Thrombopoietin,but not cytokines binding to gp130 protein-coupled receptors,activates MAPKp42/44, AKT,and STAT proteins in normal human CD34+ cells,megakaryocytes, and platelets

OBJECTIVE: The development of megakaryocytes is regulated by thrombopoietin (TPO), which binds to the c-mpl receptor, and by several other cytokines such as interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), cilliary neurotropic factor (CNTF), and oncostatin (OSM), which bind to gp130 protein-coupled receptors. We attempted to identify signal transduction pathways activated by these factors in normal human megakaryocytes. MATERIALS AND METHODS: To better understand the role of these factors in normal human megakaryopoiesis we studied their effect on 1) purified human bone marrow-derived CD34+ cells, 2) human alpha(IIb)beta3+ cells (shown by immunophenotypical and morphological criteria to be megakaryoblasts), which had been expanded ex vivo from CD34+ cells in chemically defined artificial serum, and 3) gel-filtered human peripheral blood platelets. Further, in an attempt to correlate the influence of these factors on cell proliferation and survival with activation of signal transduction pathways, we evaluated their effect on the phosphorylation of MAPK p42/44 and activation of PI-3K-AKT and JAK-STAT proteins in these various cell types. RESULTS: Using serum-free liquid cultures, we found that only TPO and IL-6 protected CD34+ cells and megakaryocytes from undergoing apoptosis (decrease in annexin-V binding, PARP cleavage, and activation of caspase-3). Moreover, only TPO when used alone and IL-6 only when used in combination with TPO, stimulated the growth of human colony-forming unit-megakaryocytes (CFU-Meg) in semisolid serum-free medium. We also observed that while TPO efficiently activated various signaling pathways in CD34+ cells, megakaryocytes, and platelets (MAPK p42/44, PI-3K-AKT, STAT proteins), IL-6 stimulated phosphorylation of STAT-1, -3, and -5 proteins only in CD34+ cells and megakaryoblasts. To our surprise, none of the other gp130 protein-related cytokines tested (IL-11, LIF, CNTF, and OSM) activated these signaling pathways in CD34+ cells, megakaryoblasts, or platelets. CONCLUSIONS: Our signal transduction studies explain why TPO, by simultaneously activating several signaling pathways, is the most potent megakaryopoietic regulator and why of all five gp130 protein-related cytokines tested, only IL-6, through activation of STAT proteins, plays a role in normal human megakaryopoiesis.     

Hematopoietic abnormalities in mice deficient in gp130-mediated STAT signaling

OBJECTIVE: Studies on mice lacking the common receptor subunit gp130 reveal that activation of gp130-dependent signaling pathways is essential for normal fetal and adult hematopoiesis. However, the extent to which hematopoiesis is dependent upon activation of a particular gp130 signaling pathway, namely STAT1/3 or SHP2/MAPK, is unknown. This study examined the specific contribution of gp130-mediated STAT1/3 signaling to the regulation of hematopoiesis. MATERIALS AND METHODS: Hematopoiesis was examined at various developmental stages in mice homozygous for a targeted carboxy-terminal truncation mutation in gp130 (gp130(delta)/(delta)) that deletes all STAT1/3 binding sites, thereby abolishing gp130-mediated STAT1/3 activation. RESULTS: Adult gp130(delta)/(delta) mice have increased numbers of immature colony-forming unit spleen progenitor cells in the bone marrow and spleen, elevated numbers of committed myeloid progenitor cells in the spleen and peripheral blood, and leukocytosis. Increased progenitor cell production was observed in gp130(delta)/(delta) fetal livers from 14 days of gestation onward. In contrast, the circulating platelet count was reduced by 30% in gp130(delta)/(delta) mice, without any corresponding decrease in the number of bone marrow and splenic megakaryocytes. In liquid cultures, megakaryocytes from gp130(delta)/(delta) mice are smaller than those from wild-type mice and do not increase in size upon stimulation with interleukin-6 or interleukin-11. Administration of either interleukin-6 or interleukin-11 to gp130(delta)/(delta) mice failed to increase platelet numbers, despite an increase in the production of megakaryocytes. CONCLUSIONS: Collectively, these results reveal that gp130-mediated STAT1/3 activation is required to maintain the normal balance of hematopoietic progenitors during fetal and adult hematopoiesis. Furthermore, they suggest two distinct roles for gp130-mediated STAT1/3 activation in hematopoiesis, one restricting the production of immature hematopoietic progenitor cells and the other promoting the functional maturation of megakaryocytes to produce platelets.     

