Interleukin-6-induced JAK2/STAT3 signaling pathway in endothelial cells is suppressed by hemodynamic flow

Endothelial cells (ECs) are constantly exposed to shear stress, the action of which triggers signaling pathways and cellular responses. During inflammation, cytokines such as IL-6 increase in plasma. In this study, we examined the effects of steady flow on IL-6-induced endothelial responses. ECs exposed to IL-6 exhibited STAT3 activation via phosphorylation of Tyr705. However, when ECs were subjected to shear stress, shear force-dependent suppression of IL-6-induced STAT3 phosphorylation was observed. IL-6 treatment increased the phosphorylation of JAK2, an upstream activator of STAT3. Consistently, shear stress significantly reduced IL-6-induced JAK2 activation. Pretreatment of ECs with an inhibitor of MEK1 did not alter this suppression by shear stress, indicating that extracellular signal-regulated kinase (ERK1/2) was not involved. However, pretreatment of ECs with an endothelial nitric oxide synthase inhibitor (nitro-L-arginine methyl ester) attenuated this inhibitory effect of shear stress on STAT3 phosphorylation. Shear stress-treated ECs displayed decreased nuclear transmigration of STAT3 and reduced STAT3 binding to DNA. Intriguingly, ECs exposed to IL-6 entered the cell cycle, as evidenced by increasing G₂/M phase, and shear stress to these ECs significantly reduced IL-6-induced cell cycle progression. STAT3-mediated IL-6-induced cell cycle was confirmed by the inhibition of the cell cycle in ECs infected with adenovirus carrying the inactive mutant of STAT3. Our study clearly shows that shear stress exerts its inhibitory regulation by suppressing the IL-6-induced JAK2/STAT3 signaling pathway and thus inhibits IL-6-induced EC proliferation. This shear force-dependent inhibition of IL-6-induced JAK2/STAT3 activation provides new insights into the vasoprotective effects of steady flow on ECs against cytokine-induced responses. shear stress; nitric oxide; cell cycle     

α(1A)-adrenergic receptor differentially regulates STAT3 phosphorylation through PKCϵ and PKCδ in myocytes

Previous studies demonstrated α?-adrenergic receptors (ARs) increase STAT3 activation in transfected and non-cardiac primary cell lines. However, the mechanism used by α?-ARs resulting in STAT3 activation is unknown. While other G-protein-coupled receptors (GPCRs) can couple to STAT3, these mechanisms demonstrate coupling through SRC, TYK, Rac, or complex formation with Gq and used only transfected cell lines. Using normal and transgenic mice containing constitutively active mutations (CAM) of the α(1A)-AR subtype, neonatal mouse myocytes and whole hearts were analyzed for the mechanism to couple to STAT3 activation. α?-ARs stimulated time-dependent increases in p-SRC, p-JAK2, and p-STAT3 in normal neonatal myocytes. Using various kinase inhibitors and siRNA, we determined that the α(1A)-AR coupled to STAT3 through distinct and unique pathways in neonatal myocytes. We found that PKC? inhibition decreased p-ERK and p-Ser STAT3 levels without affecting p-Tyr STAT3. In contrast, we found that PKCδ inhibition affected p-SRC and p-JAK2 resulting in decreased p-Tyr and p-Ser STAT3 levels. We suggest a novel α(1A)-AR mediated PKC?/ERK pathway that regulates the phosphorylation status of STAT3 at Ser-727 while PKCδ couples to SRC/JAK2 to affect Tyr-705 phosphorylation. Furthermore, this pathway has not been previously described in a GPCR system that couples to STAT3. Given cell survival and protective cardiac effects induced by PKC, STAT3 and ERK signaling, our results could explain the neuroprotective and cardiac protective pathways that are enhanced with α(1A)-AR agonism.     

α1A-Adrenergic receptor differentially regulates STAT3 phosphorylation through PKCϵ and PKCδ in myocytes

