H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.     

Hydrogen sulfide induces human colon cancer cell proliferation: Role of Akt,ERK and p21

H₂S (hydrogen sulfide), regarded as the third gaseous transmitter, is implicated in ulcerative colitis and colorectal cancers. The present study investigates the effects of H₂S on cell proliferation in human colon cancer HCT 116 cells and SW480 cells. We identified the two key enzymes, CBS and CSE, for H₂S synthesis in HCT 116 cells. An exogenously administered H₂S donor NaHS induced cell proliferation in a concentration‐dependent manner, with optimal proliferative concentration at 200 μmol/l. NaHS administration increased Akt and ERK phosphorylation. Blockade of Akt and ERK activation attenuated NaHS‐induced cell proliferation. Cell‐cycle analysis showed that NaHS treatment for 6 h decreased the proportion of cells in G₀–G₁ phase and increased the proportion of cells in S phase. Protein expressions of Cyclin D1 and PCNA (proliferating cell nuclear antigen) were not altered, but the cyclin‐dependent kinase inhibitor p21^Waf1/Cip1 was inhibited significantly by NaHS treatment. NaHS significantly reduced NO metabolite levels. In conclusion, NaHS induced human colon cancer cell proliferation. This effect might be mediated by the increase of Akt and ERK phosphorylation and the decrease of p21^Waf1/Cip1 expression and NO production. The results suggested a role for H₂S in human colonic cancer development.     

Involvement of MEKK1/ERK/P21Waf1/Cip1 signal transduction pathway in inhibition of IGF-I-mediated cell growth response by methylglyoxal

The abnormal accumulation of methylglyoxal (MG), a physiological glucose metabolite, is strongly related to the development of diabetic complications by affecting the metabolism and functions of organs and tissues. These disturbances could modify the cell response to hormones and growth factors, including insulin-like growth factor-1 (IGF-I). In this study, we investigated the effect of MG on IGF-I-induced cell proliferation and the mechanism of the effect in two cell lines, a human embryonic kidney cell line (HEK293), and a mouse fibroblast cell line (NIH3T3). MG rendered these cells resistant to the mitogenic action of IGF-I, and this was associated with stronger and prolonged activation of ERK and over-expression of P21(Waf1/Cip1). The synergistic effect of MG with IGF-I in activation of ERK was completely abolished by PD98059 but not by a specific PI3K inhibitor, LY294002, or a specific PKC inhibitor, bisindolylmaleimide. Blocking of Raf-1 activity by expression of a dominant negative form of Raf-1 did not reduce the enhancing effect of MG on IGF-I-induced activation of ERK. However, transfection of a catalytically inactive form of MEKK1 resulted in inactivation of the MG-induced activation of ERK and partial inhibition of the enhanced activation of ERK and over-expression of p21(Waf1/Cip1) induced by co-stimulation of MG and IGF-I. These results suggested that the alteration of intracellular milieu induced by MG through a MEKK1-mediated and PI3K/PKC/Raf-1-independent pathway resulted in the modification of cell response to IGF-I for p21(Waf1/Cip1)-mediated growth arrest, which may be one of the crucial mechanisms for MG to promote the development of chronic clinical complications in diabetes.     

5-aza-2'-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation

In addition to its demethylating function, 5-aza-2'-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 microm) induces inhibition of cell proliferation as well as increased p53/p21(Waf1/Cip1) expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21(Waf1/Cip1) expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21(Waf1/Cip1) expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21(Waf1/Cip1) expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.     

Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation

Epigallocatechin-3-gallate (EGCG), a tea polyphenol, inhibits the proliferation of many cancer cell lines; however, the antiproliferative mechanism(s) are not well-characterized. The objective of this study is to identify the cellular signaling mechanism(s) responsible for the antiproliferative effects of EGCG in the PC-3 prostate cancer cell line. EGCG inhibited PC-3 cell proliferation in a concentration-dependent manner with an IC(50) value of 39.0 microM, but had no effect on the proliferation of a nontumorigenic prostate epithelial cell line (RWPE-1). Treatment of PC-3 cells with EGCG (0-50 microM) resulted in time and concentration-dependent activation of the extracellular signal-regulated kinase (ERK1/2) pathway. EGCG treatment did not induce ERK1/2 activity in RWPE-1 cells. The activation of ERK1/2 by EGCG was not inhibited using PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK), the immediate upstream kinase responsible for ERK1/2 activation; suggesting a MEK-independent signaling mechanism. Pretreatment of PC-3 cells with a phosphoinositide-3 kinase (PI3K) inhibitor partially reduced both EGCG-induced ERK1/2 activation and the antiproliferative effects of this polyphenol. These results suggest that ERK1/2 activation via a MEK-independent, PI3-K-dependent signaling pathway is partially responsible for the antiproliferative effects of EGCG in PC-3 cells.     

