The amelioration and reconstitution of the excessive growth phenotype in SOCS2–/–
mice in response to GH provides compelling evidence that SOCS2 plays an important role in the negative regulation of GH signaling. The exact site of SOCS2 action in the somatotrophic axis has been controversial, as SOCS2 has been shown to bind to the IGF-1 receptor in yeast 2-hybrid studies (23
), and the phenotype of SOCS2
knockout mice has some similarities to that of IGF-1 transgenic mice (19
). However, there are no data to indicate that IGF-1 induces SOCS2 mRNA expression in vitro, and IGF-1 signaling is not perturbed in SOCS2-
deficient embryonic fibroblasts (27
). The hypothesis that SOCS2 is a negative regulator of GH signaling is supported by data presented here and published results showing the following: SOCS2-GH receptor interactions in vitro and in vivo (28
); the role of STAT5b in the development of the SOCS2–/–
phenotype; modestly prolonged GH signaling in SOCS2
-deficient hepatocytes (27
); and increases in size of non–IGF-1 responsive tissues such as the liver (17
). Furthermore, detection of significant differences in the liver gene expression profiles in the SOCS2+/+Ghrhrlit/lit
mice 2 hours after GH injection almost certainly precludes the role of other growth factors in mediating these differences. We do not believe that there is sufficient time for GH signaling to induce another growth factor to then act upon its own signaling system to induce SOCS2 mRNA expression, and then for the subsequent protein to exert an effect such that it could be detected in mRNA.
The hyperresponsiveness of these genes to GH in hepatic tissue, as well as the identification of new GH-regulated genes in the double knockout, further emphasize that SOCS2 is an important regulator of GH signaling and a therapeutic target for the enhancement of the growth-promoting effects of GH. The magnitude of organ and tissue growth in SOCS2-deficient mice in response to GH was significant, but size differences were also tissue dependent. Tissue-specific sensitivities to GH, the level and timing of SOCS2 expression in that tissue, gender, and sexual dimorphisms all appeared to be modifying factors in controlling target sensitivity to GH. This is emphasized in the pregnancy-driven enhancement of body size in SOCS2–/–Ghrhrlit/lit females. Although the mechanism behind this phenomenon is not completely clear, it is plausible that pregnant female mice are exposed to placentally derived GH or prolactin, to which SOCS2–/–Ghrhrlit/lit mice are hyperresponsive.
The unique nature of SOCS2 action is further emphasized in the microarray studies. SOCS2–/–Ghrhrlit/lit
mice clearly have an enhanced GH-induced liver gene expression response compared to SOCS2+/+Ghrhrlit/lit
mice, although the magnitude and type of response do not simply fit a model of a completely deregulated GH receptor signaling action. IGF-1 is a classic GH-induced gene of the liver, and it has been shown to be a contributor to somatic growth (42
). The overexpression of IGF-1 in SOCS2–/–Ghrhrlit/lit
mice may contribute to their increased growth response when administered GH. The appearance of other GH-regulated genes such as PPAR
α and Spi2.2
fits the proposed model, but the alterations of other genes that were previously thought to be unresponsive to GH (e.g., PIM-3
) indicate that SOCS2 function is more complicated than first thought. Similar observations have been made in other SOCS knockout models where IL-6 stimulation of SOCS3-deficient livers led to exaggerated gene expression of IL-6–responsive genes, as compared to wild-type expression, but it also let to the induction of other genes thought to be classically IFN-γ–inducible genes (12
). The magnitude of change in GH-induced expression with a lack of SOCS2 was not as large as might be predicted from that observed in SOCS3
-deficient livers stimulated with IL-6 (14
), but these small changes in signaling and expression may be large when considering that they would be cumulative over an animal’s somatic growth period.
Our results demonstrate that all 3 domains of SOCS2 may play a role in protein function. The SH2 domain of a number of SOCS proteins plays a central role in their function and SOCS2 is no different. Whereas mutating the conserved arginine in the SOCS1 SH2 domain was sufficient to eliminate the inhibitory activity of SOCS1 (43
), SOCS2 required additional mutations to become inactive. The reasons for this are unclear, but it may reflect redundancy in SOCS2 generated by its ability to bind a number of different residues on the GH receptor. The SOCS box of SOCS2 plays a central role in the negative regulation of GH signaling. This is intriguing, as removal of the SOCS box from other SOCS molecules does not impede inhibitory function in overexpression studies, although the SOCS box of SOCS1 has been shown to play an important role physiologically (44
). Consequently, this may reflect a difference in the mechanism of SOCS2 action, with a strong reliance on ubiquitination and degradation to terminate GH signaling, rather than on inhibition of receptor function. Consistent with this interpretation, SOCS1 and SOCS3 contain a kinase inhibitory region that is absent in SOCS2 (45
We have shown here that it is necessary to delete both the Y487 and Y595 residues to remove the inhibitory effects of SOCS2 on GH signaling. Mutations of these 2 sites have previously been shown to result in the prolongation of the GH-activated JAK/STAT5 pathway. SHP2 binds these residues and is thought to act as the negative regulator of the receptor (39
). However, when the function of SHP2 is directly tested in overexpression systems, it acts as a positive regulator of GH signaling (46
). In retrospect, the data supporting a role for SHP2 as a negative regulator generated using GH receptor tyrosine mutations could now be reinterpreted as SOCS2 mediating the inhibition directly or acting to inhibit SHP2 action (39
). This model has a number of similarities to that for SOCS3 and SHP2 binding to the gp130 receptor (47
). The observations of significant increases in luciferase output of the GH mutant receptors when stimulated with hormone, as well as increased basal levels without stimulation, are also seen in similar studies in which the SOCS3 binding site of gp130 was mutated (48
). A possible explanation for this phenomenon is that basal receptor activity also utilizes low levels of SOCS2 for negative control, and mutation of SOCS2 binding residues causes a proportional increase in luciferase output compared to the stimulated levels. Presuming that SOCS2 protein levels are low in unstimulated cells, it is possible that other negative regulators can interact with the receptor through these tyrosines. Another candidate is PTP1B, a tyrosine phosphatase known to interact with the GH receptor and has been confirmed to have negative actions on GH signaling (50
). A negative action of SHP2 on certain tissues cannot be excluded, and the fact that SOCS2 and SHP2 both bind the same sites on the GH receptor sets the scene for potential interplay between these molecules. It is anticipated that further characterization of these phosphatases and SOCS2 interactions in the negative regulation of the GH receptor will decipher the nature of interplay that may exist between these molecules and provide further insights into the exact mechanism by which SOCS2 inhibits GH signaling.
Converting the information gathered in this manuscript into a clinical application is the next step. The α-screen assay technology used in this work forms the basis of a high-throughput screen, and we are currently using this approach to screen chemical compound libraries for small-molecule inhibitors of SOCS2-GH receptor interactions. An alternate approach would be to downregulate SOCS2 mRNA expression by using antisense or siRNA approaches, which are becoming more prevalent in strategies to target intracellular molecules. It is hoped that such approaches may be useful in the development of new strategies targeting growth disorders by replacing or amplifying the effects of exogenous GH administration.