Helicobacter pylori and mitogen-activated protein kinases regulate the cell cycle, proliferation and apoptosis in gastric epithelial cells

Background and Aims:  Helicobacter pylori infection activates mitogen-activated protein kinases (MAPK) and modulates cell proliferation and apoptosis. However, the relationship between H. pylori infection and MAPK signaling in controlling cell proliferation and apoptosis is not clear, nor has the role of MAPK on the gastric epithelial cell cycle and proliferation been established. Therefore, we investigated the effects of H. pylori infection and MAPK inhibition on these processes.
Methods:  Gastric epithelial cell lines (AGS and MKN45) were infected with H. pylori and/or treated with MAPK inhibitors. Cell cycle and apoptosis were measured by flow cytometry. Cell cycle proteins and proliferation were monitored by western blot and cell count, respectively.
Results:  Infection with H. pylori resulted in dose-dependent MAPK activation, cell cycle arrest, reduced proliferation and increased apoptosis. The effect of H. pylori and MAPK at various cell cycle checkpoints was noted: MEK1/2 and p38 inhibition increased H. pylori -induced cell cycle G₁ arrest, while JNK inhibition reduced G₁ arrest. MEK1/2 inhibition increased p21, p27 and cyclin E and JNK inhibition additionally increased cyclin D1 expression. Both inhibitors decreased cell proliferation. All inhibitors enhanced apoptosis after H. pylori infection. We also detected MAPK cross-talk in AGS cells: p38 and JNK inhibitors increased ERK activation. The p38 inhibitor increased JNK and the MEK1/2 inhibitor decreased JNK activation only during H. pylori infection.
Conclusions:  These results suggest H. pylori and MAPK differentially regulate the cell cycle, proliferation and apoptosis in gastric epithelial cells. The imbalance between H. pylori infection and MAPK activation likely contributes to the H. pylori -induced pathogenesis.     

p16(INK4a) induces differentiation and apoptosis in erythroid lineage cells

OBJECTIVE: Hematopoiesis is regulated by proliferation, differentiation, and death. p16(INK4a) has been reported to regulate apoptosis and differentiation of diverse cells, as well as arresting the cell cycle at G1 phase. The aim of this study is to explore the properties of p16 in apoptosis and differentiation of erythroid cells. METHODS: We transfected the INK4a gene to K562 cells, which defect the INK4a gene, and compared the effect of enforced expression of p16(INK4a) with that of various additives, topoisomerase I inhibitor (SN 38), interferon-alpha, phosphatidyl-inositol-3 kinase inhibitor (LY294002), and serum deprivation, which arrest cell cycle at different phases. We also investigated the role of p16(INK4a) in normal day-6 human erythroid colony-forming cells by transfecting the INK4a gene. RESULTS: p16(INK4a) induced cell cycle arrest at the G0/G1 phase, and promoted erythroid differentiation in viable K562 cells, but induced apoptosis in K562 cells with incomplete differentiation. The apoptosis induced by p16 was accompanied with downregulation of bcl-x and nuclear NF-kappaB. These findings were not observed in K562 cells treated with various additives. p16(INK4a) decreased the cell viability and promoted apoptosis in day-9 ECFC. CONCLUSION: We propose that p16(INK4a) plays a role in maintaining homeostasis during erythroid differentiation, and that the mechanisms for this effect are not confined to those inducing cell cycle arrest.     

