Autocrine growth hormone production prevents apoptosis and inhibits differentiation in C2C12 myoblasts.

Although C2C12 myoblasts express low levels of growth hormone receptor (GHR), we failed to see any effect of exogenous growth hormone (GH) on cell proliferation or differentiation. C2C12 cells stably overexpressing (sixfold) more in GHR (C2C12(GHR)) grew faster than parental cells in media containing 2% serum, and proliferated while parental cells died, in the absence of serum. These effects were independent of exogenous GH but were inhibited by anti-GH and anti-insulin-like growth factor (anti-IGF-1) antibodies, consistent with a local production of GH, which we confirmed by RT-PCR and radioimmunoassay. In C2C12(GHR) cells, we observed an increased activation of the Janus kinase 2 (Jak2), signal transducers and activator of transcription 5 (Stat5), mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) upon acute GH stimulation. GHR overexpression also inhibited the formation of myotubes and the expression of markers for myoblast differentiation. Taken together, our data suggest that GH acts as an autocrine factor in C2C12 cells, to enhance proliferation and to inhibit differentiation.

Jak2 and proteasome activities control the availability of cell surface growth hormone receptors during ligand exposure.

Several mechanisms participate in the down-regulation of growth hormone receptor (GHR) signalling under ligand exposure. In CHO cells expressing GHR, we show that ligand stimulation induces degradation of the total cell GHR content. Experiments with 125I-hGH indicate that ligand-bound internalized receptors are not immediately replaced. Using cell surface biotinylation, we demonstrate for the first time that, concomitantly with the degradation of cell surface receptors, GHRs from the intracellular compartments are also degraded. We thus suggest that under prolonged ligand exposure, some GHRs are targeted to the cell surface, while others are routed to degradation compartments. Inhibitors of Jak2 and of the proteasome partially inhibited degradation of cell surface receptors, while these compounds completely inhibit the degradation of intracellular GHRs, resulting in their accumulation. We therefore propose that Jak2 and proteasome activities control the amount of intracellular GHRs, and thus the availability of receptors at the cell surface, during ligand exposure.