N-acetylcysteine reduces chemokine release via inhibition of p38 MAPK in human airway smooth muscle cells.

Reactive oxygen species are involved in the activation of several mitogen-activated protein kinases (MAPKs), key-players in the production of several cytokines. Therefore the current study investigated whether N-acetylcysteine (NAC), an antioxidative agent, inhibits the interleukin (IL)-1beta-induced expression and production of eotaxin and monocyte chemotactic protein (MCP)-1 in human airway smooth muscle cells (HASMC). NAC (10 mM) decreased the expression of eotaxin and MCP-1, by 46 +/- 11% (n=7) and 87 +/- 4% (n=6), respectively; the eotaxin release was inhibited by 75 +/- 5% (n=7), whereas the MCP-1 release was decreased by 69 +/- 41% (n=10). NAC (1 mM) also decreased the IL-1beta-induced activation of p38 MAPK. Compared with unstimulated cells, a four-fold increase in 8-isoprostane production in IL-1beta-stimulated HASMC was observed, which could be inhibited by NAC in a concentration-dependent way, with a maximum inhibition of 39 +/- 12%, with 1 mM NAC. The present study demonstrated that N-acetylcysteine inhibits the interleukin-1beta-induced eotaxin and monocyte chemotactic protein 1 expression and production due to a decreased activation of p38 mitogen-activated protein kinase. This study has also shown that N-acetylcysteine decreases the interleukin-1beta-induced production of reactive oxygen species, as suggested by a reduction in the 8-isoprostane production.     

Modulation by cAMP of IL-1beta-induced eotaxin and MCP-1 expression and release in human airway smooth muscle cells.

Inflammatory cells, such as eosinophils, seem to be key players in the inflammatory process of asthma. These cells are attracted by chemokines, for example eotaxin and monocyte chemotactic protein (MCP-1). In this study, the authors investigated whether eotaxin and MCP-1 expression and release in human airway smooth muscle cells could be modulated by an increase in intracellular cyclic adenosine monophosphate (cAMP) concentration. The possible involvement of cAMP-dependent protein kinase A (PKA) was also studied. Forskolin, a direct stimulator of adenylyl cyclase, decreased the interleukin (IL)-1beta-induced eotaxin and MCP-1 release by 73+/-8 and 65+/-6%, respectively. 8Bromo-cAMP, a cAMP analogue, similarly decreased the chemokine production by 58+/-9 and 63+/-8% for eotaxin and MCP-1, respectively. Prostaglandin E2, known as an activator of the prostanoid receptors EP2 and EP4, which are positively coupled to adenylyl cyclase, also decreased the IL-1beta-induced eotaxin and MCP-1 production by 57+/-17 and 53+/-4%, respectively. H-89, an inhibitor of PKA, was able to inhibit the decrease in eotaxin and MCP-1 protein release induced by forskolin. Using Western-blot analysis, no effect of cAMP was found on the IL-1beta-induced p38 mitogen-activated protein kinase, extracellular signal-related kinase or cJun N-terminal kinase activation. This study shows that an increase in intracellular cyclic adenosine monophosphate concentration may decrease the interleukin-1beta-induced eotaxin and monocyte chemotactic protein-1 expression and production. This can be inhibited by addition of H-89, an inhibitor of cyclic adenosine monophosphate-dependent protein kinase. No decrease was observed in interleukin-1beta-induced p38 mitogen-activated protein kinase, extracellular signal-related kinase or cJun N-terminal kinase activation. These findings may be important for the further development of new anti-inflammatory drugs.     

Interleukin-1 beta up-regulates tissue inhibitor of matrix metalloproteinase-1 mRNA and phosphorylation of c-jun N-terminal kinase and p38 in hepatic stellate cells