Neighbor of Brca1 gene (Nbr1) functions as a negative regulator of postnatal osteoblastic bone formation and p38 MAPK activity

The neighbor of Brca1 gene (Nbr1) functions as an autophagy receptor involved in targeting ubiquitinated proteins for degradation. It also has a dual role as a scaffold protein to regulate growth-factor receptor and downstream signaling pathways. We show that genetic truncation of murine Nbr1 leads to an age-dependent increase in bone mass and bone mineral density through increased osteoblast differentiation and activity. At 6 mo of age, despite normal body size, homozygous mutant animals (Nbr1^tr/tr) have ~50% more bone than littermate controls. Truncated Nbr1 (trNbr1) co-localizes with p62, a structurally similar interacting scaffold protein, and the autophagosome marker LC3 in osteoblasts, but unlike the full-length protein, trNbr1 fails to complex with activated p38 MAPK. Nbr1^tr/tr osteoblasts and osteoclasts show increased activation of p38 MAPK, and significantly, pharmacological inhibition of the p38 MAPK pathway in vitro abrogates the increased osteoblast differentiation of Nbr1^tr/tr cells. Nbr1 truncation also leads to increased p62 protein expression. We show a role for Nbr1 in bone remodeling, where loss of function leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis.     

Involvement of p38 mitogen-activated protein kinase signaling in transformed growth of a cholangiocarcinoma cell line

Although mitogen-activated protein kinase (MAPK) pathways play a key role in cell growth, their role in mediating the altered growth phenotype of transformed cells remains unclear. The p44/p42 MAPK signaling cascades are activated by mitogenic stimulation of human cholangiocytes. In contrast, the p38 MAPK pathway is activated by mitogen stimulation of malignant, but not nonmalignant cholangiocytes. Thus, our aims were to determine the role of p38 MAPK signaling in mediating the growth phenotype of transformed cholangiocytes. KMCH-1 malignant human cholangiocytes required the presence of serum for proliferation, but were able to grow in reduced serum conditions. Inhibition of p38 MAPK decreased serum-dependent proliferation of KMCH-1 cells. Furthermore, inhibition of p38 MAPK, but not of p44/p42 MAPK, reduced anchorage-independent growth of KMCH-1 cells. Although both p38 and p44/p42 MAPK are activated in response to mitogens, they have divergent effects on anchorage-independent growth. Inhibition of p38 MAPK, but not of p44/p42 MAPK signaling, decreased cell cycle progression and increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIPl). However, expression of p27(KIP1) or p16(INK4A) was not altered by either pathway. Thus, mitogen activation of p38 MAPK decreases expression of p21(WAF1/CIP1) and mediates growth independent of anchorage signals, whereas mitogen activation of p44/p42 MAPK mediates an anchorage signal-dependent growth pathway. These data provide a link between aberrant stress-activated cell signaling and the altered growth phenotype of transformed cells that may be important for the development of therapies to limit transformed cell growth.     

淫羊藿苷通过雌激素受体和p38MAPK信号诱导MC3T3-E1Subclone14细胞的分化

目的观察淫羊藿苷(ICA)对MC3T3-E1Subclone14前体成骨细胞株活力、分化的影响,以及雌激素受体(ER)信号、p38MAPK信号在分化过程中的作用。方法 WST-8方法检测MC3T3-E1Subclone14细胞活力;pNPP法检测细胞碱性磷酸酶活性(ALP);ELISA检测I型胶原(ColI)和骨钙素(BGP);Western印迹法检测p38MAPK的蛋白磷酸化;并分别用ICI182780阻断ER受体或SB203580阻断p38MAPK信号后检测ICA对细胞ALP、Col I、BGP的影响;Western印迹法检测ICI182780阻断ER受体信号后,ICA对p38MAPK蛋白磷酸化的影响。结果 ICA(10-7、10-6、10-5mol/L)对细胞活力与对照组比较,在统计学上无显著差异(P>0.05);ICA可以浓度依赖性的提高细胞的ALP、Col I和BGP和矿化结节数量(P<0.01,P<0.05);ICI182780阻断ER受体信号后,10-5mol/L浓度的ICA促细胞分化的特性明显下降(P<0.01);ICA可以浓度组依赖性的提高细胞p38MAPK的蛋白磷酸的水平(P<0.01);SB203580阻断p38MAPK信号后,10-5mol/L浓度的ICA促分化的特性下降(P<0.01);ICI182780阻断ER受体信号后,10-5mol/L浓度ICA促p38MAPK磷酸化明显减弱(P<0.01)。结论 ICA可以促进MC3T3-E1Subclone14细胞分化,ER受体信号、p38MAPK信号在促分化过程中起着重要作用,ER受体信号通路在p38MAPK信号通路的上游。     

