Neuroprotective properties of ciliary neurotrophic factor for cultured adult rat dorsal root ganglion neurons

We observed that recombinant ciliary neurotrophic factor (CNTF) enhanced survival and neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. Among other neurotrophic factors (NGF and GDNF) and interleukin (IL)-6 cytokine members [IL-6, LIF, cardiotrophin-1, and oncostatin M (OSM)] at the same concentration (50 ng/ml), CNTF, as well as LIF and OSM, displayed high efficacy for the promotion of the number of viable neurons and neurite-bearing cells. CNTF enhanced the number of neurite-bearing cells in both small neurons (soma diameter <30 mum) and large neurons (soma diameter >/=30 mum), whereas NGF and GDNF promoted that in only small neurons. Western blot analysis revealed that CNTF induced phosphorylation of STAT3, Akt, and ERK1/2 in the neurons. Furthermore, the neurite outgrowth-promoting activity of CNTF was diminished by co-treatment with Janus kinase (JAK) 2 inhibitor, AG490; STAT3 inhibitor, STA-21; phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor, LY294002; and mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, in a concentration-dependent manner. Its survival-promoting activity was also affected by AG490, STA-21, and LY294002 at higher concentrations, but not by PD98059. These findings suggest the involvement of JAK2/STAT3, PI3K/Akt, and MEK/ERK signaling pathways in CNTF-induced neurite outgrowth, where the former two pathways are thought to play major roles in mediating the survival response of neurons to CNTF.     

Interleukin-1beta-induced iNOS expression in human lung carcinoma A549 cells: involvement of STAT and MAPK pathways

For understanding of signaling molecules important in lung cancer growth and progression, IL-1beta effect was analyzed on iNOS expression and key signaling molecules in human lung carcinoma A549 cells and established the role of specific signaling molecules by using specific chemical inhibitors. IL-1beta exposure (10 ng/ml) induced strong iNOS expression in serum starved A549 cells. Detailed molecular analyses showed that IL-1beta increased expression of phosphorylated STAT1 (Tyr701 and Ser727) and STAT3 (Tyr705 and Ser727) both in total cell lysates and nuclear lysates. Further, IL-1beta exposure strongly activated MAPKs (ERK1/2, JNK1/2 and p38) and Akt as well as increased nuclear levels of NF-kappaB and HIF-1alpha in A549 cells. Use of specific chemical inhibitors for JAK1 kinase (piceatannol), JAK2 kinase (AG-490), MEK1/2 (PD98059) and JNK1/2 (SP600125) revealed that IL-1beta-induced iNOS expression involved signaling pathways in addition to JAK-STAT and ERK1/2-JNK1/2 activation. Overall, these results suggested that instead of specific pharmacological inhibitors, use of chemopreventive agents with broad spectrum efficacy to inhibit IL-1beta-induced signaling cascades and iNOS expression would be a better strategy towards lung cancer prevention and/or treatment.     

Sodium orthovanadate suppresses palmitate-induced cardiomyocyte apoptosis by regulation of the JAK2/STAT3 signaling pathway

Elevated circulatory free fatty acids (FFAs) especially saturated FFAs, such as palmitate (PA), are detrimental to the heart. However, mechanisms responsible for this phenomenon remain unknown. Here, the role of JAK2/STAT3 in PA-induced cytotoxicity was investigated in cardiomyocytes. We demonstrate that PA suppressed the JAK2/STAT3 pathway by dephosphorylation of JAK2 (Y1007/1008) and STAT3 (Y705), and thus blocked the translocation of STAT3 into the nucleus. Conversely, phosphorylation of S727, another phosphorylated site of STAT3, was increased in response to PA treatment. Pretreatment of JNK inhibitor, but not p38 MAPK inhibitor, inhibited STAT3 (S727) activation induced by PA and rescued the phosphorylation of STAT3 (Y705). The data suggested that JNK may be another upstream factor regulating STAT3, and verified the important function of P-STAT3 (Y705) in PA-induced cardiomyocyte apoptosis. Sodium orthovanadate (SOV), a protein tyrosine phosphatase inhibitor, obviously inhibited PA-induced apoptosis by restoring JAK2/STAT3 pathways. This effect was diminished by STAT3 inhibitor Stattic. Collectively, our data suggested a novel mechanism that the inhibition of JAK2/STAT3 activation was responsible for palmitic lipotoxicity and SOV may act as a potential therapeutic agent by targeting JAK2/STAT3 in lipotoxic cardiomyopathy treatment.     

