Growth hormone-stimulated insulin-like growth factor-1 expression in rainbow trout (Oncorhynchus mykiss) hepatocytes is mediated by ERK, PI3K-AKT, and JAK-STAT

Growth hormone (GH) initiates many of its growth-promoting actions by binding to GH receptors (GHR) and stimulating the synthesis and secretion of insulin-like growth factor-1 (IGF-1) from the liver and other sites. In this study, we used hepatocytes isolated from rainbow trout as a model system in which to determine the molecular signaling events of GH in fish. GH directly stimulated the phosphorylation of ERK, protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K), JAK2, and STAT5 in hepatocytes incubated in vitro. Activation of ERK, Akt, JAK2, and STAT5 was rapid, occurring within 5-10 min, and was concentration dependent. GH-induced ERK activation was completely blocked by the ERK pathway inhibitor, U0126, and the JAK2 inhibitor, 1,2,3,4,5,6-hexabromocyclohexane (Hex), and was partially blocked by the PI3K inhibitor LY294002. GH-stimulated Akt activation was completely blocked by LY294002 and Hex, but was not affected by U0126; whereas, STAT5 activation by GH was blocked only by Hex, and was not affected by either U0126 or LY294002. GH stimulated hepatic expression of IGF-1 mRNA as well as the secretion of IGF-1, effects that were partially or completely blocked by U0126, LY294002, and Hex. These results indicate that GHR linkage to the ERK, PI3K/Akt, or STAT pathways in trout liver cells requires activation of JAK2, and that GH-stimulated IGF-1 synthesis and secretion is mediated through the ERK, PI3K/Akt, and JAK-STAT pathways.     

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.     

Somatostatin inhibits hepatic growth hormone receptor and insulin-like growth factor I mRNA expression by activating the ERK and PI3K signaling pathways

Previously, we reported that somatostatins (SS) inhibit organismal growth by reducing hepatic growth hormone (GH) sensitivity and by inhibiting insulin-like growth factor I (IGF-I) production. In this study, we used hepatocytes isolated from rainbow trout to elucidate the mechanism(s) associated with the extrapituitary growth-inhibiting actions of SS. SS-14, a predominant SS isoform, stimulated tyrosine phosphorylation of several endogenous proteins, including extracellular signal-regulated kinase (ERK), a member the mitogen-activated protein kinase (MAPK) family, and protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K). SS-14 specifically stimulated the phosphorylation of both ERK 1/2 and Akt in a concentration-dependent fashion. This activation occurred within 5-15 min, then subsided after 1 h. The ERK inhibitor U0126 retarded SS-14-stimulated phosphorylation of ERK 1/2, whereas the PI3K inhibitor LY294002 blocked SS-14-stimulated phosphorylation of Akt. SS-14-inhibited expression of GH receptor (GHR) mRNA was blocked by U0126 but not by LY294002. By contrast, U1026 had no effect on SS-14 inhibition of GH-stimulated IGF-I mRNA expression, whereas LY294002 partially blocked the inhibition of GH-stimulated IGF-I mRNA expression by SS-14. These results indicate that SS-14-inhibited GHR expression is mediated by the ERK signaling pathway and that the PI3K/Akt pathway mediates, at least in part, SS-14 inhibition of GH-stimulated IGF-I expression.     

Growth hormone-induced differential desensitization of STAT5, ERK,and Akt phosphorylation

Secretion of growth hormone (GH) in adult male rats is characterized by high peak and undetectable trough levels, both of which are required for male-specific pattern of liver gene expression and GH-induced phosphorylation of STAT5. The present study suggests that regulation of GH receptor (GHR) levels in rat hepatoma cells by repeated GH stimulation determines GH responsiveness via the JAK2/STAT5 pathway. A short exposure to GH rapidly reduced GHR levels which resulted in an equal desensitization of the JAK2/STAT5 pathway. Recovery of GH-induced STAT5 phosphorylation correlated with the time-dependent recovery of GHR levels during incubation in the absence of GH. Acute GH also induced phosphorylation of ERK1/2 and Akt, and this induction was also inhibited by prior exposure to GH. However, unlike the JAK2/STAT5 pathway, the effect of GH to activate the MEK/ERK and phosphatidylinositol 3-kinase/Akt pathways did not recover following prolonged incubation in the absence of GH. Thus, GH administration desensitizes the JAK2/STAT5 pathway, possibly because of down-regulation of GHR, whereas an additional post-receptor mechanism is required for the prolonged refractoriness of the MEK/ERK and phosphatidylinositol 3-kinase/Akt pathways toward a second GH stimulation. Our study suggests that both receptor and post-receptor mechanisms are important in GH-induced homologous desensitization.     