Role of p27(Kip1) in human intestinal cell differentiation

BACKGROUND & AIMS: Growth arrest and differentiation are generally considered to be temporally and functionally linked phenomena in the intestinal epithelium. METHODS: To delineate the mechanism(s) responsible for the loss of proliferative potential as committed intestinal cells start to differentiate, we have analyzed the regulation of G(1)-phase regulatory proteins in relation to differentiation in the intact epithelium as well as in well-established intestinal cell models that allow the recapitulation of the crypt-villus axis in vitro. RESULTS: With intestinal cell differentiation, we have observed an induction of the cell cycle inhibitors p21(Cip), p27(Kip1), and p57(Kip2) expression with an increased association of p27(Kip1) and p57(Kip2) with cyclin-dependent kinase 2 (Cdk2). At the same time, there was an accumulation of the hypophosphorylated form of the pRb proteins and a strong decline in Cdk2 activity. Stable expression of a p27(Kip1) antisense complementary DNA in Caco-2/15 cells did not prevent growth arrest induced by confluence, but repressed villin, sucrase-isomaltase, and alkaline phosphatase expression. CONCLUSIONS: Our results indicate that the growth arrest that precedes differentiation involves the activation of Rb proteins and the inhibition of Cdk2. Furthermore, intestinal cell differentiation apparently requires a function of p27(Kip1) other than that which leads to inhibition of Cdks.     

Melatonin induces cell cycle arrest and apoptosis in hepatocarcinoma HepG2 cell line

Abstract: Melatonin reduces proliferation in many different cancer cell lines. However, studies on the oncostatic effects of melatonin in the treatment of hepatocarcinoma are limited. In this study, we examined the effect of melatonin administration on HepG2 human hepatocarcinoma cells, analyzing cell cycle arrest, apoptosis and mitogen‐activated protein kinase (MAPK) signalling pathways. Melatonin was dissolved in the cell culture media in 0.2% dimethyl sulfoxide and administered at different concentrations for 2, 4, 6, 8 and 10 days. Melatonin at concentrations 1000–10,000 μm caused a dose‐ and time‐dependent reduction in cell number. Furthermore, melatonin treatment induced apoptosis with increased caspase‐3 activity and poly(ADP‐ribose) polymerase proteolysis. Proapoptotic effects of melatonin were related to cytosolic cytochrome c release, upregulation of Bax and induction of caspase‐9 activity. Melatonin treatment also resulted in increased caspase‐8 activity, although no significant change was observed in Fas‐L expression. In addition, JNK 1,‐2 and ‐3 and p38, members of the MAPK family, were upregulated by melatonin treatment. Growth inhibition by melatonin altered the percentage or cells in G0–G1 and G2/M phases indicating cell cycle arrest in the G2/M phase. The reduced cell proliferation and alterations of cell cycle were coincident with a significant increase in the expression of p53 and p21 proteins. These novel findings show that melatonin, by inducing cell death and cell cycle arrest, might be useful as adjuvant in hepatocarcinoma therapy.     

Cell cycle exit during terminal erythroid differentiation is associated with accumulation of p27(Kip1) and inactivation of cdk2 kinase