Opposing roles of gp130-mediated STAT-3 and ERK-1/ 2 signaling in liver progenitor cell migration and proliferation

Gp130-mediated IL-6 signaling may play a role in oval cell proliferation in vivo. Levels of IL-6 are elevated in livers of mice treated with a choline-deficient ethionine-supplemented (CDE) diet that induces oval cells, and there is a reduction of oval cells in IL-6 knockout mice. The CDE diet recapitulates characteristics of chronic liver injury in humans. In this study, we determined the impact of IL-6 signaling on oval cell-mediated liver regeneration in vivo. Signaling pathways downstream of gp130 activation were also dissected. Numbers of A6(+ve) liver progenitor oval cells (LPCs) in CDE-treated murine liver were detected by immunohistochemistry and quantified. Levels of oval cell migration and proliferation were compared in CDE-treated mouse strains that depict models of gp130-mediated hyperactive ERK-1/2 signaling (gp130(deltaSTAT)), hyperactive STAT-3 signaling (gp130(Y757F) and Socs-3(-/deltaAlb)) or active ERK-1/2 as well as active STAT-3 signaling (wild-type). The A6(+ve) LPC numbers were increased with IL-6 treatment in vivo. The gp130(Y757F) mice displayed increased A6(+ve) LPCs numbers compared with wild-type and gp130(deltaSTAT) mice. Numbers of A6(+ve) LPCs were also increased in the livers of CDE treated Socs-3(-/deltaAlb) mice compared with their control counterparts. Lastly, inhibition of ERK-1/2 activation in cultured oval cells increased hyper IL-6-induced cell growth. For the first time, we have dissected the gp130-mediated signaling pathways, which influence liver progenitor oval cell proliferation. Conclusion: Hyperactive STAT-3 signaling results in enhanced oval cell numbers, whereas ERK-1/2 activation suppresses oval cell proliferation.     

IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation

Interleukin (IL)-6 signaling through its soluble receptor (IL-6 transsignaling) directs transition between innate and acquired immune responses by orchestrating the chemokine-directed attraction and apoptotic clearance of leukocytes. Through analysis of mononuclear cell infiltration in WT and IL-6-deficient mice during peritoneal inflammation, we now report that IL-6 selectively governs T cell infiltration by regulating chemokine secretion (CXCL10, CCL4, CCL5, CCL11, and CCL17) and chemokine receptor (CCR3, CCR4, CCR5, and CXCR3) expression on the CD3+ infiltrate. Although blockade of IL-6 trans-signaling prevented chemokine release, chemokine receptor expression remained unaltered suggesting that this response is regulated by IL-6 itself. To dissect the signaling events promoting T cell migration, inflammation was established in knock-in mice expressing mutated forms of the universal signal-transducing element for IL-6-related cytokines gp130. In mice (gp130Y757F/Y757F) deficient in SHP2 and SOCS3 binding, but presenting hyperactivation of STAT1/3, T cell recruitment and CCL5 expression was enhanced. Conversely, both of these parameters were suppressed in mice with ablated gp130-mediated STAT1/3 activation (gp130DeltaSTAT/DeltaSTAT). T cell migration was related to STAT3 activity, because monoallelic deletion of Stat3 in gp130(Y757F/Y757F) mice (gp130Y757F/Y757F:Stat3+/-) corrected the exaggerated responses observed in gp130Y757F/Y757F mice. Consequently, STAT3 plays a defining role in IL-6-mediated T cell migration.     