Previous studies demonstrated α₁-adrenergic receptors (ARs) increase STAT3 activation in transfected and non-cardiac primary cell lines. However, the mechanism used by α₁-ARs resulting in STAT3 activation is unknown. While other G-protein-coupled receptors (GPCRs) can couple to STAT3, these mechanisms demonstrate coupling through SRC, TYK, Rac, or complex formation with Gq and used only transfected cell lines. Using normal and transgenic mice containing constitutively active mutations (CAM) of the α_1A-AR subtype, neonatal mouse myocytes and whole hearts were analyzed for the mechanism to couple to STAT3 activation. α₁-ARs stimulated time-dependent increases in p-SRC, p-JAK2, and p-STAT3 in normal neonatal myocytes. Using various kinase inhibitors and siRNA, we determined that the α_1A-AR coupled to STAT3 through distinct and unique pathways in neonatal myocytes. We found that PKC? inhibition decreased p-ERK and p-Ser STAT3 levels without affecting p-Tyr STAT3. In contrast, we found that PKCδ inhibition affected p-SRC and p-JAK2 resulting in decreased p-Tyr and p-Ser STAT3 levels. We suggest a novel α_1A-AR mediated PKC?/ERK pathway that regulates the phosphorylation status of STAT3 at Ser-727 while PKCδ couples to SRC/JAK2 to affect Tyr-705 phosphorylation. Furthermore, this pathway has not been previously described in a GPCR system that couples to STAT3. Given cell survival and protective cardiac effects induced by PKC, STAT3 and ERK signaling, our results could explain the neuroprotective and cardiac protective pathways that are enhanced with α_1A-AR agonism.     

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.     

p21-Activated Kinase 1 (PAK1) Can Promote ERK Activation in a Kinase-independent Manner

PAK1 plays an important role in proliferation and tumorigenesis, at least partially by promoting ERK phosphorylation of C-RAF (Ser-338) or MEK1 (Ser-298). We observed how that overexpression of a kinase-dead mutant form of PAK1 increased phosphorylation of MEK1/2 (Ser-217/Ser-221) and ERK (Thr-202/Tyr-204), although phosphorylation of B-RAF (Ser-445) and C-RAF (Ser-338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1, and MEK1 independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate MEK activity in a kinase-independent manner, probably by serving as a scaffold to facilitate interaction of C-RAF.     

AG490 prevents cell death after exposure of rat astrocytes to hydrogen peroxide or proinflammatory cytokines: involvement of the Jak2/STAT pathway

Janus kinases/STAT pathway mediates cellular responses to certain oxidative stress stimuli and cytokines. Here we examine the activation of Stat1 and Stat3 in rat astrocyte cultures and its involvement in cell death. H(2)O(2), interferon (INF)-gamma and interleukin (IL)-6 but not IL-10 caused cell death. Stat1 was phosphorylated on tyrosine (Tyr)-701 after exposure to H(2)O(2), INF-gamma or IL-6 but not IL-10. Tyr-705 pStat3 was observed after H(2)O(2), IL-6 and IL-10. Also, H(2)O(2) induced serine (Ser)-727 phosphorylation of Stat1 but not Stat3. The degree of Tyr-701 pStat1 by the different treatments positively correlated with the corresponding reduction of cell viability. AG490, a Jak2 inhibitor, prevented Tyr-701 but not Ser-727, Stat1 phosphorylation. Also, AG490 inhibited Tyr-705 Stat3 phosphorylation induced by H(2)O(2) and IL-6 but did not prevent that induced by IL-10. Furthermore, AG490 conferred strong protection against cell death induced by INF-gamma, IL-6 and H(2)O(2). These results suggest that Jak2/Stat1 activation mediates cell death induced by proinflammatory cytokines and peroxides. However, we found evidence suggesting that AG490 reduces oxidative stress induced by H(2)O(2), which further shows that H(2)O(2) and/or derived reactive oxygen species directly activate Jak2/Stat1, but masks the actual involvement of this pathway in H(2)O(2)-induced cell death.     

High glucose enhances TGF-β1 expression in rat bone marrow stem cells via ERK1/2-mediated inhibition of STAT3 signaling

AimsThis study was to investigate the effect of high glucose (HG) on TGF-β1 expression and the underlying mechanisms in bone marrow stem cells.Main methodsRat bone marrow multipotent adult progenitor cells (MAPCs) were cultured in normal (5.5 mM d-glucose) and HG media (25.5 mM d-glucose) for up to 14 days. l-Glucose (20 mM plus 5.5 mM d-glucose) was used as high osmolarity control. TGF-β1 expression was evaluated using quantitative RT-PCR, ELISA, and immunofluorescence staining for its mRNA and protein level in the cells and in the conditioned media. The expression and activation of ERK1/2 and STAT3 were examined in MAPCs cultured in HG media with Western blot.Key findingsMeasurable level of TGF-β1 was detected in the cells cultured in normal media. TGF-β1 expression was substantially increased in MAPCs after 36 h of culture in HG media with over 20-fold increase in the mRNA and 5-fold increase in protein level over control. Interestingly, ERK1/2 phosphorylation was significantly increased in MAPCs cultured in HG media, while in STAT3 (Tyr705), not STAT3 (Ser727), phosphorylation was dramatically decreased. Treatment of cells with the specific MEK1 inhibitor PD98059 or U0126 suppressed ERK1/2 phosphorylation and TGF-β1 expression, and completely restored the level of STAT3 (Tyr705) phosphorylation in MAPCs cultured in HG media. Treatment of the cells with the specific STAT3 phosphorylation inhibitor AG490 significantly blocked STAT3 (Tyr705) phosphorylation and increased TGF-β1 expression without change in ERK1/2 phosphorylation in MPACs.SignificanceHG increased TGF-β1 expression through inhibition of STAT3 (Tyr705) by enhanced ERK1/2 signaling in MAPCs.     

