IGF-1R Modulation of Acute GH-Induced STAT5 Signaling: Role of Protein Tyrosine Phosphatase Activity

Abstract

GH is a potent anabolic and metabolic factor that binds its cell surface receptor (GHR), activating the GHR-associated tyrosine kinase, Janus kinase 2, which phosphorylates and activates the latent transcription factor, signal transducer and activator of transcription 5 (STAT5). Some GH actions are mediated by the elaboration of IGF-1, which exerts effects by binding and activating the heterotetrameric tyrosine kinase growth factor receptor, IGF-1R. In addition to this GH-GHR-IGF-1-IGF-1R scheme, we have demonstrated in primary osteoblasts and in islet β-cells that then deletion or silencing of IGF-1R results in diminished GH-induced STAT5 phosphorylation, suggesting that the presence of IGF-1R may facilitate GH signaling. In this study, we explore potential roles for protein tyrosine phosphatase activity in modulating GH-induced signaling, comparing conditions in which IGF-1R is present or diminished. We confirm that in mouse primary osteoblasts harboring loxP sites flanking the IGF-1R gene, infection with an adenovirus that expresses the Cre recombinase results in IGF-1R deletion and diminished acute GH-induced STAT5 phosphorylation. Furthermore, we present a new model of IGF-1R silencing, in which expression of short hairpin RNA directed at IGF-1R greatly reduces IGF-1R abundance in LNCaP human prostate cancer cells. In both models, treatment with a chemical inhibitor of protein tyrosine phosphatase-1B (PTP-1B), but not one of src homology region 2 domain-containing phosphotase-1 (SHP-1) and SHP-2, reverses the loss of GH-induced STAT5 phosphorylation in cells lacking IGF-1R but has no effect in cells with intact IGF-1R. Furthermore, expression of either a dominant-negative PTP-1B or the PTP-1B-interacting inhibitory protein, constitutive photomorphogenesis 1, also rescues acute GH-induced STAT5 signaling in IGF-1R-deficient cells but has no effect in IGF-1R replete cells. By expressing a substrate-trapping mutant PTP-1B, we demonstrate that tyrosine phosphorylated Janus kinase-2 is a PTP-1B substrate only in cells lacking IGF-1R. Collectively, our data suggest that IGF-1R positively regulates acute GH signaling by preventing access of PTP-1B activity to Janus kinase 2 and thereby preventing PTP-1B-mediated suppression of GH-induced STAT5 activation.

Human GH Receptor-IGF-1 Receptor Interaction: Implications for GH Signaling

“…Osteoblasts were isolated from calvaria of newborn Igf1r flox/flox mice and cultured as described previously (31,33,34,42). LNCaP-vec and LNCaP-shIGF-1R cells were previously described and characterized, as were the conditions of cell culture (33).…”

“…LNCaP-vec and LNCaP-shIGF-1R cells were previously described and characterized, as were the conditions of cell culture (33). ␥2A-JAK2-GHR-Nluc cells were generated by the cotransfection of ␥2A-JAK2 cells (16,43) with pcDNA3.1(ϩ)/zeo-GHRNluc and a hygromycin-encoding plasmid at a weight ratio as 20:1 and followed by hygromycin selection and single-clone amplification, as described (44).…”

“…As previously described (33,34), serum starvation of LNCaP-vec and LNCaP-shIGF-1R cells and primary osteoblasts was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V; Roche Molecular Biochemicals) for serum in their respective culture media for 16 -20 hours prior to the experiments. Stimulations were performed at 37°C.…”

“…In this manuscript, we use the LNCaP human prostate cancer cell as a GH-responsive system to further assess the role of IGF-1R in human GH signaling (33,35,36). We demonstrate by coimmunoprecipitation that, as in murine cells, GH causes GHR-JAK2-IGF-1R complex formation in LNCaP cells and also further explore this GHR-IGF-1R interaction in human cells using a novel split luciferase complementation assay.…”

Minireview: Mechanisms of Growth Hormone-Mediated Gene Regulation

“…Direct cross talk of GH and IGF-1 downstream signaling has also been investigated. A complex of GHR-JAK2-IGF-1R has been described in several cell types (100 -102), and further studies have indicated that IGF-1R perpetuates GH signaling by preventing PTP1b-mediated suppression of Stat5 activation (103). Starvation and malnutrition lead to a state of GH resistance that can be considered an adaptation of fasting (104).…”