SOCS2 negatively regulates growth hormone action in vitro and in vivo

Abstract

Mice deficient in SOCS2 display an excessive growth phenotype characterized by a 30-50% increase in mature body size. Here we show that the SOCS2-/- phenotype is dependent upon the presence of endogenous growth hormone (GH) and that treatment with exogenous GH induced excessive growth in mice lacking both endogenous GH and SOCS2. This was reflected in terms of overall body weight, body and bone lengths, and the weight of internal organs and tissues. A heightened response to GH was also measured by examining GH-responsive genes expressed in the liver after exogenous GH administration. To further understand the link between SOCS2 and the GH-signaling cascade, we investigated the nature of these interactions using structure/function and biochemical interaction studies. Analysis of the 3 structural motifs of the SOCS2 molecule revealed that each plays a crucial role in SOCS2 function, with the conserved SOCS-box motif being essential for all inhibitory function. SOCS2 was found to bind 2 phosphorylated tyrosines on the GH receptor, and mutational analysis of these amino acids showed that both were essential for SOCS2 function. Together, the data provide clear evidence that SOCS2 is a negative regulator of GH signaling.

Figure 1

GH is essential for the SOCS2–/– phenotype. Growth curves for SOCS2+/+Ghrhrlit/lit, SOCS2–/–Ghrhrlit/lit, SOCS2+/+GhrhrWT/WT and SOCS2–/–GhrhrWT/WT mice of both sexes over a 12-week period. At least 10–15 male mice were used at each point for the male growth curves and 6–17 female mice are represented at each time point for the female curves.

Figure 2

SOCS2 controls growth responses to GH. (A) Male and female SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mice were weighed daily before being injected twice daily with 10 μg rpGH or saline for 28 days from 4 weeks of age. Growth curves were constructed from 5–10 male mice or 5–7 female mice per treatment group. (B) Differences in organ weights of male and female SOCS2+/+Ghrhrlit/lit (white bars) and SOCS2–/–Ghrhrlit/lit (black bars) mice are represented as a percentage increase over the mean of saline-injected mouse organ weights. *P < 0.05 vs. saline-injected mice; #P < 0.05 vs. SOCS2+/+Ghrhrlit/lit mice. Sal gland, salivary gland. (C) Picture of 6-month-old SOCS2–/–Ghrhrlit/lit mice that have had 3 pregnancies (upper) or no pregnancies (lower).

Figure 3

SOCS2 controls bone growth parameters. (A) Long bone lengths and femur morphometric data (B) were measured from 4–9 male mice of both genotypes. Data for each parameter is expressed as the percentage change with GH treatment compared to saline controls for SOCS2+/+Ghrhrlit/lit (white bars) and SOCS2–/–Ghrhrlit/lit (black bars) mice. *P < 0.05, vs. saline-treated mice; #P < 0.05 vs. SOCS2+/+Ghrhrlit/lit mice. Trabec. BMD, trabecular bone mineral density; circum., circumference.

Figure 4

SOCS2 regulates GH-induced gene expression in the liver. (A) The number of GH-regulated genes in the liver of SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mice 2 hours after GH injection was compared based on SAM analysis (5% FDR) for 4 independently replicated treatments. Individual genes are arranged along the x axis according to the value order of decreases and increases in gene expression of SOCS2–/–Ghrhrlit/lit mice. The y axis shows the log ratio of the transcript signals in GH-treated SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mice (B), and the average changes in gene expression in SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit was also examined (C). Real-time RT-PCR results from SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mouse livers showing genes that were downregulated (D) or upregulated (E). *P < 0.05.

Figure 5

SOCS2 motif control of GH signaling. (A) A schematic diagram of SOCS2 is provided to help clarify mutant constructs used and residues mutated in the SH2 domain. (B–D) 293T cells were transfected with pig GH receptor and SOCS2 (SOCS2 WT), or with the following SOCS2 mutant constructs: (B) SOCS2 with a point mutation in the SH2 domain R73K (SOCS2 D) or SOCS2 with a triple mutation at R73K, D74E, and S75C in the SH2 domain (SOCS2 KD); (C) SOCS2 lacking the 37-AA N terminus (SOCS2°NT) or SOCS2 with the N-terminal region of SOCS1 (SOCS1/2/2); or (D) SOCS2 lacking the 39-AA C terminus (SOCS2°SB) at a range of plasmid concentrations (ng). The transfected cells were then stimulated with rpGH and the luciferase activity from an LHRE-luciferase reporter was measured. Data is corrected for transfection efficiency by cotransfection of a β-galactosidase–expressing plasmid. Luciferase activity was corrected using values obtained in the absence of GH, then expressed as a percentage of wild-type activity, which was assigned a value of 100%. Experiments were performed in triplicate, and data presented here are representative of 3 independent experiments. (E) Flag-tagged SOCS2 and empty vector were transfected into 293T cells, lysed, and immunoprecipitated using antibodies against Flag. After separation on SDS-PAGE and Western transfer, blots were probed with antibodies against Elongins B and C, then stripped and reprobed with antibodies against the Flag epitope.

Figure 6

SOCS2 interacts with tyrosine residues on the GH receptor. Biosensor analysis was performed on the binding between GH receptor–derived phosphopeptides and NusA.SOCS2 SH2 protein (A) or SHP2 protein (B). Sensorgrams correspond to binding of immobilized peptide on a streptavidin sensorchip.

Figure 7

SOCS2 effects are mediated through Tyr487 and Tyr595. (A) 293T cells were transfected with wild-type or mutated GH receptor constructs, then they were starved of (white bars) or stimulated with rpGH (black bars), and the luciferase activity from an LHRE-luciferase reporter was measured. Data were corrected for transfection efficiency by cotransfection of a β-galactosidase–expressing plasmid. *P < 0.05, significant difference between stimulated receptors. (B–D) 293T cells were transfected with wild-type GH receptor, (B) Y595F GH receptor, (C) Y487F GH receptor, or (D) Y487,595F GH receptor and increasing concentrations of SOCS2 plasmid (ng). Data were corrected for transfection efficiency by cotransfection of a β-galactosidase–expressing plasmid. Luciferase activity was corrected using values obtained in the absence of GH, then expressed as a percentage of wild-type GH receptor activity without SOCS2 expression, which was assigned a value of 100%. Experiments were performed in triplicate and data presented here are representative of 3 independent experiments. Expression of SOCS2 was confirmed by Western blotting of cell lysate with antibodies against the Flag epitope at the N terminus of the SOCS2 expression construct.