We have previously described two families (H and M) with
GH binding protein-positive
Laron Syndrome (LS), proposed to have one or more post GHR signaling defects. In the present study, we have examined whether the signal transducers and activators of transcription (STAT) pathway is activated by GH in skin fibroblast cultures established from these LS children, to determine the level(s) at which GH insensitivity has occurred. On immunoblots, both normal and LS fibroblasts express JAK2 and STATs 1, 3, and 5. GH induced rapid
tyrosine phosphorylation of a
protein at approximately 93 kDa in normal fibroblasts, and Western blotting with STAT-specific
antibodies revealed STAT5 activation (phosphorylation) by GH. To determine further the identity and the
DNA binding characteristics of the STAT
proteins that were activated by GH, EMSAs were performed using three
DNA elements known to bind STAT
proteins; m67, the high affinity c-sis-inducible
element (SIE), the
interferon response element (IRE), and the lactogenic
hormone-responsive region (LHRR). GH failed to induce protein binding to the SIE or IRE in normal skin fibroblasts but did induce the formation of a specific complex with the LHRR. Induction by GH of this LHRR/
protein complex, which could be supershifted partially by anti-STAT1
antisera and completely by anti-STAT5
antisera, was transient, maximal between 10 and 30 min and reduced by 60 min. GH also induced distinct LHRR/
protein complexes in mouse 3T3-F442A fibroblasts and in human IM-9 lymphocytes, but supershift analysis revealed that these complexes contained STAT5 but not STAT1. Whereas no binding to the LHRR was observed in GH-treated H fibroblasts, GH induced binding to this
element in M fibroblasts. These results demonstrate that 1) the JAK-STAT pathway is activated by GH in normal fibroblasts and that STATs 1 and 5 have a role in GH-dependent signaling in these cells; 2) GH activation of
DNA/STAT binding is cell type- and species-specific; and 3) GH failed to activate the STAT pathway in H fibroblasts but induced STAT signaling in M fibroblasts, indicating that the site of GH resistance in the latter is likely to be located within another GH signaling pathway. These fibroblast cultures therefore provide unique models with which to further our understanding of the mechanisms of human GH signaling.