An agonist-induced conformational change in the growth hormone receptor determines the choice of signalling pathway

The growth and metabolic actions of growth hormone (GH) are believed to be mediated through the GH receptor (GHR) by JAK2 activation. The GHR exists as a constitutive homodimer, with signal transduction by ligand-induced realignment of receptor subunits. Based on the crystal structures, we identify a conformational change in the F'G' loop of the lower cytokine module, which results from binding of hGH but not G120R hGH antagonist. Mutations disabling this conformational change cause impairment of ERK but not JAK2 and STAT5 activation by the GHR in FDC-P1 cells. This results from the use of two associated tyrosine kinases by the GHR, with JAK2 activating STAT5, and Lyn activating ERK1/2. We provide evidence that Lyn signals through phospholipase C gamma, leading to activation of Ras. Accordingly, mice with mutations in the JAK2 association motif respond to GH with activation of hepatic Src and ERK1/2, but not JAK2/STAT5. We suggest that F'G' loop movement alters the signalling choice between JAK2 and a Src family kinase by regulating TMD realignment. Our findings could explain debilitated ERK but not STAT5 signalling in some GH-resistant dwarfs and suggest pathway-specific cytokine agonists.     

Insulin reverses growth hormone-induced homologous desensitization

Growth hormone (GH) is secreted in a pulsatile pattern to promote body growth and metabolism. GH exerts its function by activating several signaling pathways, including JAK2/STAT and MEK/ERK. ERK1/2 activation by GH plays important roles in gene expression, cell proliferation, and growth. We previously reported that in rat H4IIE hepatoma cells after an initial GH exposure, a second GH exposure induces STAT5 phosphorylation but not ERK1/2 phosphorylation (Ji, S., Frank, S. J., and Messina, J. L. (2002) J. Biol. Chem. 277, 28384-28393). In this study the mechanisms underlying GH-induced homologous desensitization were investigated. A second GH exposure activated the signaling intermediates upstream of MEK/ERK, including JAK2, Ras, and Raf-1. This correlated with recovery of GH receptor levels, but was insufficient for GH-induced phosphorylation of MEK1/2 and ERK1/2. Insulin restored the ability of a second GH exposure to induce phosphorylation of MEK1/2 and ERK1/2 without altering GH receptor levels or GH-induced phosphorylation/activation of JAK2 and Raf-1. GH and insulin synergized in promoting cell proliferation. Further investigation suggested that insulin increased the amount of MEK bound to KSR (kinase suppressor of Ras) and restored GH-induced tyrosine phosphorylation of KSR. Previous GH exposure also induced desensitization of STAT1 and STAT3 phosphorylation, but this desensitization was not reversed by insulin. Thus, insulin-regulated resensitization of GH signaling may be necessary to reset the complete response to GH after a normal, physiologic pulse of GH.     

Protein tyrosine phosphatase 1B attenuates growth hormone-mediated JAK2-STAT signaling