抑瘤素M受体对慢性自身免疫性荨麻疹发病机制的影响

本研究旨在探讨抑瘤素M受体(OSMR)在慢性自身免疫性荨麻疹(CAU)发病机制中的作用。本研究分别检测30例CAU患者及30名健康受试者的皮肤组织中OSMR、JAK和STAT3的表达,研究显示OSMR、JAK和STAT3在CAU患者皮肤组织中高表达(p<0.05)。转染OSMR-siRNA可显著降低CAU模型小鼠血清炎症因子IL-1、IL-6和IFN-γ水平,而转染JAK/STAT3信号通路激动剂Tyr705则可显著升高炎症因子水平(p<0.05)。转染OSMR-siRNA可显著降低CAU小鼠瘙痒次数、瘙痒时间和嗜酸性粒细胞计数,而转染Tyr705则可显著升高CAU小鼠瘙痒次数、瘙痒时间和嗜酸性粒细胞计数(p<0.05)。转染OSMR-siRNA促进了CAU小鼠上皮细胞的增殖能力,并抑制了细胞凋亡(p<0.05)。而转染Tyr705则抑制了CAU小鼠上皮细胞的增殖能力,并促进了细胞凋亡(p<0.05)。转染OSMR-siRNA下调了上皮细胞中OSMR、JAK和STAT3的表达,而转染Tyr705则上调了OSMR、JAK和STAT3的表达(p<0.05)。总之,本研究表明OSMR基因在CAU患者皮肤组织中高表达。OSMR基因沉默可通过抑制JAK/STAT3信号通路来抑制炎症因子表达及嗜酸性粒细胞数量,促进上皮细胞增殖并抑制细胞凋亡。     

Leptin induces IL-8 expression via leptin receptor, IRS-1, PI3K, Akt cascade and promotion of NF-kappaB/p300 binding in human synovial fibroblasts

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-8 production caused by leptin in both rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). RASF and OASF expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-8 production. Leptin-mediated IL-8 production was attenuated by OBRl receptor antisense oligonucleotide, JAK2 inhibitor or STAT3 small interference RNA (siRNA). Transfection with insulin receptor substrate (IRS)-1 siRNA or dominant-negative mutant of p85 and Akt or pretreatment with phosphatidylinositol 3-kinase inhibitor (Ly294002 and wortmannin), Akt inhibitor, NF-kappaB inhibitor (PDTC) and NF-kappaB inhibitor peptide also inhibited the potentiating action of leptin. Stimulation of RASF with leptin activated IkappaB kinase alpha/beta (IKK alpha/beta), p65 phosphorylation at Ser(276), p65 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Moreover, pretreatment with p300 inhibitor (curcumin) also blocked IL-8 expression. The binding of p65 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of histone H3 acetylation on the IL-8 promoter was enhanced by leptin, which was inhibited by wortmannin, Akt inhibitor or IRS-1 siRNA. These results suggest that leptin increased IL-8 production in synovial fibroblast via the OBRl/JAK2/STAT3 pathway, as well as the activation of IRS1/PI3K/Akt/NF-kappaB-dependent pathway and the subsequent recruitment of p300.     

STAT3 mutations correlated with hyper-IgE syndrome lead to blockage of IL-6/STAT3 signalling pathway

Of all the causes identified for the disease hyper-immunoglobulinemia E syndrome (HIES), a homozygous mutation in tyrosine kinase2 (TYK2) and heterozygous mutations in STAT3 are implicated the defects in Jak/STAT signalling pathway in the pathogenesis of HIES. Mutations of STAT3 have been frequently clinically identified in autosomaldominant (AD) HIES patients' cells, and therefore, the genotype of STAT3 has been associated with the phenotype of HIES. Here, we conducted studies on the functional loss of the seven specific STAT3 mutations correlated with ADHIES. Using STAT3-null human colon carcinoma cell line A4 cells, we generated seven mutants of STAT3 bearing single mutations clinically identified in AD-HIES patients' cells and studied the functional loss of these mutants in IL- 6-Jak/STAT3 signalling pathway. Our results show that five STAT3 mutants bearing mutations in the DNA-binding domain maintain the phosphorylation of Tyr705 and the ability of dimerization while the other two with mutations in SH2 domain are devoid of the phosphorylation of Try705 and abrogate the dimerization in response to IL-6. The phosphorylation of Ser727 in these mutants shows diversity in response to IL-6. These mutations eventually converge on the abnormalities of the IL-6/Gp130/Jak2-mediated STAT3 transactivation on target genes, indicative of the dysregulation of JAK/STAT signalling present in HIES.     

Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c-fos promoter activation, cell mitogenicity, and transformation

The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.