AIM: To study the relationship between interleu-kin-1beta (IL-1β) up-regulating tissue inhibitor of matrix metalloproteinase-1 (TIMMP-1) mRNA expression and phosphorylation of both c-jun N-terminal kinase (JNK) and p38 in rat hepatic stellate cells (HSC). METHODS: RT-PCR was performed to measure the expression of TIMMP-1 mRNA in rat HSC. Western blot was performed to measure IL-1β-induced JNK and p38 activities in rat HSC. RESULTS: TIMMP-1 mRNA expression (1.191±0.079) was much higher after treatment with IL-1β(10 ng/mL) for 24 h than in control group (0.545±0.091) (P<0.01). IL-1βactivated JNK and p38 in a time-dependent manner. After stimulation with IL-1βfor 0, 5, 15, 30, 60 and 120 min, the JNK activity was 0.982±0.299, 1.501±0.720, 2.133±0.882, 3.360±0.452, 2.181±0.789, and 1.385±0.368, respectively. There was a significant difference in JNK activity at 15 min (P<0.01), 30 min (P<0.01) and 60 min (P<0.01) in comparison to that at 0 min. The p38 activity was 1.061±0.310, 2.050±0.863, 2.380±0.573, 2.973±0.953, 2.421±0.793, and 1.755±0.433 at the 6 time points (0, 5, 15, 30, 60 and 120 min) respectively. There was a significant difference in p38 activity at 5 min (P<0.05), 15 min (P<0.01), 30 min (P<0.01) and 60 min (P<0.01) compared to that at 0 min. TIMMP-1 mRNA expression trended to decrease in 3 groups pretreated with different concentrations of SP600125 (10μmol/L, 1.022±0.113; 20μmol/L, 0.869±0.070; 40μmol/L, 0.666±0.123). Their decreases were all significant (P<0.05, P<0.01,P<0.01) in comparison to control group (without SP600125 treatment, 1.163±0.107). In the other 3 groups pretreated with different concentrations of SB203580 (10μmol/L, 1.507±0.099; 20μmol/L, 1.698±0.107; 40μmol/L, 1.857±0.054), the expression of TIMMP-1 mRNA increased. Their levels were higher than those in the control group (without SB203580 treatment, 1.027±0.061) with a significant statistical significance CONCLUSION: IL-1βhas a direct action on hepatic fi-brosis by up-regulating TIMMP-1 mRNA expression in rat HSC. JNK and p38 mitogen-activated protein kinases (MAPKs) are involved in IL-1β-induced TIMMP-1 gene expression, and play a distinct role in this process, indicating that p38 and JNK pathways cooperatively mediate TIMP-1 mRNA expression in rat HSC.     

Inhibitors of mitogen-activated protein kinases differentially regulate eosinophil-activating cytokine release from human airway smooth muscle

Airway smooth muscle (ASM) is a potential source of multiple proinflammatory cytokines during airway inflammation. In the present study, we examined a requirement for mitogen-activated protein (MAP) kinase activation for interleukin (IL)-1beta-stimulated GM-CSF, RANTES, and eotaxin release. IL-1beta induced concentration-dependent phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs), p38 MAP kinase, and c-Jun amino-terminal kinase (SAPK/JNK). p42/p44 ERK and p38 MAP kinase phosphorylation peaked at 15 min and remained elevated up to 4 h. SAPK/JNK phosphorylation also peaked at 15 min but fell to baseline within 60 min. SB 203580 selectively inhibited IL-1beta-stimulated activation of p38 MAP kinase; U 0126 was selective against p42/p44 ERK activity. SB 202474, an inactive analog, had no effect on p42/p44 ERK, p38 MAP kinase, or SAPK/JNK activation, or on eotaxin or RANTES release. Eotaxin release was inhibited by SB 203580 and U 0126, whereas RANTES release was prevented by U 0126 only. GM-CSF release was inhibited by U 0126 but enhanced by SB 203580. These data indicate that RANTES release is dependent on p42/p44 ERK activation but occurs independently of p38 MAP kinase activity. Eotaxin release, however, is dependent on both p38 MAP kinase- and p42/p44 ERK-dependent mechanisms. GM-CSF release is p42/p44 ERK dependent and is tonically suppressed by a mechanism that is partially dependent on p38 MAP kinase, though direct inhibition of cyclooxygenase (COX) activity due to poor inhibitor selectivity may also contribute.     

