Physical and functional interaction of growth hormone and insulin-like growth factor-I signaling elements.

GH and IGF-I are critical regulators of growth and metabolism. GH interacts with the GH receptor (GHR), a cytokine superfamily receptor, to activate the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), and initiate intracellular signaling cascades. IGF-I, produced in part in response to GH, binds to the heterotetrameric IGF-I receptor (IGF-IR), which is an intrinsic tyrosine kinase growth factor receptor that triggers proliferation, antiapoptosis, and other biological actions. Previous in vitro and overexpression studies have suggested that JAKs may interact with IGF-IR and that IGF-I stimulation may activate JAKs. In this study, we explore interactions between GHR-JAK2 and IGF-IR signaling pathway elements utilizing the GH and IGF-I-responsive 3T3-F442A and 3T3-L1 preadipocyte cell lines, which endogenously express both the GHR and IGF-IR. We find that GH induces formation of a complex that includes GHR, JAK2, and IGF-IR in these preadipocytes. The assembly of this complex in intact cells is rapid, GH concentration dependent, and can be prevented by a GH antagonist, G120K. However, it is not inhibited by the kinase inhibitor, staurosporine, which markedly inhibits GHR tyrosine phosphorylation. Moreover, complex formation does not appear dependent on GH-induced activation of the ERK or phosphatidylinositol 3-kinase signaling pathways or on the tyrosine phosphorylation of GHR, JAK2, or IGF-IR. These results suggest that GH-induced formation of the GHR-JAK2-IGF-IR complex is governed instead by GH-dependent conformational change(s) in the GHR and/or JAK2. We further demonstrate that GH and IGF-I can synergize in acute aspects of signaling and that IGF-I enhances GH-induced assembly of conformationally active GHRs. These findings suggest the existence of previously unappreciated relationships between these two hormones.

Novel effects of On-Chung-Eum, the traditional plant medicine, on cytokine production in human mononuclear cells from Behçet's.

Plant medications have been used as treatment in various kinds of systemic inflammatory disorder such as Behçet's disease (BD). We investigated the roles of On-Chung-Eum (OCE), a traditional plant medicine, in cytokine regulation of BD. The effects of OCE on cytokine production from phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) of Behçet's patients and control subjects were measured by ELISA. PBMC from patients with active BD produced higher levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) compared to control. OCE significantly inhibited the production of TNF-alpha, IL-1beta and interferon-gamma (INF-gamma), compared to absence of OCE. The inhibitory effects of OCE showed in a dose-dependent manner, and OCE had better effects than immunosuppressive drug, cyclosporin A. OCE is able to effectively inhibit proinflammatory cytokines and immunoregulatory Th1 cytokine. OCE treatment for BD patients may help the improvement of symptoms through cytokine modulation.

Growth hormone-induced phosphorylation of epidermal growth factor (EGF) receptor in 3T3-F442A cells. Modulation of EGF-induced trafficking and signaling.

Growth hormone (GH) promotes signaling by causing activation of the non-receptor tyrosine kinase, JAK2, which associates with the GH receptor. GH causes phosphorylation of epidermal growth factor receptor (EGFR; ErbB-1) and its family member, ErbB-2. For EGFR, JAK2-mediated GH-induced tyrosine phosphorylation may allow EGFR to serve as a scaffold for GH signaling. For ErbB-2, GH induces serine/threonine phosphorylation that dampens basal and EGF-induced ErbB-2 kinase activation. We now further explore GH-induced EGFR phosphorylation in 3T3-F442A, a preadipocytic fibroblast cell line that expresses endogenous GH receptor, EGFR, and ErbB-2. Using a monoclonal antibody that recognizes ERK consensus site phosphorylation (PTP101), we found that GH caused PTP101-reactive phosphorylation of EGFR. This GH-induced EGFR phosphorylation was prevented by MEK1 inhibitors but not by a protein kinase C inhibitor. Although GH did not discernibly affect EGF-induced EGFR tyrosine phosphorylation, we observed by immunoblotting a substantial decrease of EGF-induced EGFR degradation in the presence of GH. Fluorescence microscopy studies indicated that EGF-induced intracellular redistribution of an EGFR-cyan fluorescent protein chimera was markedly reduced by GH cotreatment, in support of the immunoblotting results. Notably, protection from EGF-induced degradation and inhibition of EGF-induced intracellular redistribution afforded by GH were both prevented by a MEK1 inhibitor, suggesting a role for GH-induced ERK activation in regulating the trafficking itinerary of the EGF-stimulated EGFR. Finally, we observed augmentation of early aspects of EGF signaling (EGF-induced ERK2 activation and EGF-induced Cbl tyrosine phosphorylation) by GH cotreatment; the GH effect on EGF-induced Cbl tyrosine phosphorylation was also prevented by MEK1 inhibition. These data indicate that GH, by activating ERKs, can modulate EGF-induced EGFR trafficking and signaling and expand our understanding of mechanisms of cross-talk between the GH and EGF signaling systems.

A role for Grb2-associated binder-1 in growth hormone signaling.

GH signaling begins with activation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, Janus kinase-2. GH-induced Janus kinase-2 activation leads to engagement of several signaling pathways, including the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase, phosphoinositol 3-kinase, and signal transducer and activator of transcription-5 (STAT5) pathways. Previous work suggests that ERK activation in response to GH may be modulated by several proteins acting as docking molecules, including the epidermal growth factor receptor (EGFR) and insulin receptor substrate-1. In this study we investigate potential roles for the pleckstrin homology (PH) domain-containing insulin receptor substrate-like protein, Grb-2-associated binder-1 (Gab1), in GH signaling. We find in 3T3-F442A preadipocytes that GH promotes tyrosine phosphorylation of Gab1 and its association with SHP2, an Src homology 2-containing cytoplasmic tyrosine phosphatase. The Grb2 adapter protein, in contrast, is specifically coimmunoprecipitated with Gab1, even in the absence of GH exposure. Using a COS-7 cell transient reconstitution system, we observed that GH-induced Gab1 tyrosine phosphorylation is dependent on the Gab1 PH domain, whereas GH-induced coimmunoprecipitation of SHP2 requires tyrosine 627 of Gab1, as previously reported for EGF-induced Gab1-SHP2 association. Deletion of the Gab1 PH domain significantly attenuates GH-induced ERK activation and trans-activation of a c-fos enhancer-driven reporter construct compared with wild-type Gab1 in this system. In contrast, GH-induced STAT5 tyrosine phosphorylation and STAT5-dependent trans-activation are similar in cells expressing wild-type or PH domain-deleted Gab1. Notably, neither the ERK nor the STAT5 GH-dependent signaling outcome is affected by expression of the Gab1 mutant with tyrosine 627 changed to phenylalanine. Finally, we observed GH-dependent translocation of a wild-type, but not a PH domain-deleted, Gab1-green fluorescent protein chimera from the cytoplasm to the plasma membrane. Our results suggest selective involvement of Gab1 in GH-induced ERK activation and implicate the Gab1 PH domain as critical in this involvement.