New Role of JAK2/STAT3 Signaling in Endothelial Cell Oxidative Stress Injury and Protective Effect of Melatonin

Previous studies have shown that the JAK2/STAT3 signaling pathway plays a regulatory role in cellular oxidative stress injury (OSI). In this study, we explored the role of the JAK2/STAT3 signaling pathway in hydrogen peroxide (H₂O₂)-induced OSI and the protective effect of melatonin against (H₂O₂)-induced injury in human umbilical vein endothelial cells (HUVECs). AG490 (a specific inhibitor of the JAK2/STAT3 signaling pathway) and JAK2 siRNA were used to manipulate JAK2/STAT3 activity, and the results showed that AG490 and JAK2 siRNA inhibited OSI and the levels of p-JAK2 and p-STAT3. HUVECs were then subjected to H₂O₂ in the absence or presence of melatonin, the main secretory product of the pineal gland. Melatonin conferred a protective effect against H₂O₂, which was evidenced by improvements in cell viability, adhesive ability and migratory ability, decreases in the apoptotic index and reactive oxygen species (ROS) production and several biochemical parameters in HUVECs. Immunofluorescence and Western blotting showed that H₂O₂ treatment increased the levels of p-JAK2, p-STAT3, Cytochrome c, Bax and Caspase3 and decreased the levels of Bcl2, whereas melatonin treatment partially reversed these effects. We, for the first time, demonstrate that the inhibition of the JAK2/STAT3 signaling pathway results in a protective effect against endothelial OSI. The protective effects of melatonin against OSI, at least partially, depend upon JAK2/STAT3 inhibition.     

Inhibition of JAK2/STAT3 signalling induces colorectal cancer cell apoptosis via mitochondrial pathway

Abnormalities in the JAK2/STAT3 pathway are involved in the pathogenesis of colorectal cancer (CRC), including apoptosis. However, the exact mechanism by which dysregulated JAK2/STAT3 signalling contributes to the apoptosis has not been clarified. To investigate the role of both JAK2 and STAT3 in the mechanism underlying CRC apoptosis, we inhibited JAK2 with AG490 and depleted STAT3 with a small interfering RNA. Our data showed that inhibition of JAK2/STAT3 signalling induced CRC cellular apoptosis via modulating the Bcl-2 gene family, promoting the loss of mitochondrial transmembrane potential (Δψm) and the increase of reactive oxygen species. In addition, our results demonstrated that the translocation of cytochrome c (Cyt c), caspase activation and cleavage of poly (ADP-ribose) polymerase (PARP) were present in apoptotic CRC cells after down-regulation of JAK2/STAT3 signalling. Moreover, inhibition of JAK2/STAT3 signalling suppressed CRC xenograft tumour growth. We found that JAK2/STAT3 target genes were decreased; meanwhile caspase cascade was activated in xenograft tumours. Our findings illustrated the biological significance of JAK2/STAT3 signalling in CRC apoptosis, and provided novel evidence that inhibition of JAK2/STAT3 induced apoptosis via the mitochondrial apoptotic pathway. Therefore, JAK2/STAT3 signalling may be a potential target for therapy of CRC.     

Leukemia inhibitory factor enhances bFGF-induced IL-6 synthesis in osteoblasts: involvement of JAK2/STAT3

We previously showed that basic fibroblast growth factor (bFGF) stimulates release of vascular endothelial growth factor (VEGF) and synthesis of interleukin-6 (IL-6) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of leukemia inhibitory factor (LIF) on the release of VEGF and IL-6 in these cells. LIF did not affect the bFGF-stimulated VEGF release. On the contrary, LIF, which alone had little effect on IL-6 release, significantly enhanced the bFGF-stimulated IL-6 release. The amplifying effect of LIF on the IL-6 release was dose dependent in the range between 0.01 and 10 ng/ml. AG490, an inhibitor of JAK2, suppressed the amplifying effect of LIF. LIF induced the phosphorylation of STAT3. AG490 inhibited the LIF-induced STAT3 phosphorylation. Taken together, our results strongly suggest that LIF enhances bFGF-stimulated IL-6 synthesis via JAK2/STAT3 pathway in osteoblasts.     

JAK-STAT参与血管平滑肌细胞血管紧张素原和心钠素基因的转录激活

为探讨Janus蛋白酪氨酸激酶2-转导及转录激活因子5（JAK2-STAT5）途径在介导血管紧张素Ⅱ（AngⅡ）诱导的血管平滑肌细胞（VSMC）血管舒-缩肽表达调节的作用及分子机制, 以Ang Ⅱ为诱发因素刺激培养的大鼠VSMC, 用免疫共沉淀、Western印迹分析和激光共聚焦显微镜观察STAT5磷酸化及其核转位, 用电泳迁移率改变分析（EMSA）确定STAT5与血管活性肽基因调控区顺式调控元件的结合活性. 结果显示, STAT5磷酸化水平分别于Ang Ⅱ刺激10 min和12 h出现两个高峰, 增加的磷酸化STAT5主要分布在细胞核内. Ang Ⅱ诱导的STAT5活化与核转位可被JAK2的特异抑制剂AG490所抑制. EMSA结果显示, 用Ang Ⅱ刺激VSMC后, 核蛋白与含有血管紧张素原基因启动子STAT5识别序列的探针结合活性显著升高,而核蛋白与含有心钠素（ANF）基因启动子STAT5识别序列的探针结合活性则呈下降趋势, 核蛋白与两种探针的结合活性均可被JAK2抑制剂AG490所消除, 并且加入抗STAT5抗体后均可出现滞后的超迁移带. 结果提示, Ang Ⅱ通过激活JAK2-STAT5介导信号向胞核内传递, STAT5与相应的顺式元件结合是启动血管紧张素原和心钠素基因表达所必需的转录调控机制之一.     

