is dependent on activation of ERK 1/2 by specific components within the cag island. Differential induction of MMP-7 by H. pylori cag(+) isolates may explain in part the augmentation in gastric cancer risk associated with these strains.     

Down-regulation of the dual-specificity phosphatase MKP-1 suppresses tumorigenicity of pancreatic cancer cells

BACKGROUND & AIMS: In both pancreatic cancer and chronic pancreatitis, there is enhanced expression of mitogenic growth factors and their tyrosine kinase receptors, which have the capacity to activate mitogen-activated protein kinase (MAPK). In view of the important role of MAPK kinase phosphatase (MKP)-1 in the regulation of MAPK activation, the expression and functional role of MKP-1 was analyzed. METHODS: Pancreatic tissues were analyzed by Northern blotting, Western blotting, and immunohistochemistry. Pancreatic cancer cells were transfected with a full-length MKP-1 antisense construct. Growth characteristics and tumorigenicity in vivo and the effects of mitogenic growth factors on cell growth and MAPK activation were determined in transfected and control cells. RESULTS: MKP-1 messenger RNA (mRNA) levels were increased in pancreatic cancer and chronic pancreatitis (CP) tissues. Moderate to strong MKP-1 immunoreactivity was present in the cancer cells, ductal cells of pancreatic intraepithelial neoplasia, and in tubular complexes in CP. Down-regulation of MKP-1 resulted in decreased anchorage-dependent and -independent growth of pancreatic cancer cells, and decreased tumorigenicity in a nude mouse tumor model. MKP-1 down-regulation led to decreased proliferation and sustained MAPK activation in response to mitogens. CONCLUSIONS: Suppression of MKP-1 expression reduces the tumorigenicity of pancreatic cancer cells in vivo, suggesting that MKP-1 contributes to enhanced mitogenic signaling in pancreatic cancer cells.     

Bile acids induce cyclooxygenase-2 expression via the epidermal growth factor receptor in a human cholangiocarcinoma cell line

BACKGROUND & AIMS: Although bile acids have been implicated in colon cancer development, their role in biliary tract carcinogenesis remains unexplored. Because receptor tyrosine kinases and cyclooxygenase (COX)-2 have been implicated in carcinogenesis, we examined the hypothesis that bile acids modulate these enzymes in KMBC cells, a human cholangiocarcinoma cell line. METHODS: The effect of bile acids on epidermal growth factor receptor (EGFR) stimulation, mitogen-activated protein kinase (MAPK) activation, and COX-2 expression was evaluated. RESULTS: Bile acids both induced EGFR phosphorylation and enhanced COX-2 protein expression. Bile acid-induced EGFR phosphorylation was associated with subsequent activation of MAPK p42/44, p38, and c-Jun-N-terminal kinase (JNK). The MAPK inhibitors, PD098059 for MAP or extracellular signal-regulated kinase 1, SB203580 for p38, and BAY 37-9751 for Raf-1, blocked COX-2 induction by bile acids. However, inhibition of JNK activity did not block bile acid-mediated COX-2 induction. CONCLUSIONS: The results show that EGFR is activated by bile acids and functions to induce COX-2 expression by an MAPK cascade. This induction of COX-2 may participate in the genesis and progression of cholangiocarcinomas.     

HB-EGF enhances restitution after intestinal ischemia/reperfusion via PI3K/Akt and MEK/ERK1/2 activation

BACKGROUND & AIMS: Early recovery of intestinal function after injury occurs by restitution, a complex process with a poorly understood molecular basis. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent chemotactic factor that is induced during ischemia/reperfusion in vivo and intestinal wounding in vitro. The role of HB-EGF in intestinal restitution and the underlying intracellular signaling pathways involved were investigated. METHODS: Adult rats were subjected to intestinal ischemia, with histologic and biochemical damage assessed during the first 3 hours of reperfusion. The effect of recombinant HB-EGF (rHB-EGF) on structural and functional recovery of the intestine by restitution was evaluated in vivo. Scrape wounding of intestinal epithelial cell monolayers was used to elucidate the mechanisms of intrinsic and rHB-EGF-induced restitution. RESULTS: Early structural recovery occurred within 3 hours of reperfusion and was attributed to restitution rather than proliferation. HB-EGF treatment significantly improved structural recovery and accelerated functional recovery of the gut barrier. In vivo restitution was preceded by activation of Akt and extracellular signal-regulated kinase (ERK) 1/2, which were accelerated and enhanced by HB-EGF treatment. Blocking of ErbB-1, phosphatidylinositol 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase/ERK kinase (MEK)/ERK activity resulted in significant reduction in intrinsic and HB-EGF-induced restitution in vitro. Endogenous HB-EGF was shown to play an essential role in wound-induced ErbB-1 and ERK1/2 activation and in intrinsic restitution. CONCLUSIONS: Endogenous HB-EGF, ErbB-1, PI3K/Akt, and MEK/ERK are involved in intrinsic restitution. rHB-EGF enhances restitution in vivo and in vitro in a PI3K/Akt- and MEK/ERK1/2-dependent fashion.     

Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 microM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.     

Melatonin stimulates calmodulin phosphorylation by protein kinase C

Calmodulin (CaM)-dependent processes can be modulated by the availability of Ca(+2), the subcellular distribution of both CaM and its target proteins, CaM antagonism, and post-translational modifications such as CaM phosphorylation. Melatonin, the pineal secretory product synthesized during the dark phase of the photoperiod is an endogenous CaM antagonist. This indolamine causes CaM subcellular redistribution in epithelial MDCK and MCF-7 cells, and selectively activates protein kinase C alpha (PKC alpha) in neuronal N1E-115 cells. In the present work we have characterized the phosphorylation of CaM mediated by PKC alpha and its stimulation by melatonin in an in vitro reconstituted enzyme system. Additionally, the participation of MAPK and ERKs, downstream kinases of the PKC signaling pathway, was explored utilizing MDCK cell extracts as source of these kinases. Phosphorylation of CaM was characterized in the whole cells by MDCK cell metabolic labeling with [(32)P]-orthoposhospate, and CaM separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, as well as by immunocolocalization of phosphorylated threonine/serine residues and CaM in cultured cells incubated with melatonin. Our results show that melatonin increased CaM phosphorylation by PKC alpha with an EC(50) of 10(-8) m in the presence of the phorbol ester, phorbol-12-myristate-13-acetate (PMA) in the in vitro reconstituted enzyme system. An increase in phosphorylated CaM was also observed in cells cultured with melatonin, or PMA for 2 hr, while, PKC, MAPK, or ERK inhibitors abolished CaM phosphorylation elicited by melatonin in MDCK cell extracts. Our data show that melatonin can stimulate phosphorylation of CaM by PKC alpha in the in vitro reconstituted system and suggest that in MDCK cells this phosphorylation is accomplished by PKC. Modification of CaM by melatonin can be another route to inhibit CaM interaction with its target enzymes.