Regulation of Glucose Transport and c-fos and egr-1 Expression in Cells with Mutated or Endogenous Growth Hormone Receptors*

Abstract

To identify mechanisms by which GH receptors (GHR) mediate downstream events representative of growth and metabolic responses to GH, stimulation by GH of c-fos and egr-1 expression and glucose transport activity were examined in Chinese hamster ovary (CHO) cells expressing mutated GHR. In CHO cells expressing wild-type GHR(GHR(1-638)), GH stimulated the expression of c-fos and egr-1, and stimulated 2-deoxyglucose uptake, responses also mediated by endogenous GHR in 3T3-F442A cells. Deletion of the proline-rich box 1 of GHR (GHR(deltaP)) abrogated all of these responses to GH, indicating that box 1, a site of association of GHR with the tyrosine kinase JAK2, is crucial for these GH-stimulated responses. As the C-terminal half of the cytoplasmic domain of GHR is required for GH-stimulated calcium flux and for stimulation of spi-2.1 transcription, GHR lacking this sequence (GHR(1-454)) were examined. Not only did GHR(1-454) mediate stimulation of c-fos and egr-1 expression and 2-deoxyglucose uptake, but they also mediated GH-stimulated transcriptional activation via Elk-1, a transcription factor associated with the c-fos Serum Response Element. Thus, the C-terminal half of the cytoplasmic domain of GHR is not required for GH-stimulated c-fos transcription, suggesting that increased calcium is not required for GH-stimulated c-fos expression. In CHO cells lacking all but five N-terminal residues of the cytoplasmic domain (GHR(1-294)), GH did not induce c-fos or egr-1 expression or stimulate 2-deoxyglucose uptake. Further, in 3T3-F442A fibroblasts with endogenous GHR, GH-stimulated c-fos expression and 2-deoxyglucose uptake were reduced by the tyrosine kinase inhibitors herbimycin A, staurosporine, and P11. Herbimycin A and staurosporine inhibit JAK2 and tyrosyl phosphorylation of all proteins stimulated by GH, whereas P11 inhibits the GH-dependent tyrosyl phosphorylation of only some proteins, including extracellular signal regulated kinases ERK1 and -2, but not JAK2. Taken together, these results implicate association of GHR with JAK2 and GH-stimulated tyrosyl phosphorylation of an additional cellular protein in GH-stimulated glucose transport and c-fos and egr-1 expression. These studies also indicate that, in contrast to spi-2.1, the N-terminal half of the cytoplasmic domain of GHR is sufficient to mediate stimulation of c-fos and egr-1 expression and Elk-1 activation, supporting multiple mechanisms for GH signaling to the nucleus.

Endogenous CCAAT/Enhancer Binding Protein β and p300 Are Both Regulated by Growth Hormone to Mediate Transcriptional Activation

“…Quantitative real-time PCR (QT-PCR) confirms that c-fos mRNA expression peaks 30 min after GH treatment in 3T3-F442A and CHO-GHR cells, then subsides within 1 h and is almost undetectable at later times (Fig. 1A), consistent with previous Northern analysis (17,37,38). The pattern of Fig.…”

“…The transcription of c-fos is stimulated by GH rapidly and transiently in various cell types, including 3T3-F442A preadipocytes (17,18,37) and Chinese hamster ovary (CHO) cell lines stably expressing GH receptors (CHO-GHR) (38). Quantitative real-time PCR (QT-PCR) confirms that c-fos mRNA expression peaks 30 min after GH treatment in 3T3-F442A and CHO-GHR cells, then subsides within 1 h and is almost undetectable at later times (Fig.…”

Growth Enhancement in Suppressor of Cytokine Signaling 2 (SOCS-2)-Deficient Mice Is Dependent on Signal Transducer and Activator of Transcription 5b (STAT5b)

“…3, A and B). The responsiveness of the cells to IGF-I and GH was confirmed by blotting for early growth response factor-I (egr-1) that is induced by both of these growth factors (33,34). SOCS-2 Ϫ/Ϫ or wild-type hepatocytes were starved for 12 h and then stimulated for various periods with 50 ng/ml GH.…”

Reduced cardiotropic response to insulin in spontaneously hypertensive rats: role of peroxisome proliferator-activated receptor-γ-initiated signaling

“…Glucose transport assay An additional experiment was performed to measure myocardial glucose transport by [ 14 C]2-deoxyglucose method, as described previously [18]. In brief, isolated cardiomyocytes from WKY rats and SHR with or without treatment with RSG were washed twice with KrebsRinger phosphate buffer (KRP), followed by 30-min incubation with or without insulin (10 4 pmol/l) in KRP containing 1% bovine serum albumin (BSA).…”