Thyroid-stimulating hormone induces a Wnt-dependent, feed-forward loop for osteoblastogenesis in embryonic stem cell cultures

Abstract

We have shown that the anterior pituitary hormone, thyroid-stimulating hormone (TSH), can bypass the thyroid to exert a direct protective effect on the skeleton. Thus, we have suggested that a low TSH level may contribute to the bone loss of hyperthyroidism that has been attributed traditionally to high thyroid hormone levels. Earlier mouse genetic, cell-based, and clinical studies together have established that TSH inhibits osteoclastic bone resorption. However, the direct influence of TSH on the osteoblast has remained unclear. Here, we have used a model system developed from murine ES cells, induced to form mature mineralizing osteoblasts, and show that TSH stimulates osteoblast differentiation primarily through the activation of protein kinase Cδ and the up-regulation of the noncanonical Wnt components frizzled and Wnt5a. We predict that a TSH-induced, fast-forward short loop in bone marrow permits Wnt5a production, which, in addition to enhancing osteoblast differentiation, also stimulates osteoprotegerin secretion to attenuate bone resorption by neighboring osteoclasts. We surmise that this loop should uncouple bone formation from bone resorption with a net increase in bone mass, which is what has been observed upon injecting TSH.

Fig. 1.

Murine ES cell cultures can be induced to form osteoblasts. ES cell culture in differentiation medium (DF) (Materials and Methods) resulted in (A) a marked reduction in the expression of the stem cell marker genes Rex-1, Sox-2, and Oct-4 at day 30 compared with day 2 (by qPCR). *P < 0.005, **P < 0.0001, ***P < 0.0004. (B) The appearance of alkaline phosphatase (ALP)-positive colonies and CFU-fs. (C) The expression of osteoblast genes, namely ALP, type 1 collagen, osteocalcin (OC), and osteoprotegerin (OPG). Data are representative of two separate experiments.

Fig. 2.

TSH receptors (TSHRs) are expressed in differentiating murine ES cell cultures. RT-PCR (A) and flow cytometry (B) were performed on ES cell cultures after 30 d of incubation in differentiation medium (DF) (Materials and Methods). (A) DF stimulated, with or without TSH (10 mU/mL) and TSHR mRNA expression in long-term cultures. FRTL5 cells treated with 5 hormone was used as the positive control and GAPDH as the loading control. (B) Approximately 73% of cells were TSHR-positive when stained with anti-TSHR antibody (M1) (blue) compared with an isotype IgG control (red) (Materials and Methods).

Fig. 3.

TSH stimulates osteoblastogenesis and mineralization. (A and B) Von Kossa-positive and Alizarin-positive colony formation in ES cell cultures incubated in differentiating medium (DF) for 30 d (Materials and Methods). (a) Untreated and (b) enhanced mineralization with differentiation and (c) influence of TSH (1 mU/mL). TSH failed to induce differentiation of ES cells in the absence of DF (not illustrated). (C and D) Quantity of calcium and phosphate in the colonies was enhanced with DF and TSH (1 mU/mL). (E) DF and TSH (0, 1, 5, and/or 10 mU/mL) enhanced the expression of (a) collagen type-1, (b) alkaline phosphatase (AP), and (c) osteocalcin (OC) at day 30 as assessed by qPCR.

Fig. 4.

TSH stimulates noncannonical Wnt signaling. The effects of differentiation media (DF) and TSH (0, 5, or 10 mU/mL) on components of the Wnt-signaling pathway (assessed by real time PCR), namely Lrp5 (A), Lrp6 (B), β-catenin (C), Frizzled (Frz) (D), Wnt5a (E), and osteoprotegerin (OPG) (F) in 30-d ES cell cultures. *P < 0.05 by ANOVA one-way analysis. (G) Densitometric values from Western blots showing the up-regulation of Wnt5a but not of β-catenin in the cell lysates of cultures treated with DF+/TSH+. β-Actin is shown as the loading control.

Fig. 5.

TSH stimulates protein kinase Cδ phosphorylation. (A) Differentiation medium (DF) (Materials and Methods) with and without TSH (0 and 1 mU/mL) triggered the phosphorylation of protein kinase Cδ (PKC-δ), but not of protein kinase A (PKA) as shown in this Western blot. β-Actin was the loading control. n = 2. (B and C) PKC-δ inhibitor, rotterlin (10 μM), inhibited the increase in type1 collagen and osteopontin (OPN) expression triggered by TSH (1 mU/mL) as assessed by qPCR.

Fig. 6.

TSH induces a local fast-forward loop for the regulation of bone remodeling. TSH stimulates Wnt5a secretion that enhances osteoblastogenesis and mineralization, but also, through its action on frizzled (Frz), stimulates the production of osteoprotegerin (OPG). OPG, in turn, inhibits bone resorption through its attenuation of RANKL signaling. These effects underscore the bone mass-enhancing effects of TSH that we have noted in vivo (13).