Endometrial stromal cells undergoing decidualization down-regulate their properties to produce proinflammatory cytokines in response to interleukin-1 beta via reduced p38 mitogen-activated protein kinase phosphorylation

The decidualized endometrium plays a role in regulating trophoblast invasion for successful implantation and maintenance of pregnancy. IL-1 beta, a proinflammatory cytokine, has been suggested to play a role in this process. Recently, several lines of evidence indicate the importance of p38 MAPK in various inflammatory responses. We investigated whether endometrial stromal cells (ESC) change their inflammatory responses to IL-1 beta as related to p38 MAPK phosphorylation during the process of decidualization. ESC were decidualized by the treatment with progesterone for 9 d, as determined as such by an increase in the production of prolactin and cAMP by the cells. Whereas IL-1 beta increased the production of IL-6, IL-8, and monocyte chemotactic protein-1, and expression of cyclooxygenase-2 mRNA in ESC cultured without treatment, the stimulatory effects of IL-1 beta were reduced in the decidualized cells. Treatment with SB202190, a p38 MAPK inhibitor, also reduced the stimulatory effects of IL-1 beta in nondecidualized ESC. P38 MAPK phosphorylation was increased by IL-1 beta in nondecidualized ESC, whereas the IL-1 beta-induced increase was suppressed in the decidualized cells. Treatment with 8-bromo-cAMP reduced IL-1 beta-induced phosphorylation of p38 MAPK in nondecidualized ESC. In contrast, treatment with H89, a protein kinase A inhibitor, reversed a reduction in IL-1 beta-induced p38 MAPK phosphorylation in the decidualized cells. In summary, decidualization seems to be a process during which endometrial cells diminish their response to IL-1 beta, a known key factor for implantation, leading to the down-regulation of inflammation-like events, which may be relevant to controlled trophoblast invasion. The altered property of decidualized cells is thought to be caused by attenuation of IL-1 beta-induced p38 MAPK phosphorylation in a way that involves the activation of the cAMP/protein kinase A pathway.     

Possible contributions of reactive oxygen species and mitogen-activated protein kinase to renal injury in aldosterone/salt-induced hypertensive rats

Studies were performed to test the hypothesis that reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) contribute to the pathogenesis of aldosterone/salt-induced renal injury. Rats were given 1% NaCl to drink and were treated with one of the following combinations for 6 weeks: vehicle (0.5% ethanol, SC, n=6); aldosterone (0.75 microg/H, SC, n=8); aldosterone plus a selective mineralocorticoid receptor antagonist; eplerenone (0.125% in chow, n=8); aldosterone plus an antioxidant; and tempol (3 mmol/L in drinking solution, n=8). The activities of MAPKs, including extracellular signal-regulated kinases (ERK)1/2, c-Jun-NH2-terminal kinases (JNK), p38MAPK, and big-MAPK-1 (BMK1) in renal cortical tissues were measured by Western blot analysis. Aldosterone-infused rats showed higher systolic blood pressure (165+/-5 mm Hg) and urinary excretion of protein (106+/-24 mg/d) than vehicle-infused rats (118+/-3 mm Hg and 10+/-3 mg/d). Renal cortical mRNA expression of p22phox, Nox-4, and gp91phox, measured by real-time polymerase chain reaction, was increased in aldosterone-infused rats by 2.3, 4.3, and 3.0-fold, respectively. Thiobarbituric acid-reactive substances (TBARS) content in renal cortex was also higher in aldosterone (0.23+/-0.02) than vehicle-infused rats (0.09+/-0.01 nmol/mg protein). ERK1/2, JNK, and BMK1 activities were significantly elevated in aldosterone-infused rats by 3.3, 2.3, and 3.0-fold, respectively, whereas p38MAPK activity was not changed. Concurrent administration of eplerenone or tempol to aldosterone-infused rats prevented the development of hypertension (127+/-2 and 125+/-5 mm Hg), and the elevations of urinary excretion of protein (10+/-2 and 9+/-2 mg/day) or TBARS contents (0.08+/-0.01 and 0.11+/-0.01 nmol/mg protein). Furthermore, eplerenone and tempol treatments normalized the activities of ERK1/2, JNK, and BMK1. These data suggest that ROS and MAPK play a role in the progression of renal injury induced by chronic elevations in aldosterone.     

Mechanisms of interleukin 1beta-induced human airway smooth muscle hyporesponsiveness to histamine. Involvement of p38 MAPK NF-kappaB

We have investigated the effect of IL-1beta on histamine H(1)-receptor (H(1)R)-mediated inositol phosphate (IP) accumulation in human airway smooth muscle cells (HASMC) and on histamine-induced contraction of human bronchial rings. Stimulation of HASMC for 24 h with IL-1beta resulted in significant loss of histamine-induced IP formation, which was associated with a reduction of histamine- induced contraction of IL-1beta-treated human bronchial rings. An inhibitor of NF-kappaB activation, pyrrolidine dithiocarbamate, and a p38 MAPK inhibitor, blocked the IL-1beta-induced H(1)R desensitization, whereas anisomycin, an SAPK/JNK and p38 MAPK activator, mimicked the effect of IL-1beta. IL-1beta has been demonstrated to induce cox-2 expression and PGE(2) synthesis. In our study, indomethacin a cox antagonist, completely inhibited the effect of IL-1beta on H(1)R, whereas exogenously added PGE(2) was able to desensitize H(1)R. Furthermore, H-89, a selective PKA inhibitor, antagonized the effect of IL-1beta. Here, we have demonstrated that IL-1beta desensitizes H(1)R, which involves the activation of p38 MAPK and NF-kappaB, leading to the expression of cox-2 and the synthesis of PGE(2). PGE(2) increases intracellular cAMP resulting in PKA activation, which phosphorylates and functionally uncouples H(1)R. Our results suggest that IL-1beta protects airway smooth muscle against histamine-induced contractile responses and that bronchial hyperreactivity to histamine is not associated with proinflammatory cytokine-induced enhancement in H(1)R signaling.     

Regulation of monocyte chemoattractant protein 1 (MCP-1) release by explants of human visceral adipose tissue

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a chemokine involved in monocyte recruitment during inflammation whose plasma level is elevated in obesity. OBJECTIVE: The present studies were designed to examine the release of MCP-1 in primary culture by explants of visceral adipose tissue from morbidly obese women. RESULTS: Most of the MCP-1 released by adipose tissue explants was derived from the nonfat cells in adipose tissue. The release of MCP-1 by adipose tissue explants was upregulated almost five-fold between 3 and 48 h of incubation. Approximately half of this upregulation was due to the release of endogenous tumor necrosis factor alpha (TNFalpha) and IL-1beta based on the ability of a combination of a soluble TNFalpha receptor (etanercept) and a blocking antibody against IL-1beta to reduce MCP-1 release. The release of MCP-1 over 48 h was unaffected by insulin or dexamethasone but significantly reduced by the combination of both agents. MCP-1 release was reduced by 60% in the presence of an inhibitor of the nuclear factor kappaB (NF-kappaB) pathway. There were no significant effects of inhibitors of p44/42 mitogen-activated protein kinase (ERK), Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways on MCP-1 release. However, inhibition of MCP-1 release in the presence of inhibitors of both the p38 MAPK and NF-kappaB pathways was greater than that seen with only the NF-kappaB inhibitor. DISCUSSION: The present data shows that MCP-1 formation is upregulated over a 48-h incubation of primary explants of visceral adipose tissue. Half of this upregulation is dependent upon endogenous TNFalpha and Il-1beta and involves the p38 MAPK and NF-kappaB pathways.     

Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis

OBJECTIVE: To investigate whether stress- and mitogen-activated protein kinases (SAPK/MAPK), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, are significantly activated in rheumatoid arthritis (RA) synovial tissue compared with their activation in degenerative joint disease; to assess the localization of SAPK/MAPK activation in rheumatoid synovial tissue; and to search for the factors leading to stress kinase activation in human synovial cells. METHODS: Immunoblotting and immunohistology by antibodies specific for the activated forms of SAPK/MAPK were performed on synovial tissue samples from patients with RA and osteoarthritis (OA). In addition, untreated and cytokine-treated human synovial cells were assessed for SAPK/MAPK activation and downstream signaling by various techniques. RESULTS: ERK, JNK, and p38 MAPK activation were almost exclusively found in synovial tissue from RA, but not OA, patients. ERK activation was localized around synovial microvessels, JNK activation was localized around and within mononuclear cell infiltrates, and p38 MAPK activation was observed in the synovial lining layer and in synovial endothelial cells. Tumor necrosis factor alpha, interleukin-1 (IL-1), and IL-6 were the major inducers of ERK, JNK, and p38 MAPK activation in cultured human synovial cells. CONCLUSION: Signaling through SAPK/MAPK pathways is a typical feature of chronic synovitis in RA, but not in degenerative joint disease. SAPK/MAPK signaling is found at distinct sites in the synovial tissue, is induced by proinflammatory cytokines, and could lead to the design of highly targeted therapies.     

