Thyroid hormone-mediated growth and differentiation of growth plate chondrocytes involves IGF-1 modulation of beta-catenin signaling

Abstract

Thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulation of the Wnt/beta-catenin signaling pathway. Insulin-like growth factor 1 (IGF-1) has been described as a stabilizer of beta-catenin, and thyroid hormone is a known stimulator of IGF-1 receptor expression. The purpose of this study was to test the hypothesis that IGF-1 signaling is involved in the interaction between the thyroid hormone and the Wnt/beta-catenin signaling pathways in regulating growth plate chondrocyte proliferation and differentiation. The results show that IGF-1 and the IGF- receptor (IGF1R) stimulate Wnt-4 expression and beta-catenin activation in growth plate chondrocytes. The positive effects of IGF-1/IGF1R on chondrocyte proliferation and terminal differentiation are partially inhibited by the Wnt antagonists sFRP3 and Dkk1. T(3) activates IGF-1/IGF1R signaling and IGF-1-dependent PI3K/Akt/GSK-3beta signaling in growth plate chondrocytes undergoing proliferation and differentiation to prehypertrophy. T(3)-mediated Wnt-4 expression, beta-catenin activation, cell proliferation, and terminal differentiation of growth plate chondrocytes are partially prevented by the IGF1R inhibitor picropodophyllin as well as by the PI3K/Akt signaling inhibitors LY294002 and Akti1/2. These data indicate that the interactions between thyroid hormone and beta-catenin signaling in regulating growth plate chondrocyte proliferation and terminal differentiation are modulated by IGF-1/IGF1R signaling through both the Wnt and PI3K/Akt signaling pathways. While chondrocyte proliferation may be triggered by the IGF-1/IGF1R-mediated PI3K/Akt/GSK3beta pathway, cell hypertrophy is likely due to activation of Wnt/beta-catenin signaling, which is at least in part initiated by IGF-1 signaling or the IGF-1-activated PI3K/Akt signaling pathway.

Fig. 1

Thyroid hormone treatment increases Igf1r expression in rat growth plate chondrocytes. (A, C) Quantitative real-time RT-PCR analysis of Igf1r and Igf1 mRNA expression in pellet cultures of growth plate chondrocytes treated with 100 ng/mL of T₃ for 1 to 8 days. The expression in T₃-treated cells was normalized to the expression in control cells. (B) Immunoblotting of IGF1R protein from whole-cell lysates of growth plate chondrocytes treated with or without T₃ (100 ng/mL) for 2 to 7 days. Actin was used as an internal control. (D) Detection of IGF-1 protein in culture medium of growth plate chondrocytes treated with or without T₃ for 1 to 8 days. (E, F) Cyclin D1 mRNA and BrdU incorporation in growth plate cells treated with or without T₃ for 1 to 8 days. (G, H) Col10a1 mRNA and alkaline phosphatase activity in growth plate chondrocytes treated with or without T₃ for 1 to 8 days. *p < .05 versus control cells.

Fig. 2

The inhibitor of IGF1R decreases T₃-induced Wnt/β-catenin activation, cell proliferation, and terminal differentiation in growth plate chondrocytes. (A) Quantitative RT-PCR analysis of Wnt4 mRNA expression in growth plate chondrocytes treated with T₃ (100 ng/mL) and/or the IGF1R inhibitor picropodophyllin (PPP, 1 µM) for 5 days. The expression in T₃-treated cells was normalized to the expression in control cells. (B) Immunoblotting of active β-catenin protein from whole-cell lysates of growth plate chondrocytes treated with T₃ and/or PPP for 7 days. Actin was used as an internal control. (C) Detection of BrdU incorporation of growth plate chondrocytes treated with T₃ and/or PPP for 5 days. (D) Cyclin D1 mRNA expression in growth plate chondrocytes treated with T₃ and/or PPP for 5 days. (E, F) Col10a1 mRNA and alkaline phosphatase activity in growth plate chondrocytes treated with T₃ and/or PPP for 5 days. *p < .05 versus the cells treated with T₃ alone.