An essential role for gp130 in neointima formation following arterial injury

Interleukin (IL)-6 induced vascular smooth muscle cell (VSMC) motility in a dose-dependent manner. In addition, IL-6 stimulated tyrosine phosphorylation of gp130, resulting in the recruitment and activation of STAT-3. IL-6-induced VSMC motility was found to be dependent on activation of gp130/STAT-3 signaling. IL-6 also induced cyclin D1 expression in a time- and gp130/STAT-3-dependent manner in VSMCs. Suppression of cyclin D1 levels via the use of its small interfering RNA molecules inhibited IL-6-induced VSMC motility. Furthermore, balloon injury induced IL-6 expression both at mRNA and protein levels in rat carotid artery. Balloon injury also caused increased STAT-3 phosphorylation and cyclin D1 expression, leading to smooth muscle cell migration from the media to the intimal region. Blockade of gp130/STAT-3 signaling via adenovirus-mediated expression of dngp130 or dnSTAT-3 attenuated balloon injury-induced STAT-3 phosphorylation and cyclin D1 induction, resulting in reduced smooth muscle cell migration from media to intima and decreased neointima formation. Together, these observations for the first time suggest that IL-6/gp130/STAT-3 signaling plays an important role in vascular wall remodeling particularly in the settings of postangioplasty and thereby in neointima formation.     

Pathologic consequences of STAT3 hyperactivation by IL-6 and IL-11 during hematopoiesis and lymphopoiesis

We have previously demonstrated that STAT3 hyperactivation via the interleukin 6 (IL-6) cytokine family receptor gp130 in gp130 (Y757F/Y757F) mice leads to numerous hematopoietic and lymphoid pathologies, including neutrophilia, thrombocytosis, splenomegaly, and lymphadenopathy. Because IL-6 and IL-11 both signal via a gp130 homodimer, we report here a genetic approach to dissect their individual roles in these pathologies. Neutrophilia and thrombocytosis were absent in gp130 (Y757F/Y757F) mice lacking either IL-6 (gp130 (Y757F/Y757F): IL-6 (-/-)) or the IL-11 receptor alpha subunit (gp130 (Y757F/Y757F): IL-11Ralpha1 (-/-)), and this was associated with a normalized bone marrow compartment. The elevated myelopoiesis and megakaryopoiesis in bone marrow of gp130 (Y757F/Y757F) mice was attributable to an increase by either IL-6 or IL-11 in the STAT3-driven impairment of transforming growth factor beta (TGF-beta) signaling, which is a suppressor of these lineages. In contrast, the absence of IL-6, but not IL-11 signaling, prevented the splenomegaly, abnormal lymphopoiesis, and STAT3 hyperactivation in lymphoid organs of gp130 (Y757F/Y757F) mice. Furthermore, hyperactivation of STAT3 in lymphoid organs was associated with increased expression of IL-6Ralpha, and IL-6Ralpha expression was reduced in gp130 (Y757F/Y757F): Stat3 (+/-) mice displaying normal levels of STAT3 activity. Collectively, these data genetically define distinct roles of IL-6 and IL-11 in driving pathologic hematopoietic and lymphoid responses mediated by STAT3 hyperactivation.     

Requirement of gp130 signaling for the AGM hematopoiesis

OBJECTIVE: Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region during mouse development and remarkably expands in the liver at a later stage of ontogeny. gp130 is a signal transducing receptor component shared by all the IL-6 family cytokines, whose gene ablation in mouse results in the significant reduction in the fetal liver hematopoiesis. The present study aims to evaluate the role of gp130 signaling in the fetal mouse AGM hematopoiesis. METHODS AND MATERIALS: Mouse AGM regions from the wild-type and gp130-deficient mice on embryonic day 11.5 were dissociated and cultured with a mixture of cytokines, including one which activates gp130. Wild-type human gp130 and its mutant constructs were introduced into cultured gp130-deficient AGM cells using retrovirus system. To further analyze gp130 downstream signaling, a dominant-negative mutant of STAT3 was also introduced. RESULTS: The gp130 deficiency in the culture of fetal mouse AGM cells resulted in the failure of the expansion of the c-kit(+), Sca-1(+), and lineage markers(-) population. Such failure was rescued by introduction of a wild-type gp130 expression construct but not its mutant constructs having no ability to activate STAT3. In the normal AGM cell culture, introduction of a dominant-negative form of STAT3 in which Y(705) was changed to phenylalanine suppressed the expansion of hematopoietic cell colonies. CONCLUSION: gp130 plays an indispensable role in the expansion of hematopoietic precursor cells in the fetal mouse AGM. In particular, the activation of STAT3 by gp130 is found to be important in this process.