Induction of an interferon-gamma Stat3 response in nerve cells by pre-treatment with gp130 cytokines

Many cytokines mediate their effects through Jak/STAT signaling pathways providing many opportunities for cross-talk between different cytokines. We examined the interaction between two cytokine families, gp130-related cytokines and interferon-gamma (IFN-gamma), which are coexpressed in the nervous system during acute trauma and pathological conditions. Typical nerve cells show an IFN-gamma response that is restricted to activating STAT1, with minor activation of STAT3. IFN-gamma elicited a pronounced STAT3 response in cells pre-treated for 5-7 h with ciliary neurotrophic factor (CNTF), leukemia inhibitory factor or interleukin-6. CNTF or interleukin-6 induced an IFN-gamma STAT3 response in a variety of cells including SH-SY5Y human neuroblastoma, HMN-1 murine motor neuron hybrid cells, rat sympathetic neurons and human hepatoma HepG2 cells. The enhancement was measured as an increase in tyrosine phosphorylated STAT3, in STAT3-DNA binding and in STAT-luciferase reporter gene activity. The enhanced STAT3 response was not due to an increase in overall STAT3 levels but was dependent upon ongoing protein synthesis. The induction by CNTF was inhibited by the protein kinase C inhibitor, BIM, and the MAPK-kinase inhibitor, U0126. Further, H-35 hepatoma cells expressing gp130 receptor chimeras lacking either the SHP-2 docking site or the Box 3 STAT binding sites failed to enhance the IFN-gamma STAT3 response. These results provide evidence for an interaction between gp130 and IFN-gamma cytokines that can significantly alter the final cellular response to IFN-gamma.     

Parallel signaling pathways in endothelin-1-induced proliferation of U373MG astrocytoma cells

Endothelin-1 (ET-1) is a potent mitogen for many cells, especially when its levels are elevated under pathological conditions, as seen in tumor cell progression and astroglial activation in neuropathies. While ET-1 is known to cause astroglial proliferation, in the present study, multiple signaling pathways involved in ET-1-mediated astrocyte proliferation were characterized. Treatment with PD98059 and U0126 (MEK inhibitors) inhibited not only ET-1-induced cell proliferation but also ET-1-activated phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in U373MG astrocytoma cells. Whereas the nonselective protein kinase C (PKC) inhibitor chelerythrine attenuated ET-1-induced cell proliferation, it was unable to block ET-1-induced ERK phosphorylation. However, ET-1 did not activate conventional or novel PKCs and did not elevate intracellular calcium. In addition, U73122 (a selective phospholipase C inhibitor), FTI-277 (an H-Ras inhibitor), as well as protein tyrosine kinase inhibitors also did not abolish ET-1-induced ERK1/2 phosphorylation. ET-1 treatment increased the activity of total Ras but not H-Ras. The phosphoinositide 3-kinase (PI3K) pathway appeared to be involved in signal transduction induced by ET-1, but it did not appear to participate in cross talk with the mitogen-activated protein kinase (MAPK) pathway. Activated ET receptors did not propagate signals either through protein tyrosine kinases or transactivation of EGF receptor tyrosine kinases, which typically trigger Ras-Raf-MAPK pathways. The results indicate that ET-1 stimulates cell proliferation by the activation of MAPK-, PKC-, and PI3K-dependent pathways that appear to function in a parallel manner. There is no apparent, direct "cross talk" between these pathways in U373MG cells, but rather, they might act on the independent but necessary components of the mitogenic effects of ET-1.     