Fig. 3

IGF-1/IGF1R promotes Wnt/β-catenin signaling, cell proliferation, and terminal differentiation of growth plate chondrocytes. (A) Quantitative RT-PCR analysis of Wnt4 mRNA expression in rat growth plate chondrocytes infected with Ad-IGF1R or control adenovirus at an MOI of 50 and treated for 5 days with or without IGF-1 (50 ng/mL). (B) Immunoblotting of β-catenin from whole-cell lysates of growth plate chondrocytes treated with or without Ad-IGF1R and IGF-1 for 5 days. Actin was used as an internal control. (C, D) Runx2 mRNA and cyclin D1 mRNA expression in growth plate chondrocytes treated with Ad-IGF1R and/or IGF-1 for 5 days. (E) BrdU incorporation in growth plate chondrocytes treated with Ad-IGF1R and/or IGF-1 for 5 days. (F) Col10a1 mRNA in growth plate chondrocytes treated with Ad-IGF1R and/or IGF-1 for 8 days. The data were expressed as the fold increase over the results of the cells infected with control adenovirus. *p < .05 versus control cells.

Fig. 4

The effects of IGF-1/IGF1R on β-catenin signaling, cell proliferation, and terminal differentiation of growth plate chondrocytes are suppressed by inhibition of Wnt signaling. (A) Immunoblotting of β-catenin from whole-cell lysates of growth plate chondrocytes treated with Ad-IGF1R (MOI of 50) and IGF-1 (50 ng/mL) in the presence or absence of Wnt antagonists sFRP3 (100 ng/mL) and Dkk1 (100 ng/mL) for 5 days. (B, C) Quantitative real-time RT-PCR of Runx2 mRNA (B) and cyclin D1 mRNA expression (C) in rat growth plate chondrocytes treated with Ad-IGF1R and IGF-1 for 5 days in the presence or absence of sFRP3 and Dkk1. (D) Col10a1 mRNA expression in growth plate chondrocytes treated with Ad-IGF1R and IGF-1 for 8 days in the presence or absence of sFRP3 and Dkk1. *p < .05 versus the cells treated with Ad-IGF1R and IGF-1.

Fig. 5

Thyroid hormone promotes IGF-1-activated PI3K/Akt signaling and PI3K/Akt-dependent β-catenin signaling in growth plate chondrocytes. (A) Immunoblotting of phosphorylated Akt (pAkt), phosphorylated GSK-3β (pGSK-3β), and active β-catenin protein from whole-cell lysates of growth plate chondrocytes treated with or without T₃ (100 ng/mL) for 2 to 7 days. Actin was used as an internal control. (B) Immunoblotting of pAkt, pGSK-3β, and β-catenin protein of growth plate chondrocytes treated with T₃ for 5 days in the presence or absence of PI3K signaling inhibitor LY294002 (20 µM) and Akt signaling inhibitor Akti1/2 (1 µM). (C–E) Wnt4 mRNA (C), Runx2 mRNA (D), and cyclin D1 mRNA (E) expression of growth plate chondrocytes treated with T₃ for 5 days in the presence or absence of LY294002 and Akti1/2. ^*p < .05 versus the cells treated with T₃ alone. (F, G) Col10a1 mRNA (F) and ALP activity (G) of growth plate cells treated with T₃ for 5 days in the presence or absence of LY294002 and Akti1/2. *p < .05 versus the cells treated with T₃ alone.

Fig. 6

Schematic diagram of the proposed interactions between thyroid hormone, IGF-1/IGF1R, and β-catenin signaling pathways in regulating cell proliferation and terminal differentiation of growth plate chondrocytes. RZ = resting zone; PZ = proliferating zone; preHZ = prehypertrophic zone; HZ = hypertrophic zone.