ERK, Akt, and STAT5 are Differentially Activated by the Two Growth Hormone Receptor Subtypes of a Teleost Fish (Oncorhynchus Mykiss)

Abstract

Previously, we found that the teleost fish, rainbow trout, possesses two growth hormone receptor (GHR) subtypes that display distinct ligand-binding and agonist-induced regulation features. In this study, we used Chinese hamster ovary-K1 cells stably transfected individually with the two trout GHR subtypes, GHR1 and GHR2, to elucidate receptor-effector pathway linkages. Growth hormone (GH) stimulated rapid (5-10 min) phosphorylation of ERK, Akt, JAk2, and STAT5 in both GHR1- and GHR2-expressing cells; however; STAT5 was activated to a greater extent through GHR1 than through GHR2, whereas ERK and Akt were activated to a greater through GHR2 than through GHR1. Although blockade of the ERK pathway had no effect on the activation of Akt, inhibition of PI3K-Akt partially prevented activation of ERK, suggesting cross-talk between the ERK and PI3K-Akt pathways. JAK2 inhibition completely blocked activation of ERK, Akt, and STAT5, suggesting that all of these pathways link to GHR1 and GHR2 via JAK2. These findings establish important receptor-effector pathway linkages and suggest that the GHR subtypes of teleost fish may be functionally distinct.

Keywords: LY294002; U0126; growth hormone; signal transduction.

Figure 1

Growth hormone (GH) activates multiple signal pathways via rainbow trout GH receptors (GHR) transfected in Chinese hamster ovary (CHO)-K1 cells. CHO-K1 cells were stably transfected with cDNA encoding rainbow trout GHR1 and GHR2. Cells were incubated with 100 ng/ml salmonid GH for various times; after which, the cells were collected and lysed. Lysates were separated by SDS-PAGE followed by Western immunoblotting using phospho-specific (pERK 1/2, pAkt, pJAK2, pSTAT5) and total (tERK 1/2; tAkt, tJAK2, tSTAT5) antibodies.

Figure 2

Growth hormone receptors (GHR) subtypes differentially activate (A) ERK, (B) Akt, and (C) STAT5. CHO-K1 cells were stably transfected with cDNA encoding rainbow trout GHR1 and GHR2. Cells were incubated with various concentrations of salmonid GH for 10 min (control is 0 ng/ml); after which time, the cells were collected and lysed. Lysates were separated by SDS-PAGE followed by Western immunoblotting using phospho-specific (pERK 1/2, pAkt, pJAK2, pSTAT5) and total (tERK 1/2; tAkt, tJAK2, tSTAT5) antibodies. Data are expressed as percentage of control and are presented as means ± SEM (n = 8) of blots quantified with a digital imaging system; * designates groups that are significantly different from each other (P < 0.05).

Figure 3

Effects of pathway blockade on growth hormone-induced activation of (A) ERK, (B) Akt, and (C) STAT5. CHO-K1 cells were stably transfected with cDNA encoding rainbow trout GHR1 and GHR2. Transfected cells were pretreated with or without specific inhibitors [10 μM of the MEK inhibitor, U0126; 20 μM of the PI3K inhibitor, LY294002 (LY); 25 μM of the Akt inhibitor, 1L6-hydroxyymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate (Carb); 200 μM of the STAT5 inhibitor, N′-((4-oxo-4H-chroen-3-yl)methylene)nicotinohydrazide (Nico); and 50 μM of the JAK2 inhibitor, 1,2,3,4,5,6-hexabromocyclohexane (Hex)] for 2 h, then treated with or without 100 ng/ml growth hormone (GH) for 10 min (control is 0 ng/ml GH); after which time, the cells were collected and lysed. Cell lysates were separated by SDS-PAGE followed by Western immunoblotting using phospho-specific (pERK1/2, pAkt, or pSTAT) and total (tERK1/2, tAkt, or tSTAT) antibodies. Data are expressed as percentage of control and are presented as means ± SEM (n = 8). For a given GHR subtype, groups with different letters are significantly different from each other (P < 0.05); * designates a significant difference (P < 0.05) between subtypes within a given treatment.

Figure 4

Model of differential activation of signaling pathways by growth hormone receptor (GHR) subtypes. JAK2 activation is essential for propagation of signaling from both GHR1 and GHR2 to the ERK, PI3K/Akt, and STAT5 pathways (details of pathway elements are omitted for simplicity). Cross-talk occurs between the ERK and PI3K/Akt pathways, possibly through Akt activation of c-Raf in the ERK pathway.