IL-3 and IL-4 activate cyclic nucleotide phosphodiesterases 3 (PDE3) and 4 (PDE4) by different mechanisms in FDCP2 myeloid cells

In FDCP2 myeloid cells, IL-4 activated cyclic nucleotide phosphodiesterases PDE3 and PDE4, whereas IL-3, granulocyte-macrophage CSF (GM-CSF), and phorbol ester (PMA) selectively activated PDE4. IL-4 (not IL-3 or GM-CSF) induced tyrosine phosphorylation of insulin-receptor substrate-2 (IRS-2) and its association with phosphatidylinositol 3-kinase (PI3-K). TNF-alpha, AG-490 (Janus kinase inhibitor), and wortmannin (PI3-K inhibitor) inhibited activation of PDE3 and PDE4 by IL-4. TNF-alpha also blocked IL-4-induced tyrosine phosphorylation of IRS-2, but not of STAT6. AG-490 and wortmannin, not TNF-alpha, inhibited activation of PDE4 by IL-3. These results suggested that IL-4-induced activation of PDE3 and PDE4 was downstream of IRS-2/PI3-K, not STAT6, and that inhibition of tyrosine phosphorylation of IRS molecules might be one mechnism whereby TNF-alpha could selectively regulate activities of cytokines that utilized IRS proteins as signal transducers. RO31-7549 (protein kinase C (PKC) inhibitor) inhibited activation of PDE4 by PMA. IL-4, IL-3, and GM-CSF activated mitogen-activated protein (MAP) kinase and protein kinase B via PI3-K signals; PMA activated only MAP kinase via PKC signals. The MAP kinase kinase (MEK-1) inhibitor PD98059 inhibited IL-4-, IL-3-, and PMA-induced activation of MAP kinase and PDE4, but not IL-4-induced activation of PDE3. In FDCP2 cells transfected with constitutively activated MEK, MAP kinase and PDE4, not PDE3, were activated. Thus, in FDCP2 cells, PDE4 can be activated by overlapping MAP kinase-dependent pathways involving PI3-K (IL-4, IL-3, GM-CSF) or PKC (PMA), but selective activation of PDE3 by IL-4 is MAP kinase independent (but perhaps IRS-2/PI3-K dependent).     

Autocrine/Paracrine secretion of IL-6 family cytokines causes angiotensin II-induced delayed STAT3 activation

We recently reported that angiotensin II (AngII) biphasically activates the JAK/STAT pathway and induces delayed phosphorylation of STAT3 in the late stage (120 min) in cardiomyocytes. This study was designed to determine the mechanism of delayed phosphorylation of STAT3. Conditioned medium prepared from AngII-stimulated cardiomyocytes could reproduce the tyrosine phosphorylation of STAT3 at 5 min. This delayed phosphorylation was almost completely inhibited by anti-gp130 blocking antibody RX435, but not by TAK044 (ET-A/B-R antagonist), prazosin, or propranolol. AngII induced phosphorylation of gp130 in the late stage, which was temporally in parallel with the delayed phosphorylation of STAT3. AngII augmented IL-6, CT-1, and LIF mRNA expression at 30-60 min, but not CNTF expression. AngII increased IL-6 protein levels by 3-fold in the conditioned media at 2 h compared with the control. These findings indicated that AngII-induced delayed activation of STAT3 is caused by autocrine/paracrine secreted IL-6 family cytokines.     

Role of the cytokine-inducible SH2 protein CIS in desensitization of STAT5b signaling by continuous growth hormone

Growth hormone (GH)-inducible suppressors of cytokine signaling (SOCS/CIS proteins) inhibit GH receptor (GHR) signaling to STAT5b via phosphotyrosine-dependent binding interactions with the tyrosine kinase JAK2 (SOCS-1) and/or the cytoplasmic tail of GHR (CIS and SOCS-3). Presently, we investigate the mechanism of CIS inhibition and CIS's role in down-regulating GHR-JAK2 signaling to STAT5b in cells exposed to GH continuously. CIS is shown to inhibit GHR-JAK2 signaling by two distinct mechanisms: by a partial inhibition that is decreased at elevated STAT5b levels and may involve competition between CIS and STAT5b for common GHR cytoplasmic tail phosphotyrosine-binding sites; and by a time-dependent inhibition, not seen with SOCS-1 or SOCS-3, that involves proteasome action. Investigation of the latter mechanism revealed that GH stimulates degradation of CIS, but not SOCS-3. The proteasome inhibitor MG132 blocked this protein degradation and also blocked the inhibitory action of CIS, but not that of SOCS-1 or SOCS-3, on STAT5b signaling. Proteasome-dependent degradation of CIS, most likely in the form of a (GHR-JAK2)-CIS complex, is therefore proposed to be an important step in the time-dependent CIS inhibition mechanism. Finally, the down-regulation of GHR-JAK2 signaling to STAT5b seen in continuous GH-treated cells could be prevented by treatment of cells with the proteasome inhibitor MG132 or by expression of CIS-R107K, a selective, dominant-negative inhibitor of CIS activity. These findings lead us to propose that the cytokine signaling inhibitor CIS is a key mediator of the STAT5b desensitization response seen in cells and tissues exposed to GH chronically, such as adult female rat liver.     

