The Thyroid Hormone Transporter MCT10 Is a Novel Regulator of Trabecular Bone Mass and Bone Turnover in Male Mice

Abstract

Thyroid hormones (TH) are essential for skeletal development and adult bone homeostasis. Their bioavailability is determined by specific transporter proteins at the cell surface. The TH-specific transporter monocarboxylate transporter 8 (MCT8) was recently reported as a regulator of bone mass in mice. Given that high systemic triiodothyronine (T3) levels in Mct8 knockout (KO) mice are still able to cause trabecular bone loss, alternative TH transporters must substitute for MCT8 function in bone. In this study, we analyzed the skeletal phenotypes of male Oatp1c1 KO and Mct10 KO mice, which are euthyroid, and male Mct8/Oatp1c1 and Mct8/Mct10 double KO mice, which have elevated circulating T3 levels, to unravel the role of TH transport in bone. MicroCT analysis showed no significant trabecular bone changes in Oatp1c1 KO mice at 4 weeks and 16 weeks of age compared with wild-type littermate controls, whereas 16-week-old Mct8/Oatp1c1 double KO animals displayed trabecular bone loss. At 12 weeks, Mct10 KO mice, but not Mct8/Mct10 double KO mice, had decreased trabecular femoral bone volume with reduced osteoblast numbers. By contrast, lack of Mct10 in 24-week-old mice led to trabecular bone gain at the femur with increased osteoblast numbers and decreased osteoclast numbers whereas Mct8/Mct10 double KO did not alter bone mass. Neither Mct10 nor Mct8/Mct10 deletion affected vertebral bone structures at both ages. In vitro, osteoblast differentiation and activity were impaired by Mct10 and Mct8/Mct10-deficiency. These data demonstrate that MCT10, but not OATP1C1, is a site- and age-dependent regulator of bone mass and turnover in male mice.

Keywords: Mct10; Mct8; Oatp1c1; Slc16a10; Slc16a2; bone homeostasis; thyroid hormone transporters.

Figure 1.

Deletion of Oatp1c1 does not affect bone mass and microarchitecture. Bones from 16-week-old male wildtype (WT), Oatp1c1 KO, and Mct8/Oatp1c1 double KO (M8/O dKO) mice were examined by microCT. At the spine (A) bone volume per total volume (BV/TV), (B) trabecular number (Tb.N), (C) trabecular separation (Tb.Sp), and (D) trabecular thickness (Tb.Th) were determined. (E) Representative 3D reconstructions of the trabecular compartment of L4 vertebrae from WT and mutant mice. Additionally, (F) BV/TV, (G) Tb.N, (H) Tb.Sp, and (I) Tb.Th were determined in the distal femur. (J) Representative 3D reconstructions of the trabecular compartment of femurs. Cortical bone parameters, (K) cortical bone volume/total volume (Ct.BV/TV) and (L) cortical thickness (Ct.Th) at the femoral midshaft. (M) Femur length was measured using a caliper. As an indicator of bone strength, (N) maximal force (F_max) was determined by 3-point bending testing of femurs. Data are shown as mean ± SD. N = 11-15 per group. Statistical analysis was performed by one-way ANOVA. Statistical significance is denoted in the graphs.

Figure 2.

Deletion of Mct10 in 12-week-old mice leads to trabecular bone loss at the distal femur. Bones of 12-week-old wild-type (WT), Mct10 KO, and Mct8/Mct10 double KO (M8/M10 dKO) mice were analyzed by microCT. (A) Bone volume per total volume (BV/TV), (B) trabecular number (Tb.N), (C) trabecular separation (Tb.Sp), and (D) trabecular thickness (Tb.Th) at the spine. (E) BV/TV, (F) Tb.N, (G) Tb.Sp, and (H) Tb.Th at the distal femur. (I) cortical bone volume/total volume (Ct.BV/TV) and (J) cortical thickness (Ct.Th) analyzed at the femoral midshaft. (K) Femur length was measured using a caliper. (L) maximal force (F_max) was determined by 3-point bending test of femurs. (M) Representative 3D reconstructions of the trabecular compartment of distal femurs. Data are shown as mean ± SD. N = 9-15 per group. Statistical analysis was performed by one-way ANOVA. Statistical significance is denoted in the graphs.

Figure 3.

Deletion of Mct10 in 24-week-old mice increases trabecular bone mass at the distal femur. Bones of 24-week-old wild-type (WT), Mct10 KO, and Mct8/Mct10 double KO (M8/M10 dKO) mice were analyzed by microCT. (A) Bone volume per total volume (BV/TV), (B) trabecular number (Tb.N), (C) trabecular separation (Tb.Sp), and (D) trabecular thickness (Tb.Th) at the spine. (E) BV/TV, (F) Tb.N, (G) Tb.Sp, and (H) Tb.Th at the distal femur. (I) Cortical bone volume/total volume (Ct.BV/TV) and (J) cortical thickness (Ct.Th) at the femoral midshaft. (K) Femur length was measured using a caliper. (L) Maximal force (F_max) was determined by 3-point bending test of femurs. (M) Representative 3D reconstructions of the trabecular compartment of distal femurs. Data are shown as mean ± SD. N = 9-15 per group. Statistical analysis was performed by one-way ANOVA. Statistical significance is denoted in the graphs.

Figure 4.

Deletion of Mct10 decreases osteoblast numbers in 12-week-old mice. Bones and serum samples from 12-week-old wild-type (WT), Mct10 KO, and Mct8/Mct10 double KO (M8/M10 dKO) mice were used for histological analysis and determination of bone turnover markers. (A/B) Osteoblast number per bone perimeter (Ob.N/B.Pm) and (C/D) osteoclast number per bone perimeter (Oc.N/B.Pm) were determined in TRAP-stained sections of vertebra and femur. Serum concentration of (E) bone formation marker type 1 procollagen amino-terminal-propeptide (P1NP) and (F) bone resorption marker C-terminal telopeptide (CTX). (G) Representative images of TRAP-stained femurs. Scale bar = 500 µm. Scale bar of detailed images = 100 μm. Arrows indicate specific cell types: black arrows osteoblasts, red arrows osteoclasts. Data are shown as mean ± SD. N = 9-15 per group. Statistical analysis was performed by one-way ANOVA. Statistical significance is denoted in the graphs.

Figure 5.

Deletion of Mct10 alters bone turnover in femurs, but not vertebrae of 24-week-old mice. Bones and serum samples from 24-week-old wild-type (WT), Mct10 KO, and Mct8/Mct10 double KO (M8/M10 dKO) mice were used for histological analysis and determination of bone turnover markers. (A/B) Osteoblast number per bone perimeter (Ob.N/B.Pm) and (C/D) osteoclast number per bone perimeter (Oc.N/B.Pm) were determined in TRAP-stained sections of vertebra and femur. Serum concentration of (E) bone formation marker type 1 procollagen amino-terminal-propeptide (P1NP) and (F) bone resorption marker C-terminal telopeptide (CTX). (G) Representative images of TRAP-stained femurs. Scale bar = 500 µm. Scale bar of detailed images = 100 μm. Arrows indicate specific cell types: black arrows osteoblasts, red arrows osteoclasts. Data are shown as mean ± SD. N = 9-15 per group. Statistical analysis was performed by one-way ANOVA. Statistical significance is denoted in the graphs.

Figure 6.

Mct10 and Mct8 mRNA expression in bone tissue and bone cells. Lumbar vertebrae and femur midshafts from 6-, 12-, 24-, and 34-week-old male C57BL/6 mice. (A) Mct10 and (B) Mct8 expression was determined by real-time PCR. In vitro, Mct10 expression in primary murine (C) osteoblasts and (D) osteoclasts at indicated time points during differentiation. mRNA expression of (E) Mct10 and (F) Mct8 in osteocyte-like IDG-SW3 cells at indicated differentiation stages. Data are shown as mean ± SD. N = 3-7 per group. Statistical analysis was performed by Student’s t test between vertebrae and femurs derived from mice of the same age. Statistical significance is denoted in the graphs.

Figure 7.

Both Mct10 and Mct8/Mct10 gene deletion impair osteoblast differentiation and function in vitro. mRNA expression of (A) Mct10, (B) Mct8, and (C) the T₃ target gene Klf9 in primary murine osteoblasts from wild-type (WT), Mct10 KO, and Mct8/Mct10 double KO (M8/M10 dKO) mice differentiated over 7 days. Expression of osteoblast markers (D) Ocn, (E) Osx, and (F) Runx2. (G) Alkaline phosphatase (ALP) activity in WT, Mct10- and Mct8/Mct10-deficient primary osteoblasts after 10 days differentiation. (H) Mineralization capacity of osteoblasts quantified by Alizarin Red staining at day 10 of differentiation. (I) Representative image of Alizarin Red stained bone matrix. Data are shown as mean ± SD. N = 4-8 per group. Statistical analysis was performed by one-way ANOVA. Statistical significance is denoted in the graphs.