Dual role of MEK/ERK signaling in senescence and transformation of intestinal epithelial cells

The mitogen-activated protein kinase cascade operates downstream of Ras to convey cell-surface signals to the nucleus via nuclear translocation of ERK1 and ERK2. We and others have recently demonstrated that activation of ERK1/2 by growth factors is required for proliferation of intestinal epithelial crypt cells. However, it remained to be established whether ERK1/2 activation alone was sufficient to trigger intestinal epithelial cell (IEC) proliferation. To this aim, retrovirus encoding the hemagglutinin-tagged MAPK/ERK kinase (MEK)1 wild type (wtMEK), the upstream activator of ERK1/2, or a constitutively active mutant of MEK1 (MEK1-S218D/S222D; caMEK) were used to infect nonimmortalized human normal intestinal epithelial crypt cell cultures [human intestinal epithelial cells (HIEC)] and rodent immortalized intestinal crypt cells (IEC-6). Stable expression of caMEK but not wtMEK in HIEC led to the irreversible arrest of cellular proliferation (premature senescence). Concomitant with the onset of cell-cycle arrest was the induction of the cyclin-dependent kinase inhibitors p21(Cip), p53, and p16(INK4A). By contrast, overexpression of caMEK in IEC-6 cells induced growth factor relaxation for DNA synthesis, promoted morphological transformation and growth in soft agar, and did not affect expression of p21(Cip), p53, and p16(INK4A). We provided evidences that ERK1b, an alternatively spliced isoform of ERK1, is activated and may contribute to the deregulation of contact inhibition cell growth and transformation of these cells. Constitutive activation of MEK in IECs can produce either premature senescence or forced mitogenesis depending on the integrity of a senescence program controlled by the cell cycle inhibitors p53, p16(INK4A), and p21(CIP).     

Heregulin beta1 promotes breast cancer cell proliferation through Rac/ERK-dependent induction of cyclin D1 and p21Cip1

Accumulating evidence indicates that heregulins, EGF (epidermal growth factor)-like ligands, promote breast cancer cell proliferation and are involved in the progression of breast cancer towards an aggressive and invasive phenotype. However, there is limited information regarding the molecular mechanisms that mediate these effects. We have recently established that HRG (heregulin beta1) promotes breast cancer cell proliferation and migration via cross-talk with EGFR (EGF receptor) that involves the activation of the small GTPase Rac1. In the present paper we report that Rac1 is an essential player for mediating the induction of cyclin D1 and p21(Cip1) by HRG in breast cancer cells. Inhibition of Rac function by expressing either the Rac-GAP (GTPase-activating protein) beta2-chimaerin or the dominant-negative Rac mutant N17Rac1, or Rac1 depletion using RNAi (RNA interference), abolished the cyclin D1 and p21(Cip1) induction by HRG. Interestingly, the proliferative effect of HRG was impaired not only when the expression of Rac1 or cyclin D1 was inhibited, but also when cells were depleted of p21(Cip1) using RNAi. Inhibition of EGFR, PI3K (phosphoinositide 3-kinase; kinases required for Rac activation by HRG) or MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] also blocked the up-regulation of cyclin D1 and p21(Cip1) by HRG. In addition, we found that HRG activates NF-kappaB (nuclear factor kappaB) in a Rac1- and MEK-dependent fashion, and inhibition of NF-kappaB abrogates cyclin D1/p21(Cip1) induction and proliferation by HRG. Taken together, these findings establish a central role for Rac1 in the control of HRG-induced breast cancer cell-cycle progression and proliferation through up-regulating the expression of cyclin D1 and p21(Cip1).     

Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA

Nitric oxide (NO) acts as a vasoregulatory molecule that inhibits vascular smooth muscle cell (SMC) proliferation. Studies have illustrated that NO inhibits SMC proliferation via the extracellular signal-regulated kinase (ERK) pathway, leading to increased protein levels of the cyclin-dependent kinase inhibitor p21^Waf1/Cip1. The ERK pathway can be pro- or antiproliferative, and it has been demonstrated that the activation status of the small GTPase RhoA determines the proliferative fate of ERK signaling, whereby inactivation of RhoA influences ERK signaling to increase p21^Waf1/Cip1 and inhibit proliferation. The purpose of these investigations was to examine the effect of NO on RhoA activation/S-nitrosation and to test the hypothesis that inhibition of SMC proliferation by NO is dependent on inactivation of RhoA. NO decreases activation of RhoA, as demonstrated by RhoA GTP-binding assays, affinity precipitation, and phalloidin staining of the actin cytoskeleton. Additionally, these effects are independent of cGMP. NO decreases SMC proliferation, and gene transfer of constitutively active RhoA (RhoA^63L) diminished the antiproliferative effects of NO, as determined by thymidine incorporation. Western blots of p21^Waf1/Cip1 correlated with changes in proliferation. S-nitrosation of recombinant RhoA protein and immunoprecipitated RhoA was demonstrated by Western blotting for nitrosocysteine and by measurement of NO release. Furthermore, NO decreases GTP loading of recombinant RhoA protein. These findings indicate that inactivation of RhoA plays a role in NO-mediated SMC antiproliferation and that S-nitrosation is associated with decreased GTP binding of RhoA. Nitrosation of RhoA and other proteins likely contributes to cGMP-independent effects of NO. cell signaling; posttranslational modification; vascular disease     

15-LOX-1 inhibits p21 (Cip/WAF 1) expression by enhancing MEK-ERK 1/2 signaling in colon carcinoma cells

Currently, some controversy exists regarding the precise role of 15-lipoxygenase-1 (15-LOX-1) in colorectal carcinogenesis and other aspects of cancer biology. The aim of this study was to evaluate the effect of 15-LOX-1 on p21 (Cip/WAF 1) expression and growth regulation in human colon carcinoma cells. The effect of 13-S-hydroxyoctadecadienoic acid (HODE), a product of 15-LOX-1, on p21 (Cip/WAF 1) expression was evaluated in Caco-2 cells treated with sodium butyrate (NaBT) and/or nordihydroguaiarectic acid (NDGA), a LOX inhibitor. The effect of transfecting HCT-116 cells with 15-LOX-1 was also examined. NaBT-induced p21 (Cip/WAF 1) expression was enhanced by treatment with NDGA and 13-S-HODE reversed NaBT-induced p21 (Cip/WAF 1) expression in Caco-2 cells. Overexpression of 15-LOX-1 induced extracellular signal-related kinase (ERK) 1/2 phosphorylation, decreased p21 (Cip/WAF 1) expression, and increased HCT-116 cell growth. Treatment with NDGA decreased ERK 1/2 phosphorylation, and increased p21 (Cip/WAF 1) expression in 15-LOX-1 overexpressing HCT-116 cells. Our experimental results support the hypothesis that 15-LOX-1 may have "pro-neoplastic" effects during the development of colorectal cancer.     

Heme oxygenase inhibits human airway smooth muscle proliferation via a bilirubin-dependent modulation of ERK1/2 phosphorylation

The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 microm hemin or by 20 microm cobalt-protoporphyrin) and HO inhibition (by 25 microm tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5-20 microm) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 microm) and the antioxidant N-acetyl-cysteine (1 mm) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.     

IL-1-induced ERK1/2 activation up-regulates p21 protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21^Waf1/Cip1 (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.     

ERK1/2 and p38 cooperate to delay progression through G1 by promoting cyclin D1 protein turnover

The conditional kinase DeltaMEKK3:ER allows activation of JNK, p38 and ERK1/2 without overt cellular stress or damage and has proved useful in understanding how these pathways regulate apoptosis and cell cycle progression. We have previously shown that activation of DeltaMEKK3:ER causes a sustained G(1) cell cycle arrest which requires p21(CIP1), with ERK1/2 and p38 cooperating to promote p21(CIP1) expression. In cells lacking p21(CIP1), DeltaMEKK3:ER causes only a transient delay in cell cycle re-entry. We now show that this delay in cell cycle re-entry is due to a reduction in cyclin D1 levels. Activation of DeltaMEKK3:ER promotes the proteasome-dependent turnover of cyclin D1; this requires phosphorylation of threonine 286 (T(286)) and expression of cyclin D1T(286)A rescues the delay in G(1)/S progression. DeltaMEKK3:ER-dependent phosphorylation of T(286) does not appear to be mediated by GSK3beta but requires activation of the ERK1/2 and p38 pathways. ERK1/2 can physically associate with cyclin D1 but activation of ERK1/2 alone is not sufficient for phosphorylation of T(286). Rather, cyclin D1 phosphorylation appears to require coincident activation of ERK1/2 and p38. Thus activation of DeltaMEKK3:ER promotes a sustained G(1) cell cycle arrest by a bipartite mechanism involving the rapid destruction of cyclin D1 and the slower more prolonged expression of p21(CIP1). This has parallels with the bipartite response to ionizing radiation and p53-independent mechanisms of G(1) cell cycle arrest in simple organisms such as yeast.     

Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition

Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells.