Statins activate peroxisome proliferator-activated receptor gamma through extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase-dependent cyclooxygenase-2 expression in macrophages

Both statins and peroxisome proliferator-activated receptor (PPAR)gamma ligands have been reported to protect against the progression of atherosclerosis. In the present study, we investigated the effects of statins on PPARgamma activation in macrophages. Statins increased PPARgamma activity, which was inhibited by mevalonate, farnesylpyrophosphate, or geranylgeranylpyrophosphate. Furthermore, a farnesyl transferase inhibitor and a geranylgeranyl transferase inhibitor mimicked the effects of statins. Statins inhibited the membrane translocations of Ras, RhoA, Rac, and Cdc42, and overexpression of dominant-negative mutants of RhoA (DN-RhoA) and Cdc42 (DN-Cdc42), but not of Ras or Rac, increased PPARgamma activity. Statins induced extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) activation. However, DN-RhoA and DN-Cdc42 activated p38 MAPK, but not ERK1/2. ERK1/2- or p38 MAPK-specific inhibitors abrogated statin-induced PPARgamma activation. Statins induced cyclooxygenase (COX)-2 expression and increased intracellular 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) levels through ERK1/2- and p38 MAPK-dependent pathways, and inhibitors or small interfering RNA of COX-2 inhibited statin-induced PPARgamma activation. Statins also activate PPARalpha via COX-2-dependent increases in 15d-PGJ(2) levels. We further demonstrated that statins inhibited lipopolysaccharide-induced tumor necrosis factor alpha or monocyte chemoattractant protein-1 mRNA expression, and these effects by statins were abrogated by the PPARgamma antagonist T0070907 or by small interfering RNA of PPARgamma or PPARalpha. Statins also induced ATP-binding cassette protein A1 or CD36 mRNA expression, and these effects were suppressed by small interfering RNAs of PPARgamma or PPARalpha. In conclusion, statins induce COX-2-dependent increase in 15d-PGJ(2) level through a RhoA- and Cdc42-dependent p38 MAPK pathway and a RhoA- and Cdc42-independent ERK1/2 pathway, thereby activating PPARgamma. Statins also activate PPARalpha via COX-2-dependent pathway. These effects of statins may explain their antiatherogenic actions.     

脱氢表雄酮通过丝裂活化蛋白激酶途径促进成骨细胞增殖的研究

目的研究脱氢表雄酮(DHEA)促进成骨细胞增殖的作用和机制。方法取新生2～4d的BALB/c小鼠颅骨,用酶消化法进行成骨细胞原代培养,以细胞形态学和碱性磷酸酶(ALP)染色鉴定。取传1代成骨细胞给予不同浓度DHEA,在不同培养时间后,应用光镜、电镜、四唑盐(MTT)法及流式细胞术(FCM)检测细胞的生长和增殖;同时用免疫印迹法检测磷酸化细胞外信号调控激酶(p44/42)的表达。结果10-8～10-6mol/L的DHEA可明显促进小鼠成骨细胞的生长和增殖,经10-7mol/LDHEA处理3d后的细胞,增殖指数明显增加(P<001);在10-9～10-6mol/L浓度范围内,DHEA能明显提高p44/42的表达,以10-8mol/L浓度的作用最佳(P<001),该作用可被U0126阻断。结论DHEA能够促进小鼠成骨细胞生长和增殖,其作用与激活丝裂活化蛋白激酶信号途径有关。     

Insulin-mediated cell proliferation and survival involve inhibition of c-Jun N-terminal kinases through a phosphatidylinositol 3-kinase- and mitogen-activated protein kinase phosphatase-1-dependent pathway

We previously reported that long term treatment with insulin led to sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells overexpressing insulin receptors. Here we investigated the signaling molecules involved in insulin inhibition of JNKs, focusing on phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of JNK inhibition for insulin-mediated proliferation and survival. Insulin inhibition of JNKs was mediated by PI 3-K, as it was blocked by wortmannin and LY294002 and required the de novo synthesis of a phosphatase(s), as it was abolished by orthovanadate and actinomycin D. MKP-1 was a good candidate because 1) insulin stimulation of MKP-1 expression correlated with insulin inhibition of JNKs; 2) insulin stimulation of MKP-1 expression, like insulin inhibition of JNKs, was mediated by PI 3-K; and 3) the transient expression of an antisense MKP-1 RNA reduced the insulin inhibitory effect on JNKs. The overexpression of a dominant negative JNK1 mutant increased insulin stimulation of DNA synthesis and mimicked the protective effect of insulin against serum withdrawal-induced apoptosis. The overexpression of wild-type JNK1 or antisense MKP-1 RNA reduced the proliferative and/or antiapoptotic responses to insulin. Altogether, these results demonstrate that insulin inhibits JNKs through a PI 3-K- and MKP-1-dependent pathway and provide evidence for a key role for JNK inhibition in insulin regulation of proliferation and survival.     

小檗碱对脂多糖诱导的COX-2表达的影响

目的:研究中药小檗碱对脂多糖(LPS)诱导的COX-2表达的影响。方法:取人外周静脉血分离及培养单个核细胞,分为3组:空白对照组、LPS组、LPS+小檗碱组。分别在培养后30 min,6 h,12 h,24 h提取细胞,行RT-PCR法测定COX-2 mRNA水平,Western blot法测定p38MAPK,p-p38MAPK,ERK,p-ERK及COX-2蛋白水平。同时加入选择性p38MAPK抑制剂(SB203580),以及特异性ERK抑制剂(PD098059)分别测定COX-2 mRNA及蛋白水平。结果:与空白对照组相比,LPS组COX-2 mRNA及蛋白表达明显增强(P<0.01)。与LPS组相比,LPS+小檗碱组COX-2 mRNA及蛋白表达明显抑制(P<0.05),且随着浓度增加,抑制作用更明显,在给药后12 h,小檗碱对COX-2抑制作用最强。与LPS组相比,LPS+小檗碱组P38MAPK活性水平差异无统计学意义(P>0.05)。但是与LPS组相比,LPS+小檗碱组ERK活性水平差异有统计学意义(P<0.05)。加入p38MAPK抑制剂以及ERK抑制剂之后,COX-2 mRNA及蛋白水平降低明显...     

氟通过胞外信号调控激酶通路对成骨细胞增殖作用的影响

目的 观察氟通过胞外信号调控激酶(ERK)通路对成骨细胞(MC3T3-E1)增殖作用的影响.方法 取小鼠成骨细胞(MC3T3-E1)进行体外培养,加入不同浓度的氟(Fˉ,0、200、400、600、1000、2000、4000、8000、10 000 μmol/L)培养24、48 h,甲基噻唑蓝(MTT)法筛选出促进细胞增殖的最适浓度.根据最适浓度,将成骨细胞单纯随机分为3组:对照组(Fˉ,0 μmol/L)、染氟组(Fˉ,400 μmol/L)、染氟抑制组(Fˉ,400 μmol/L+PD9805,10 μmmol/L).培养48 h后应用流式细胞术检测各组细胞周期;Western bolt法和免疫荧光法检测各组磷酸化ERK(P-ERK)蛋白表达.结果 400 μmol/L的氟是促进成骨细胞增殖的最适合浓度.与对照组比较[(76.12±10.08)％、(2.06±0.31)％],染氟组G0/G1期细胞数[(63.04±8.12)％]明显减少,S期细胞数[(9.13±2.08)％]明显增多(P均＜ 0.05);而染氟抑制组G0/G1期细胞数[(92.11±9.01)％]明显增多(P＜0.05).Western blot结果表明,与对照组[(100.00±0.00)％]比较,染氟组成骨细胞P-ERK蛋白表达水平[(131.24±13.88)％]明显增高(P＜0.05),染氟抑制组P-ERK蛋白表达[(91.33±9.68)％]未见明显改变(P＞0.05);免疫荧光法检测结果与Western blot法相似.结论 400 μmol/L氟可以促进成骨细胞增殖,ERK通路在氟促进成骨细胞的增殖作用中起到了关键的作用.     

High insulin exacerbates neutrophil-endothelial cell adhesion through endothelial surface expression of intercellular adhesion molecule-1 via activation of protein kinase C and mitogen-activated protein kinase

Aims/hypothesis. The association of insulin resistance and compensatory hyperinsulinaemia with increased coronary events in diabetic patients is poorly understood. There are few publications about the direct atherogenic actions of insulin on the endothelium compared with those on vascular smooth muscle cells. The aim of this study was to elucidate whether high insulin directly affects neutrophil-endothelial cell adhesion and surface expression of endothelial adhesion molecules. We also examined what intracellular mechanisms are involved in these events. Methods. Studies of adhesion between neutrophils from healthy volunteers and human umbilical vein endothelial cells incubated in insulin-rich medium were carried out. Adhered neutrophils were quantified by measuring their myeloperoxidase activities and surface expression of endothelial adhesion molecules was examined using an enzyme immunoassay. Results. High insulin enhanced neutrophil-endothelial cell adhesion with an increase in the expression of intercellular adhesion molecule-1 but not E-selectin or P-selectin. Both phenomena were attenuated by pretreatment with protein kinase C inhibitors and a mitogen activated protein kinase inhibitor. Conclusions/interpretation. These results suggest that hyperinsulinaemia causes vascular injury by directly exacerbating neutrophil-endothelial cell adhesion through increasing endothelial expression of intercellular adhesion molecule-1 via activation of protein kinase and mitogen activated protein kinase pathways. [Diabetologia (2002) 45: ▪–▪] Received: 5 September 2001 and in revised form: 26 November 2001     

Effects of Edaravone on protein expression of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase signaling pathway in elderly patients with acute ischemic stroke

<正>Objective To investigate the effects of Edaravone on cognitive dysfunction and on protein expression of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) signaling pathway in elderly patients with acute ischemic stroke. Methods A total of 100 elderly patients with acute ischemic cerebral stroke admitted to our hospital from January 2011 to     

Structural determinants of agonist-induced signaling and regulation of the angiotensin AT1 receptor

Angiotensin II (Ang II) regulates aldosterone secretion by stimulating inositol phosphate production and Ca(2+) signaling in adrenal glomerulosa cells via the G(q)-coupled AT(1) receptor, which is rapidly internalized upon agonist binding. Ang II also binds to the heptahelical AT(2) receptor, which neither activates inositol phosphate signaling nor undergoes receptor internalization. The differential behaviors of the AT(1) and AT(2) receptors were analyzed in chimeric angiotensin receptors created by swapping the second (IL2), the third (IL3) intracellular loops and/or the cytoplasmic tail (CT) between these receptors. When transiently expressed in COS-7 cells, the chimeric receptors showed only minor alterations in their ligand binding properties. Measurements of the internalization kinetics and inositol phosphate responses of chimeric AT(1A) receptors indicated that the CT is required for normal receptor internalization, and IL2 is a determinant of G protein activation. In addition, the amino-terminal portion of IL3 is required for both receptor functions. However, only substitution of IL2 impaired Ang II-induced ERK activation, suggesting that alternative mechanisms are responsible for ERK activation in signaling-deficient mutant AT(1) receptors. Substitution of IL2, IL3, or CT of the AT(1A) receptor into the AT(2) receptor sequence did not endow the latter with the ability to internalize or to mediate inositol phosphate signaling responses. These data suggest that the lack of receptor internalization and inositol phosphate signal generation by the AT(2) receptor is a consequence of its different activation mechanism, rather than the inability of its cytoplasmic domains to couple to intracellular effectors.     

Dehydroepiandrosterone improves murine osteoblast growth and bone tissue morphometry via mitogen-activated protein kinase signaling pathway independent of either androgen receptor or estrogen receptor

Dehydroepiandrosterone (DHEA) may be a promising agent for postmenopausal osteoporosis (PMO), but its mechanism to modulate osteoblasts (OBs) is yet to be explained. To elucidate the effects of DHEA treatment on the ovariectomized (OVX) mice and its mechanisms, we evaluated the morphology of mice bone tissue and expression of proliferating cell nuclear antigen (PCNA) in the vertebrae-derived OB after having treated the OVX animals with DHEA. The results showed that DHEA administration increased the expression of PCNA in OB and changed the bone tissue morphometry of the PMO model. To further investigate this mechanism, the OB was isolated from neonatal mice calvariae by the enzyme-digested assay, exposed to DHEA, and then analyzed for ultrastructure, DNA content, early apoptotic cells, and phosphorylation of extracellular signal-regulated kinase 1/2. It was found that DHEA promoted proliferation and inhibited apoptosis of OB significantly, via mitogen-activated protein kinase signaling pathway independent of either androgen receptor or estrogen receptor, suggesting that it may exert roles via a DHEA-specific receptor directly, not by way of conversion to androgens or estrogens.     

Chronic ethanol ingestion increases expression of the angiotensin II type 2 (AT2) receptor and enhances tumor necrosis factor-alpha- and angiotensin II-induced cytotoxicity via AT2 signaling in rat alveolar epithelial cells

BACKGROUND: Alcohol abuse increases the risk of acute lung injury in critically ill patients. We have shown that alveolar epithelial cell (AEC) apoptosis in response to inflammatory mediators, including tumor necrosis factor-alpha (TNF-alpha), parallels endotoxin-mediated acute lung injury in ethanol-fed rats. Although angiotensin II mediates TNF-alpha-induced apoptosis of AECs in vitro, its role in ethanol-mediated susceptibility to AEC apoptosis is unknown. METHODS: Adult male rats were fed the Lieber-DeCarli diet for 6 weeks. AECs were isolated, and TNF-alpha- and angiotensin II-induced cytotoxicity (by terminal transferase-mediated dUTP nick end labeling staining) was determined with or without the addition of the angiotensin-converting enzyme inhibitor (lisinopril) or a selective blocker of the angiotensin II type 1 receptor (AT(1)) or type 2 receptor (AT(2)). Finally, the relative expression of the AT(1) and AT(2) receptors in AECs was determined by Western blot analysis. RESULTS: TNF-alpha-induced cytotoxicity, but not angiotensin II-induced cytotoxicity, was prevented by lisinopril, indicating that de novo angiotensin II synthesis is required for TNF-alpha-induced apoptosis in these cells. Both TNF-alpha- and angiotensin II-induced cytotoxicity in AECs from control-fed and ethanol-fed rats were inhibited by the selective AT(2) blocker, PD123319, but not by the selective AT(1) blocker, losartan. In parallel, ethanol ingestion doubled AT(2) expression in AECs (by Western blot) but had no significant effect on AT(1) receptor expression. CONCLUSIONS: Chronic ethanol ingestion increases AT(2) expression in the alveolar epithelium and enhances TNF-alpha- and angiotensin II-induced cytotoxicity, both of which act via AT(2). Together, these findings suggest that selective AT(2) receptor inhibition could limit the development of acute lung injury in alcoholic patients.