Cross-talk between alpha(1B)-adrenergic receptor (alpha(1B)AR) and interleukin-6 (IL-6) signaling pathways. Activation of alpha(1b)AR inhibits il-6-activated STAT3 in hepatic cells by a p42/44 mitogen-activated protein kinase-dependent mechanism

Treatment of primary rat hepatocytes or tranfected HepG2 cells with the alpha(1B)-adrenergic receptor (alpha(1B)AR) agonist phenylephrine (PE) significantly inhibited interleukin 6 (IL-6)-induced STAT3 binding, tyrosine phosphorylation, and IL-6-induced serum amyloid A mRNA expression. Western analyses and in vitro kinase assays indicate that this inhibition is not due to either down-regulation of STAT3 protein expression nor inactivation of upstream-located JAK1 and JAK2. Blocking the new RNA and protein syntheses antagonized the inhibitory effect of PE on IL-6-activated STAT3, suggesting synthesis of an inhibitory factor(s) is involved. The inhibitory effect of PE on IL-6 activation of STAT3 was also abolished by the tyrosine phosphatase inhibitor sodium vanadate, indicating involvement of protein tyrosine phosphatases. Furthermore, preincubation of the cells with the specific MEK1 inhibitor PD98059 or a dominant negative MEK1 reversed the inhibitory effect of PE, and expression of constitutively activated MEK1 alone abolished IL-6-activated STAT3. Taken together, these data indicate that PE inhibits IL-6 activation of STAT3 in hepatic cells by a p42/44 mitogen-activated protein kinase-dependent mechanism, and tyrosine phosphatases are involved. This inhibitory cross-talk between the alpha(1B)AR and IL-6 signaling pathways implicates the alpha(1B)AR involvement in regulating the IL-6-mediated inflammatory responses.     

Effects of inhibitors of signal transduction pathways on transforming growth factor B1 and osteogenic protein-1-induced insulinlike growth factor binding protein-3 expression in human bone cells

Signal transduction initiated by TGFB1 and OP-1 was studied in MG63 human osteosarcoma cells and in normal human bone cells (HBCs) in the presence of inhibitors of signal transduction events, using insulinlike growth factor binding protein-3 (IGFBP-3) production as an end point. Treatment of serum-free MG63 cells and normal HBCs with TGFB1 increased IGFBP-3 protein level several fold in the conditioned medium. This effect of TGFB1 was mediated by increased de novo synthesis because mRNA level increased to the same extent as protein level and TGFB1 treatment had very little effect on IGFBP-3 protease activity. The stimulatory effect of TGFB1 on IGFBP-3 production was inhibited in a dose-dependent manner by pretreatment with staurosporine, a protein kinase C inhibitor, or with vanadate, a phosphotyrosyl protein phosphatase inhibitor in both MG63 cells and normal HBCs. In addition, pretreatment with okadoic acid, an inhibitor of serine/threonine protein phosphatase, counteracted TGFB1 induction of IGFBP-3 production. Interestingly, pretreatment of MG63 cells or HBCs with staurosporine, vanadate, or okadoic acid augmented OP-1 stimulation of IGFBP-3 production. Staurosporine- or vanadate-induced changes in IGFBP-3 protein levels in the presence of TGFB1 and OP-1 were associated with corresponding changes in IGFBP-3 mRNA levels in MG63 cells. These findings are consistent with the hypothesis that TGFB1 and OP-1 increase IGFBP-3 expression via distinct intracellular signal transduction pathways. J. Cell. Physiol. 173:28–35, 1997. © 1997 Wiley-Liss, Inc.