Oxidative Stress Inhibits Insulin-like Growth Factor-I Induction of Chondrocyte Proteoglycan Synthesis through Differential Regulation of Phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK Signaling Pathways

The ability of insulin-like growth factor I (IGF-I) to stimulate cartilage matrix synthesis is reduced in aged and osteoarthritic cartilage. Aging and osteoarthritis are associated with an increase in reactive oxygen species, which we hypothesized would interfere with normal IGF-I signaling. We compared IGF-I signaling in normal and osteoarthritic human articular chondrocytes and investigated the effects of oxidative stress induced by tert-butylhydroperoxide (tBHP). In normal human chondrocytes, IGF-I initiated a strong and sustained phosphorylation of IRS-1 (Tyr-612) and Akt (Ser-473) and transient ERK phosphorylation. In contrast, in osteoarthritic chondrocytes, which possessed elevated basal IRS-1 (Ser-312) and ERK phosphorylation, IGF-I failed to stimulate IRS-1 (Tyr-612) or Akt phosphorylation. In normal human chondrocytes, tBHP triggered strong IRS-1 (Ser-312 and Ser-616) and ERK phosphorylation and inhibited IGF-I-induced IRS-1 (Tyr-612) and Akt phosphorylation. Lentivirus-mediated overexpression of constitutively active (CA) Akt significantly enhanced proteoglycan synthesis, whereas both dominant negative Akt and CA MEK inhibited proteoglycan synthesis. CA Akt also promoted type II collagen and Sox9 expression, whereas tBHP treatment and CA MEK inhibited aggrecan, collagen II, and Sox9 mRNA expression. In osteoarthritic chondrocytes, the antioxidants Mn(III) tetrakis(4-benzoic acid)porphyrin and N-acetylcysteine increased the ratio of Akt to ERK phosphorylation and promoted IGF-I-mediated proteoglycan synthesis. Chemical inhibition of ERK significantly enhanced IGF-I phosphorylation of Akt and alleviated tBHP inhibition of Akt phosphorylation. These results demonstrate opposing roles for phosphatidylinositol 3-kinase-Akt and MEK-ERK in cartilage matrix synthesis and suggest that elevated levels of reactive oxygen species cause chondrocyte IGF-I resistance by altering the balance of Akt to ERK activity.     

The MEK-ERK Pathway Is Necessary for Serine Phosphorylation of Mitochondrial STAT3 and Ras-Mediated Transformation

Activating mutations in the RasGTPases are the most common oncogenic lesions in human cancer. Similarly, elevated STAT3 expression and/or phosphorylation are observed in the majority of human cancers. We recently found that activated Ras requires a mitochondrial rather than a nuclear activity of STAT3 to support cellular transformation. This mitochondrial activity of STAT3 was supported by phosphorylation on serine 727 (S727) in the carboxyl-terminus of STAT3. In this study we show that the H-Ras oncoprotein engages the MEK-ERK pathway to drive phosphorylation of STAT3 on S727, while phosphoinositide 3-kinase (PI3K) and mTOR activity were superfluous. Moreover, pharmacological inhibition of MEK reduced transformation by H-, K- or N-Ras. However, cells expressing a mitochondrially restricted STAT3 with a phospho-mimetic mutation at S727 were partially resistant to inhibition of the ERK pathway, exhibiting a partial rescue of anchorage-independent cell growth in the presence of MEK inhibitor. This study shows that the MEK-ERK pathway is required for activated Ras-induced phosphorylation of STAT3 on S727, that inhibition of STAT3 S727 phosphorylation contributes to the anti-oncogenic potential of MEK inhibitors, and that mitochondrial STAT3 is one of the critical substrates of the Ras-MEK-ERK- axis during cellular transformation.