Activation of JAK2 by the oxidative stress generated with oxidized low-density lipoprotein.

Atherosclerosis includes a series of cellular and molecular responses characteristic of an inflammatory disease. We provide evidence that cupric-ion-oxidized LDL (CuLDL) or endothelial cell-oxidized LDL (ELDL) induced the activation by Tyr-phosphorylation of JAK2, one of the Janus kinase involved upstream of STATs in the JAK/STAT pathway of cytokine transduction. Oxidized LDL (OxLDL) also initiated STAT1 and STAT3 Tyr-phosphorylation and translocation to the nucleus, with a more marked effect for the extensively modified CuLDL. Genistein, a nonspecific Tyr-kinase inhibitor, and AG490, a specific inhibitor of JAKs, markedly prevented the CuLDL-induced enhancement of STAT1 and STAT3 Tyr-phosphorylation and DNA-binding activity, suggesting that JAKs are the main kinases involved in STATs' activation by oxidized LDL. In addition, the lipid extract of CuLDL increased the intracellular levels of lipid peroxidation products and the Tyr-phosphorylation of JAK2, STAT1, and STAT3, whereas the antioxidant vitamin E prevented all these effects. These results demonstrate that OxLDL induces the activation by Tyr-phosphorylation of JAK2, STAT1, and STAT3 by generation of an intracellular oxidative stress by means of its lipid peroxidation products, and thus include JAK2 within the range of oxidative stress-activated kinases.     

Blockade of JAK2 activity suppressed accumulation of β‐catenin in leukemic cells

The Wnt/β‐catenin pathway has been implicated in leukemogenesis. We found β‐catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β‐Catenin can be significantly down‐regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β‐catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β‐catenin, APC, Axin, β‐Trcp, GSK3α, and GSK3β were up‐regulated within 12–16 h. However, only the protein levels of GSK3β and β‐Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490‐induced inhibition of β‐catenin can be attenuated by shRNA targeting β‐TrCP. Taken together; these results suggest that β‐Trcp plays a key role in the cross‐talk between JAK/STAT and Wnt/β‐catenin signaling in leukemia cells. J. Cell. Biochem. 111: 402–411, 2010. © 2010 Wiley‐Liss, Inc.     

Apoptotic resistance exhibited by dexamethasone-resistant murine 7TD1 cells is controlled independently of interleukin-6 triggered signaling

Interleukin-6 (IL6)-mediated signaling is known to play a role in pathogenesis and resistance in several cancers like multiple myeloma (MM). In this report we used the IL6-dependent 7TD1 murine B-cell hybridoma as an in vitro model to study the interactions between IL6-signaling pathways and the development of dexamethasone resistance. Though in initial stages, 7TD1 cells grew IL6-dependent and were sensitive to dexamethasone-induced apoptosis, chronic exposure to dexamethasone led to a dexamethasone-resistant phenotype (7TD1-Dxm) that grew independent of exogenous IL6. While IL6-mediated JAK/STAT3 and PI3K/AKT signaling was important for proliferation of both cell lines, as shown in proliferation assays using the respective pathway inhibitors, AG490 and LY294002, the resistant cells were insensitive to induction of apoptosis using the same. STAT3 was constitutively phosphorylated in resistant cells and inhibition of its dimerization induced apoptosis but did not alter their insensitivity to dexamethasone. Our results suggest a role of entities downstream of IL6-mediated JAK/STAT3 signaling in development of dexamethasone resistance by 7TD1-Dxm cells.     

Leptin stimulates tissue inhibitor of metalloproteinase-1 in human hepatic stellate cells: respective roles of the JAK/STAT and JAK-mediated H2O2-dependant MAPK pathways

Leptin is recognized as a profibrogenic hormone in the liver, but the mechanisms involved have not been clarified. The tissue inhibitor of metalloproteinase (TIMP)-1, which acts through inhibition of collagen degradation, is synthesized by activated hepatic stellate cells (HSC) in response to fibrogenic substances. The capacity of leptin to induce TIMP-1 and its signaling molecules were investigated in a human HSC cell line, LX-2. Leptin stimulated TIMP-1 protein, mRNA, and promoter activity. JAK1 and -2, as well as STAT3 and -5, were activated. After leptin, there was increased expression of tyrosine 1141-phosphorylated leptin receptor, which may contribute to STAT3 activation. AG 490, a JAK inhibitor, blocked JAK phosphorylation with concomitant inhibition of STAT activation, TIMP-1 mRNA expression, and promoter activity. Leptin also induced an oxidative stress, which was inhibited by AG 490, indicating a JAK mediation process. ERK1/2 MAPK and p38 were activated, which was prevented by catalase, indicating an H2O2-dependent mechanism. Catalase treatment resulted in total suppression of TIMP-1 mRNA expression and promoter activity. SB203580, a p38 inhibitor, prevented p38 activation and reduced TIMP-1 message half-life with down-regulation of TIMP-1 mRNA. These changes were reproduced by overexpression of the dominant negative p38alpha and p38beta mutants. PD098059, an ERK1/2 inhibitor, opposed ERK1/2 activation and TIMP-1 promoter activity, leading to TIMP-1 mRNA down-regulation. Thus, leptin has a direct action on liver fibrogenesis by stimulating TIMP-1 production in activated HSC. This process appears to be mediated by the JAK/STAT pathway via the leptin receptor long form and the H2O2-dependent p38 and ERK1/2 pathways via activated JAK.