MCP-1 induces activation of MAP-kinases ERK,JNK and p38 MAPK in human endothelial cells

Activation of vascular endothelial cells (ECs) plays an important pathogenic role in the development of atherosclerosis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant of monocytes. Besides induction of monocyte recruitment, it has been suggested that MCP-1 can also affect the cellular responses of ECs. We investigated whether MCP-1 activated the three major mitogen activated protein (MAP)-kinases extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38 MAPK. Stimulation of ECs with MCP-1 induced a time- and concentration-dependent activation of all three MAP-kinases, concentrations as low as 0.1 ng/ml were sufficient for this mechanism. MCP-1 also induced secretion of matrix metalloproteinase (MMP)-2 which along with ERK activation was inhibited by PD098059. The results demonstrate that MCP-1 can lead to direct activation of MAP kinases together with induction of MMP2 in ECs. Our data thus propose a new mechanism for the proatherogenic effect of MCP-1.     

Activated protein C,an anticoagulant polypeptide,ameliorates severe acute pancreatitis via regulation of mitogen-activated protein kinases

Background Our aim was to investigate the changes of mitogen-activated protein kinases (MAPKs) by activated protein C (APC) treatment in rats with severe acute pancreatitis (SAP), and relate them to changes in SAP severity, thus providing evidence for developing clinical therapies. Methods Sprague-Dawley rats were given an intravenous injection of saline (SAP group), APC (50 μg/kg or 10 μg/kg), or CNI1493 just before SAP induction. One group of rats underwent a sham operation (control group). Experimental samples were harvested 16 h after SAP induction. The gene expression of pancreatic MAPKs was evaluated by cDNA microarrays. The mRNA and protein/phosphorylated protein levels of p38 MAPK, extracellular signal-regulated protein kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK) and the protein levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were determined in pancreatic tissue. The severity of disease was evaluated by pancreatic histology, the pancreatic wet/dry weight ratio, and the serum amylase level. Results In rats treated with APC (50 μg/kg) or CNI1493, the severity of pancreatitis and expression of pancreatic TNF-α and IL-1β proteins were attenuated by the decreased expression and activity of p38 MAPK and JNK (vs. the SAP group, P < 0.01). The expression and activity of ERK1/2 were increased in APC-treated rats, especially in the group treated with APC 50 μg/kg (vs. the SAP or CNI1493-treated group, P < 0.01, respectively). Conclusions Inhibition of expression of pancreatic p38 MAPK and JNK and upregulation of ERK1/2 expression by APC treatment may protect against pancreatic injury, thus ameliorating severity of the disease.     

Convergence of redox-sensitive and mitogen-activated protein kinase signaling pathways in tumor necrosis factor-alpha-mediated monocyte chemoattractant protein-1 induction in vascular smooth muscle cells

Monocyte chemoattractant protein-1 (MCP-1) is an important component of the inflammatory response of the vessel wall and has been shown to be regulated by cytokines, such as tumor necrosis factor-alpha (TNF-alpha). However, the precise signaling pathways leading to MCP-1 induction have not been fully elucidated in vascular smooth muscle cells (VSMCs). Cytokine signal transduction involves protein kinases as well as reactive oxygen species (ROS). The relation between these 2 factors is not clear. In this study, we show that TNF-alpha induces a parallel phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38MAPK) and increases MCP-1 mRNA expression in cultured VSMCs. Inhibition of ERK1/2 but not p38MAPK caused a partial attenuation of MCP-1 induction (43+/-10% inhibition). Incubation of VSMCs with multiple antioxidants (diphenylene iodonium, liposomal superoxide dismutase, catalase, N-acetylcysteine, dimethylthiourea, and pyrrolidine dithiocarbamate) had no effect on TNF-alpha-mediated MCP-1 upregulation. However, simultaneous blockade of the ERK1/2 and ROS pathways by using PD098059 combined with diphenylene iodonium or N-acetylcysteine potently enhanced the ability of MAPK kinase inhibitors to abrogate MCP-1 mRNA expression (100+/-2% inhibition). Thus, parallel ROS-dependent and ERK1/2-dependent pathways converge to regulate TNF-alpha-induced MCP-1 gene expression in VSMCs. These data unmask a complex but organized integration of ROS and protein kinases that mediates cytokine-induced vascular inflammatory gene expression.     