Progression through the mammalian cell cycle is regulated by cyclins, cyclin- dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs). The function of these proteins in the irreversible growth arrest associated with terminally differentiated cells is largely unknown. The function of Cip/Kip proteins p21(Cip1) and p27(Kip1) during erythropoietin-induced terminal differentiation of primary erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus was investigated. Both p21(Cip1) and p27(Kip1) proteins were induced during erythroid differentiation, but only p27(Kip1) associated with the principal G(1) CDKs-cdk4, cdk6, and cdk2. The kinetics of binding of p27(Kip1) to CDK complexes was distinct in that p27(Kip1) associated primarily with cdk4 (and, to a lesser extent, cdk6) early in differentiation, followed by subsequent association with cdk2. Binding of p27(Kip1) to cdk4 had no apparent inhibitory effect on cdk4 kinase activity, whereas inhibition of cdk2 kinase activity was associated with p27(Kip1) binding, accumulation of hypo-phosphorylated retinoblastoma protein, and G(1) growth arrest. Inhibition of cdk4 kinase activity late in differentiation resulted from events other than p27(Kip1) binding or loss of cyclin D from the complex. The data demonstrate that p27(Kip1) differentially regulates the activity of cdk4 and cdk2 during terminal erythroid differentiation and suggests a switching mechanism whereby cdk4 functions to sequester p27(Kip1) until a specified time in differentiation when cdk2 kinase activity is targeted by p27(Kip1) to elicit G(1) growth arrest. Further, the data imply that p21(Cip1) may have a function independent of growth arrest during erythroid differentiation. (Blood. 2000;96:2746-2754)     

Inhibition of PDE4 phosphodiesterase activity induces growth suppression, apoptosis, glucocorticoid sensitivity, p53, and p21(WAF1/CIP1) proteins in human acute lymphoblastic leukemia cells

Glucocorticoids are integral to successful treatment of childhood acute lymphoblastic leukemia (ALL) and other lymphoid malignancies. A large body of data indicates that in various model systems, elevation of cyclic adenosine monophosphate (cAMP) can potentiate glucocorticoid response, although this has not been well evaluated as a potential leukemia treatment. Although cAMP analogs have been studied, little data exist regarding the potential toxicity to leukemia cells of pharmacologic elevation of cAMP levels in leukemic blasts. Using MTT assays of cell proliferation on CEM ALL cells, we found that aminophylline and other nonspecific phosphodiesterase (PDE) inhibitors suppress cell growth. This effect is replicated by the PDE4-specific PDE inhibitor rolipram, but not by specific inhibitors of the PDE1 or PDE3 classes. We found that PDE inhibitors cause increased dexamethasone sensitivity and a synergistic effect with the adenylyl cyclase activator forskolin. We observed several important cellular characteristics associated with this treatment, including elevation of cAMP, induction of p53 and p21(WAF1/CIP1) proteins, G(1) and G(2)/M cell cycle arrest, and increased apoptosis. Sensitivity to forskolin and rolipram is shared by at least 2 pediatric ALL cell lines, CEM and Reh cells. Some cell lines derived from adult-type lymphoid malignancies also show sensitivity to this treatment. These findings suggest that PDE inhibitors have therapeutic potential in human ALL and characterize the molecular mechanisms that may be involved in this response.     

Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase mediate macrophage proliferation induced by oxidized low-density lipoprotein

We previously reported that oxidized low-density lipoprotein (Ox-LDL)-induced expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) via PKC, leading to activation of phosphatidylinositol-3 kinase (PI-3K), was important for macrophage proliferation [J Biol Chem 275 (2000) 5810]. The aim of the present study was to elucidate the role of extracellular-signal regulated kinase 1/2 (ERK1/2) and of p38 MAPK in Ox-LDL-induced macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages assessed by [3H]thymidine incorporation and cell counting assays was significantly inhibited by MEK1/2 inhibitors, PD98059 or U0126, and p38 MAPK inhibitors, SB203580 or SB202190, respectively. Ox-LDL-induced GM-CSF production was inhibited by MEK1/2 inhibitors but not by p38 MAPK inhibitors in mRNA and protein levels, whereas recombinant GM-CSF-induced macrophage proliferation was inhibited by p38 MAPK inhibitors but enhanced by MEK1/2 inhibitors. Recombinant GM-CSF-induced PI-3K activation and Akt phosphorylation were significantly inhibited by SB203580 but enhanced by PD98059. Our results suggest that ERK1/2 is involved in Ox-LDL-induced macrophage proliferation in the signaling pathway before GM-CSF production, whereas p38 MAPK is involved after GM-CSF release. Thus, the importance of MAPKs in Ox-LDL-induced macrophage proliferation was confirmed and the control of MAPK cascade could be targeted as a potential treatment of atherosclerosis.     

