Abstract
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.
Highlights
We previously reported that 1) a short exposure to Growth hormone (GH) rapidly reduced GH to its receptor (GHR) levels, which resulted in desensitization of STAT5 and ERK1/2 phosphorylation, 2) in the absence of GH for 3– 6 h, GHR levels and GH-induced tyrosine phosphorylation of STAT5 (Tyr(P)-STAT5) recovered to 65–75% that in cells not pretreated with GH, and 3) unlike STAT5 phosphorylation, the ability of GH to activate the MEK/ERK pathway did not recover even after prolonged (16 h) incubation in the absence of GH (25)
When H4IIE cells were pretreated with GH for 1 h followed by 5 h in GH-free, serum-free medium, the second GH-induced Tyr(P)-JAK2 was maximally 70 –75% that induced by GH before any pretreatment (Fig. 1, A and B), correlating well with the recovery of GHR levels that we previously reported (25)
Phosphorylation of MEK1/2 and ERK1/2 Induced by a Second GH Treatment Was Not Correlated with Activation of the JAK2/ Ras/Raf-1 Pathway—When H4IIE cells were pretreated with GH for 1 h followed by 5 h in GH-free, serum-free media, the GH-induced phosphorylation of MEK1/2 (Fig. 4, A and B) and ERK1/2 (Fig. 4, C and D) was greatly diminished compared with that induced by GH before any pretreatment
Summary
Prior exposure to insulin restored the ability of a second GH treatment to induce MEK/ERK phosphorylation without changing GHR levels or GH-induced activation of JAK2 and Raf-1. To investigate the mechanisms by which GH induced desensitization of the ERK1/2 phosphorylation, H4IIE cells were pretreated with GH for 1 h, incubated in GH-free, serum-free medium for 5 h followed by a second GH treatment.
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