p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase regulate interleukin-8 and RANTES production in hyperosmolarity stimulated human bronchial epithelial cells

OBJECTIVE: We have previously shown that p38 mitogen-activated protein kinase (MAPK) regulates, at least in part, hyperosmolarity induced interleukin (IL)-8 expression in human bronchial epithelial cells (BEC). In the previous study, hyperosmolarity also activated c-Jun-NH2-terminal kinase (JNK); however, the role of the JNK signalling pathway has not been determined. In the present study, we examined the role of the JNK signalling pathway in hyperosmolarity induced IL-8 and RANTES production by BEC using the novel inhibitor of the JNK signalling pathway CEP 11004 in order to clarify these issues. METHODS: Bronchial epithelial cells that had been pre-incubated with SB 203580, CEP 11004 or a combination of these were exposed to a hyperosmolar medium and then the p38 MAPK and JNK phosphorylation activity in these cells and IL-8 and RANTES concentrations in the culture supernatants were determined. RESULTS: The results showed that: (i) hyperosmolarity induced the threonine and tyrosine phosphorylation of p38 MAPK and JNK; (ii) SB 203580, as the specific inhibitor of p38 MAPK activity, and CEP 11004 attenuated hyperosmolarity induced p38 MAPK and JNK activity, respectively; (iii) SB 203580 and CEP 11004, but not PD 98059, partially attenuated IL-8 and RANTES production; and (iv) a combination of SB 203580 and CEP 11004 attenuated IL-8 and RANTES production in an additive fashion. CONCLUSION: These results indicate that p38 MAPK and the JNK pathway regulate hyperosmolarity induced IL-8 and RANTES production by BEC.     

p38丝裂原活化蛋白激酶抑制剂SB203580对溃疡性结肠炎肠黏膜肿瘤坏死因子α表达的影响

目的探讨溃疡性结肠炎（UC）患者肠黏膜组织中磷酸化p38丝裂原活化蛋白激酶（p38 MAPK）的表达及p38 MAPK抑制剂SB203580对活动性UC患者肠黏膜组织TNFα表达的影响。方法30例活动性UC患者被纳入本研究,以15例结肠癌患者的癌旁正常组织作为对照。免疫组化法检测UC患者肠黏膜活检组织中磷酸化p38 MAPK的表达。体外组织培养条件下观察SB203580对UC患者肠黏膜组织TNFα表达的影响,ELISA法检测培养上清液中TNFα含量。结果（1）UC患者肠黏膜磷酸化p38MAPK的表达明显高于正常肠黏膜,A值分别为549．22±32．54、143．52±11．89,阳染面积分别为[（1680．61±115．30）×10^-5、（351．68±12．73）×10^-5]μm^2,P值均〈0．01。（2）与未用SB203580处理的UC组比较,处理后UC肠黏膜组织分泌TNFα的水平明显较低,分别为（549．96±107．63、72．07±20．30）ng／L（P〈0．01）。（3）与未用SB203580处理的UC组比较,处理后UC肠黏膜组织p38 MAPK下游分子活化转录因子-2（ATF2）的活性表达明显减少,A值分别为688．32±47．37、265．82±40．25,阳染面积分别为[（2489．02±193．63）×10^-5、（1213．76±204．77）×10^-5]μm^2,P值均〈0．01,但对磷酸化p38 MAPK的表达无影响,A值分别为480．34±38．87、465．64±38．69,阳染面积分别为[（1536．68±182．16）×10^-5、（1486．26±165．49）×10^-5]μm62,P值均〉0．05。结论p38 MAPK信号传导通路在UC的发病中起着重要作用,阻断该通路可减少炎性细胞因子的释放。提示p38 MAPK信号传导通路可以为UC的治疗提供一个新的靶标,SB203580有可能成为UC治疗的新药物。     

Calcium has a permissive role in interleukin-1beta-induced c-jun N-terminal kinase activation in insulin-secreting cells

