A stimulatory TSH receptor antibody enhances adipogenesis via phosphoinositide 3-kinase activation in orbital preadipocytes from patients with Graves' ophthalmopathy

Abstract

Graves' ophthalmopathy (GO) is characterized by expanded volume of the orbital tissues associated with elevated serum levels of TSH receptor (TSHR) autoantibodies. Because previous studies have demonstrated evidence of adipogenesis within the GO orbit, we sought to determine whether M22, a human monoclonal antibody directed against TSHR, enhances adipogenesis in orbital fibroblasts from patients with GO and, if so, to identify signaling mechanisms involved. GO orbital fibroblast cultures (n=10) were treated for 10 days with bovine TSH (1 or 10.0 U/l) or M22 (1 or 10 ng/ml) in serum-free adipocyte differentiation medium. Some cultures also received a phosphoinositide 3-kinase (PI3K) inhibitor or an inhibitor of cAMP production. In other experiments, confluent cultures (n=8) were treated for between 1 and 30 min with TSH (0.1-10.0 U/l) or M22 (0.1-100 ng/ml) with measurement of cAMP production or levels of phosphorylated AKT (pAKT). We found levels of adiponectin, leptin, and TSHR mRNA to be increased in GO cultures treated for 10 days with either M22 (2.6 mean fold ± 0.7; P=0.03) or TSH (13.2 ± 5.8-fold, P=0.048). In other studies, M22 and TSH stimulated cAMP production and pAKT levels in GO cells. Inhibition of PI3K activity during 10 days in culture decreased the levels of M22-stimulated mRNA encoding adiponectin (67 ± 12%; P=0.021), as well as adiponectin and CCAAT/enhancer-binding protein α protein levels. In conclusion, M22 is a pro-adipogenic factor in GO orbital preadipocytes. This antibody appears to act via the PI3K signaling cascade, suggesting that inhibition of PI3K signaling may represent a potential novel therapeutic approach in GO.

Figure 1

Effect of treatment with M22 (1·0 or 10 ng/ml), TSH (1 or 10 U/l), or the positive control IGF1 Des 1,3 analog (10 ng/ml) on adipogenesis of GO orbital preadipocytes (n=10) cultured for 10 days in insulin-free differentiation medium. Results are expressed as mean±s.d. fold increase (relative to control untreated cultures) in mRNA levels encoding adipocyte-associated genes; A) adiponectin; B) leptin; and C) TSH receptor. *P<0·05.

Figure 2

Oil red-O staining of orbital adipocytes that were cultured for 10 days in insulin-free adipocyte differentiation medium containing A) IGF1 (Des 1,3 analog; 10 ng/ml); B) untreated; C) M22 (10 ng/ml); or D) TSH (10 U/l).

Figure 3

Effect of M22 (0·1–100 ng/ml) or TSH (0·001–10 U/l) treatment for 5 min on phosphorylated AKT (pAKT) protein levels in GO orbital fibroblast cultures (n=8). ELISA results are expressed as mean±s.e.m. fold increase in pAKT levels relative to parallel untreated cultures. *P<0·05.

Figure 4

Effect of 1, 5, 10, or 30 min treatment with M22 (5 ng/ml; filled circles), TSH (10 U/l; open circles), or agent in combination with the specific PI3K inhibitor LY294002 (50 μM) on phosphorylated AKT (pAKT) protein levels in GO orbital fibroblast cultures (n=8). ELISA results are expressed as mean±s.e.m. fold increase in pAKT levels (solid lines) relative to parallel untreated cultures or to fold decrease in pAKT levels (broken lines) relative to parallel cultures treated with M22 or TSH alone. *P<0·05.

Figure 5

Western blots showing total and phosphorylated AKT (pAKT) and GAPDH protein bands in confluent GO orbital fibroblast cultures exposed to the indicated treatments for 60 min. Left: 1) no treatment; 2) M22 (10 ng/ml); 3) M22 plus LY294002 (50 μM); or 4) LY294002 alone. Right: 1) no treatment; 2) IGF1 (Des 1,3 analog 10 ng/ml; 3) IGF1 plus LY294002; or 4) LY294002 alone.

Figure 6

Effect of M22 (10 ng/ml) or TSH (10 U/l) and the specific PI3K inhibitor LY294002 on adiponectin mRNA levels in GO orbital preadipocytes (n=8) cultured for 10 days in insulin-free differentiation medium. Results are expressed as mean±s.d. fold increase in adiponectin mRNA levels relative to control untreated cultures. *P<0·05.

Figure 7

Western blots showing C/EBPα (top), adiponectin (middle), and GAPDH (bottom) protein bands in confluent GO orbital fibroblast cultures exposed throughout 10 days in culture to the indicated treatments. Lane 1) no treatment; 2) LY294002 (10 μM); 3) M22 (10 ng/ml); 4) M22 plus LY294002.