Protein p53 and inducer-mediated erythroleukemia cell commitment to terminal cell division.

Inducer-mediated murine erythroleukemia cell (MELC) differentiation provides a model for examining factors determining terminal cell differentiation. The nuclear protein, p53, has been implicated as a potential determinant of cell cycle progression and cell differentiation. In this study p53 content and synthesis, during inducer-mediated MELC differentiation, has been examined with monoclonal antibodies to p53. A decrease in p53 synthesis and content was demonstrated during induced differentiation. As determined by cell cycle fractionation, the decrease in p53 is manifest at all stages of the cell cycle. Hemin, which induces globin mRNA accumulation but not terminal cell division, fails to decrease p53 content. A MELC variant resistant to inducer-mediated commitment to terminal cell division also fails to decrease p53 levels in response to inducers. These experiments suggest that p53 is implicated in MELC cell proliferation and that an induced decrease in p53 may be responsible for G1 phase prolongation and terminal G1 arrest.     

Different levels of p38 MAP kinase activity mediate distinct biological effects in primary human erythroid progenitors

There have been conflicting reports regarding the role of p38 mitogen-activated protein kinase (MAPK) in the regulation of differentiation, proliferation and apoptosis in erythroid cell lines. We have, therefore, examined the functions of this kinase in primary human erythroid progenitors. Cells in steady-state culture showed low-level p38 MAPK activity, which decreased further within 1 h of growth factor withdrawal and increased over a limited range within minutes of re-exposure of cells to erythropoietin or stem cell factor, demonstrating the link between low-level p38 MAPK activity and the prevailing growth factor milieu. Use of the p38 MAPK-specific inhibitor SB203580 demonstrated that this level of activity was necessary for (1) optimal proliferation, (2) erythroid burst-forming unit migration and (3) full upregulation of E-cadherin and CD36 expression, but not haemoglobin A or glycophorin A expression, during human erythroid differentiation. In contrast, cells deprived of growth factors for an 8-h period, following a transient decrease in p38 MAPK activity, demonstrated sustained, substantial and caspase-independent increases in p38 MAPK activity, and its blockade using SB203580 reduced the proportion of erythroblasts undergoing apoptosis by 40 +/- 7%, demonstrating a role for p38 MAPK in apoptosis induction in human erythroblasts. Thus, in primary human erythroblasts, different environmental conditions induce different levels of p38 MAPK activity, which have distinct functions.     

The essential role of p21 in radiation-induced cell cycle arrest of vascular smooth muscle cell

The biologic mechanisms for the success and failure of intravascular radiation therapy after angioplasty have not been well studied. We investigated the molecular mechanism of radiation-induced cell cycle arrest in vascular smooth muscle cell (VSMC) and examined whether p21 knock-out is a cause of radiation failure. Using different dosages of gamma radiation, we evaluated the effect of radiation on VSMC apoptosis and cell cycle progression, and its action mechanism. Irradiation significantly retarded the growth of cultured VSMC, which was not due to induction of apoptosis but mainly due to cell cycle arrest. Radiation showed remarkable cell cycle arrest at G1 and G2 phase (G0/G1:S:G2/M phases = 61%:34%:5% with 0 Gy versus 61%:9%:30% with 16 Gy, 12 h after radiation). In immunoblot analysis and kinase assay, radiation increased the expression of p21 and decreased the expression and activity of CDK2 and 1. In contrast, radiation did not affect the expression and activity of CDK4 and 6, nor the expression of p27 and p16. When p21 was knocked out, cell cycle of VSMC was not arrested by radiation, leading to increased proliferation. These finding provide the evidence that radiation inhibits VSMC proliferation through cell cycle arrest by enhancing p21 expression and suppressing CDK1 and 2. This observation supports the key role of p21 in radiation-induced cell cycle arrest and the degree of p21 expression may be the possible mechanism of radiation failure and delayed restenosis.     