The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1beta-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic beta-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1beta activation of JNK in beta-cells are largely unknown. In this study, we investigated whether Ca(2+) plays a role for IL-1beta-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca(2+) channels suppressed IL-1beta-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1beta-stimulated activation of JNK1/2 as assessed by immunoblotting. Inhibition of IL-1beta-induced in vitro kinase activity toward c-jun after collective L- and T-type Ca(2+) channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse betaTC3 cells. Ca(2+) influx, specifically via L-type but not T-type channels, contributed to IL-1beta activation of JNK. Activation of p38 and ERK in response to IL-1beta was also dependent on L-type Ca(2+) influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca(2+) ionophore A23187, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca(2+)/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1beta-stimulated c-jun phosphorylation in INS-1 or betaTC3 cells. Our data suggest that Ca(2+) plays a permissive role in IL-1beta activation of the JNK signaling pathway in insulin-secreting cells.     

Glucose-dependent insulinotropic polypeptide promotes beta-(INS-1) cell survival via cyclic adenosine monophosphate-mediated caspase-3 inhibition and regulation of p38 mitogen-activated protein kinase

The incretin glucose-dependent insulinotropic polypeptide (GIP) is a major regulator of postprandial insulin secretion in mammals. Recent studies in our laboratory, and others have suggested that GIP is a potent stimulus for protein kinase activation, including the MAPK (ERK1/2) module. Based on these studies, we hypothesized that GIP could regulate cell fate and sought to examine the underlying mechanisms involved in GIP stimulation of cell survival. GIP potentiated glucose-induced beta-(INS-1)-cell growth to levels comparable with GH and GLP-1 while promoting cell survival in the face of serum and glucose-deprivation or treatment with wortmannin or streptozotocin. In the absence of GIP, 50% of cells died after 48 h of serum and glucose withdrawal, whereas 91 +/- 10% of cells remained viable in the presence of GIP [n = 3, P < 0.05; EC50 of 1.24 +/- 0.48 nm GIP (n = 4)]. Effects of GIP on cell survival and inhibition of caspase-3 were mimicked by forskolin, but pharmacological experiments excluded roles for MAPK kinase (Mek)1/2, phosphatidylinositol 3-kinase, protein kinase A, Epac, and Rap 1. Survival effects of GIP were ablated by the inhibitor SB202190, indicating a role for p38 MAPK. Furthermore, caspase-3 activity was also regulated by p38 MAPK, with a lesser role for Mek1/2, based on RNA interference studies. We propose that GIP is able to reverse caspase-3 activation via inhibition of long-term p38 MAPK phosphorylation in response to glucose deprivation (+/-wortmannin). Intriguingly, these findings contrasted with short-term phosphorylation of MKK3/6-->p38 MAPK-->ATF-2 by GIP. Thus, these data suggest that GIP is able to regulate INS-1 cell survival by dynamic control of p38 MAPK phosphorylation via cAMP signaling and lend further support to the notion that GIP regulation of MAPK signaling is critical for its regulation of cell fate.     

Preptin对人成骨细胞增殖和分化的影响及机制研究

目的 探讨preptin对人成骨细胞增殖和分化的影响及其信号途径.方法 体外培养人成骨细胞,用10-10、10-9、10-8和10-7mol/L preptin干预24 h,以[3H]脱氧胸腺嘧啶苷掺入法分析细胞增殖,用分光光度计法测定细胞碱性磷酸酶(ALP)活性判断细胞分化程度.Western印迹法检测细胞外信号调节激酶(ERK)、p38丝裂原活化蛋白激酶(p38MAPK)和c-Jun氨基末端激酶(JNK)的磷酸化水平.并在preptin干预前以ERK抑制剂(PD98059)、p38 MAPK抑制剂(SB203580)和JNK抑制剂(SP600125)预处理,观察preptin诱导人成骨细胞增殖和分化的途径.结果 Preptin剂量依赖地增加人成骨细胞的增殖和ALP活性,10-9mol/L浓度时达最大效应(均P<0.01).Preptin刺激人成骨细胞ERK的磷酸化,对p38MAPK和JNK无作用.PD98059阻断preptin刺激的成骨细胞增殖及ALP活性增加(均P<0.05),而SP600125和SB203580无此效应.结论 Preptin通过ERK途径促进人成骨细胞的增殖和分化.