TIMP3 Modulates GH Sensitivity through Interaction with TACE

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 88-108-GHRH, GH & IGF Biology & Signaling
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-98
Yue Zhang*1, Larry A. May1, Philip Alton Berry1, Xiangdong Wang2, Jing Jiang1 and Stuart Jonathan Frank1
1University of Alabama at Birmingham, Birmingham, AL, 2Institute of Cell Biology, Shandong University School of Medicine, Jinan, China
Growth hormone receptor (GHR) binds GH at the cell surface via its extracellular domain (ECD) and initiates intracellular signaling for anabolic and metabolic actions. We previously demonstrated that metalloproteolytic cleavage of GHR in the perimembraneous ECD stem region is catalyzed by TNF-alpha converting enzyme (TACE/ADAM17), can be induced in vitro by the phorbol ester, PMA, and renders the cell less sensitive to subsequent GH treatment (1,2). Our work in mice suggests that endotoxin also elicits this proteolytic GHR downregulation and desensitization to hepatic GH action (3). Tissue inhibitor of metalloproteinases 3 (TIMP3) is an endogenous specific TACE inhibitor. In C14 human fibrosarcoma cells that stably express GHR and JAK2, PMA induces robust proteolytic GHR loss. In contrast, in HEK-293-GHR-JAK2 cells that stably express GHR and JAK2, PMA induces minimal GHR proteolysis. To probe reasons for this disparity, we compared endogenous TACE levels in these two cell lines.  Both cells harbor two TACE forms, a precursor and a mature form lacking the prodomain. Prodomain removal is considered required for TACE activity. The mature/precursor TACE ratio in C14 cells was higher than that in HEK-293-GHR-JAK2 cells (1.23 vs. 0.56).  Notably, endogenous TIMP3 was easily immunoblotted in HEK-293-GHR-JAK2 cells, but not in C14 cells. Adenoviral expression of TIMP3 in C14 cells specifically inhibited PMA-induced GHR proteolysis. Exogenous treatment with TIMP3-containing conditioned medium similarly reduced PMA-induced GHR proteolysis in C14 cells. Conversely, siRNA-mediated knockdown of TIMP3 in HEK-293-GHR-JAK2 cells markedly enhanced PMA-induced GHR proteolysis.  We also examined the impact of TIMP3 levels on PMA’s ability to modulate GH-induced JAK2 and STAT5 signaling. In control C14 cells, GH-induced STAT5 phosphorylation was decreased ~80% by brief PMA pre-exposure, consistent with desensitization of GH signaling by inducible GHR metalloproteolysis.  After TIMP3 expression, GH-induced STAT5 phosphorylation was largely preserved (~90% of control) when C14 cells were pre-exposed to PMA. Similar results were noted for GH-induced JAK2 phosphorylation. When TIMP3 was knocked down in HEK-293-GHR-JAK2 cells, PMA pretreatment significantly reduced GH-induced STAT5 phosphorylation (~60% reduction vs. ~27% in control cells). In exploring TIMP3’s modulation of GHR’s susceptibility to proteolysis and of cellular GH sensitivity, we found that adenovirally-expressed TIMP3 was coimmunoprecipitated with TACE in C14 cells. Further, TIMP3 expression reduced the mature/precursor TACE ratio in these cells. Taken together, our data suggest TIMP3 interacts with TACE and regulates both TACE activity and the ratio of mature/precursor TACE, thereby modulating GHR susceptibility to PMA-induced proteolysis and the degree of PMA’s desensitization to GH.

(1)       Zhang Y et al., Endocrinology. 2000; 141: 4342 (2)       Guan R et al., Endocrinology. 2001; 142: 1137 (3)       Wang X et al., Mol Endocrinol. 2008; 22: 1427

Nothing to Disclose: YZ, LAM, PAB, XW, JJ, SJF

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: VA Merit Review Award (to SJF)