Cell cycle block at G1-S or G2-M phase correlates with differentiation of Caco-2 cells: effect of constitutive insulin-like growth factor II expression.

BACKGROUND & AIMS: We have previously shown that autocrine insulin-like growth factor (IGF)-II synthesis through IGF-I receptor stimulates proliferation and inhibits differentiation of Caco-2 cells. To demonstrate whether differentiation of Caco-2 cells is dependent on cell growth status, we analyzed the effect of cell cycle arrest on differentiation of wild-type and IGF-II-overexpressing cells. METHODS: Cells were treated with drugs that inhibit the progression either to S phase (l-b-D-arabinofuranosylcytosine or M phase (nocodazole). Cell differentiation was analyzed by assessing apolipoprotein A-1 and sucrase-isomaltase expression. Cell proliferation and DNA content were assessed by thymidine incorporation and fluorescence-activated cell sorter analysis, respectively. Cell cycle regulatory molecules were analyzed by assessing p21 and retinoplasma protein (pRb) expression and pRb phosphorylation. RESULTS: Cell cycle block at G1-S phase was associated with increased expression of differentiation markers in both parental and IGF-II-transfected cells. On the contrary, cell cycle arrest at G2-M phase correlated with the expression of differentiation markers in parental but not in IGF-II-transfected cells. Constitutive IGF-II-expressing cells actively incorporated thymidine and showed an increase in the proportion of cells with >4N DNA ploidy in the presence of nocodazole. Nocodazole treatment of constitutive IGF-II-expressing cells stimulated p21 expression in the presence of hyperphosphorylated pRb. CONCLUSIONS: The data show that cell cycle arrest increases differentiation of Caco-2 cells. IGF-II-mediated proliferation may prevent cell differentiation through effects on control cell checkpoint proteins.     

Cyclin kinase inhibitor p21 potentiates bile acid-induced apoptosis in hepatocytes that is dependent on p53

Prolonged activation of the mitogen-activated protein kinase (MAPK) pathway enhances expression of the cyclin kinase inhibitor p21 that can promote growth arrest and cell survival in response to cytotoxic insults. Bile acids can also cause prolonged MAPK activation that is cytoprotective against bile acid-induced cell death. Here, we examined the impact of bile acid-induced MAPK signaling and p21 expression on the survival of primary mouse hepatocytes. Deoxycholic acid (DCA) caused prolonged activation of the MAPK pathway that weakly enhanced p21 protein expression. When DCA-induced MAPK activation was blocked using MEK1/2 inhibitors, both hepatocyte viability and expression of p21 were reduced. Surprisingly, constitutive overexpression of p21 in p21+/+ hepatocytes enhanced DCA-induced cell killing. In agreement with these findings, treatment of p21-/- hepatocytes with DCA and MEK1/2 inhibitors also caused less apoptosis than observed in wild-type p21+/+ cells. Expression of p21 in p21-/- hepatocytes did not modify basal levels of apoptosis but restored the apoptotic response of p21-/- cells to those of p21+/+ cells overexpressing p21. These findings suggest that basal expression of p21 plays a facilitating, proapoptotic role in DCA-induced apoptosis. Overexpression of p21 enhanced p53 protein levels. In agreement with a role for p53 in the enhanced apoptotic response, overexpression of p21 did not potentiate apoptosis in p53-/- hepatocytes but, instead, attenuated the death response in these cells. In conclusion, our data suggest that overexpression of p21 can promote apoptosis, leading to elevated sensitivity to proapoptotic stimuli.