Protein tyrosine phosphatase-1B (PTP-1B) attenuates insulin, PDGF, EGF, and IGF-I signaling by dephosphorylating tyrosine residues located in the tyrosine kinase domain of the corresponding receptors. More recently, PTP-1B was shown to modulate the action of cytokine signaling via the nonreceptor tyrosine kinase JAK2. Transmission of the growth hormone (GH) signal also depends on JAK2, raising the possibility that PTP-1B modulates GH action. Consistent with this hypothesis, GH increased the abundance of tyrosine-phosphorylated JAK2 associated with a catalytically inactive mutant of PTP-1B. GH-induced JAK2 phosphorylation was greater in knockout (KO) than in wild-type (WT) PTP-1B embryonic fibroblasts and resulted in increased tyrosine phosphorylation of STAT3 and STAT5, while overexpression of PTP-1B reduced the GH-mediated activation of the acid-labile subunit gene. To evaluate the in vivo relevance of these observations, mice were injected with GH under fed and fasted conditions. As expected, tyrosine phosphorylation of JAK2 and STAT5 occurred readily in the livers of fed WT mice and was almost completely abolished during fasting. In contrast, resistance to the action of GH was severely impaired in the livers of fasted KO mice. These results indicate that PTP-1B regulates GH signaling by reducing the extent of JAK2 phosphorylation and suggest that PTP-1B is essential for limiting the action of GH during metabolic stress such as fasting.     

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.     

Insulin enhances growth hormone induction of the MEK/ERK signaling pathway

Growth hormone (GH) plays an important role in growth and metabolism by signaling via at least three major pathways, including STATs, ERK1/2, and phosphatidylinositol 3-kinase/Akt. Physiological concentrations of insulin promote growth probably by modulating liver GH receptor (GHR) levels in vivo, but the possible effects of insulin on GH-induced post-GHR signaling have yet to be studied. We hypothesized that short-term insulin, similar to the fluctuations that occur following feeding, affects GH-induced post-GHR signaling. Our present studies suggest that, in rat H4IIE hepatoma cells, insulin (4 h or less) selectively enhanced GH-induced phosphorylation of MEK1/2 and ERK1/2, but not GH-induced activation of STAT5 and Akt. Although insulin pretreatment altered GH-induced formation of Shc.Grb2.SOS complex, it did not significantly affect GH-induced activation of other signaling intermediates upstream of MEK/ERK, including JAK2, Ras, and Raf-1. Immunofluorescent staining indicated that insulin pretreatment facilitated GH-induced cell membrane translocation of MEK1/2. Insulin pretreatment also increased the amount of MEK association with its scaffolding protein, KSR. In summary, short-term insulin treatment of cultured, liver-derived cells selectively sensitized GH-induced MEK/ERK phosphorylation independent of JAK2, Ras, and Raf-1, but likely resulted from increased cell membrane translocation of MEK1/2. These findings suggest that insulin may be necessary for sensitization of cells to GH-induced ERK1/2 activation and provides a potential cellular mechanism by which insulin promotes growth.     

Disulfide linkage of growth hormone (GH) receptors (GHR) reflects GH-induced GHR dimerization. Association of JAK2 with the GHR is enhanced by receptor dimerization.

The growth hormone (GH) receptor (GHR) binds GH in its extracellular domain and transduces activating signals via its cytoplasmic domain. Both GH-induced GHR dimerization and JAK2 tyrosine kinase activation are critical in initiation of GH signaling. We previously described a rapid GH-induced disulfide linkage of GHRs in human IM-9 cells. In this study, three GH-induced phenomena (GHR dimerization, GHR disulfide linkage, and enhanced GHR-JAK2 association) were examined biochemically and immunologically. By using the GH antagonist, G120K, and an antibody recognizing a dimerization-sensitive GHR epitope, we demonstrated that GH-induced GHR disulfide linkage reflects GH-induced GHR dimerization. GH, not G120K, promoted both GHR disulfide linkage and enhanced association with JAK2. Measures that diminished GH-dependent JAK2 and GHR tyrosine phosphorylation diminished neither GH-induced GHR disulfide linkage nor GH-enhanced GHR-JAK2 association. By using both transient and stable expression systems, we determined that cysteine 241 (an unpaired extracellular cysteine) was critical for GH-induced GHR disulfide linkage; however, GH-induced GHR dimerization, GHR-JAK2 interaction, and GHR, JAK2, and STAT5 tyrosine phosphorylation still proceeded when this cysteine residue was mutated. We conclude GH-induced GHR disulfide linkage is not required for GHR dimerization, and activation and GH-enhanced GHR-JAK2 association depends more on GHR dimerization than on GHR and/or JAK2 tyrosine phosphorylation.