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Regulation of Bone Mass Through Pineal-Derived melatonin-MT2 Receptor Pathway

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Regulation of Bone Mass Through Pineal-Derived melatonin-MT2 Receptor Pathway

Kunal Sharan et al. J Pineal Res.

Abstract

Tryptophan, an essential amino acid through a series of enzymatic reactions gives rise to various metabolites, viz. serotonin and melatonin, that regulate distinct biological functions. We show here that tryptophan metabolism in the pineal gland favors bone mass accrual through production of melatonin, a pineal-derived neurohormone. Pineal gland-specific deletion of Tph1, the enzyme that catalyzes the first step in the melatonin biosynthesis lead to a decrease in melatonin levels and a low bone mass due to an isolated decrease in bone formation while bone resorption parameters remained unaffected. Skeletal analysis of the mice deficient in MT1 or MT2 melatonin receptors showed a low bone mass in MT2-/- mice while MT1-/- mice had a normal bone mass compared to the WT mice. This low bone mass in the MT2-/- mice was due to an isolated decrease in osteoblast numbers and bone formation. In vitro assays of the osteoblast cultures derived from the MT1-/- and MT2-/- mice showed a cell intrinsic defect in the proliferation, differentiation and mineralization abilities of MT2-/- osteoblasts compared to WT counterparts, and the mutant cells did not respond to melatonin addition. Finally, we demonstrate that daily oral administration of melatonin can increase bone accrual during growth and can cure ovariectomy-induced structural and functional degeneration of bone by specifically increasing bone formation. By identifying pineal-derived melatonin as a regulator of bone mass through MT2 receptors, this study expands the role played by tryptophan derivatives in the regulation of bone mass and underscores its therapeutic relevance in postmenopausal osteoporosis.

Keywords: bone; melatonin; osteoblasts; osteoporosis; tryptophan.

Figures

Figure 1
Figure 1
Pineal‐derived melatonin positively regulates bone mass. (A) Schematic representation of the breeding strategy to obtain Tph1 pineal −/− on 129/SvJ (melatonin‐deficient) and C3H/HeJ (melatonin‐proficient) genetic backgrounds. (B) Recombination PCR for Tph1 in tissues and serum melatonin levels in different genotypes. (C) Histological analysis of vertebra. (D) μCT analysis of tibia in 12‐wk‐old +/+, +/+; Crx‐Cre, Tph1 fl/fl and Tph1 pineal −/− mice on C3H/HeJ background. (E) Histological analysis of vertebra of 12‐wk‐old WT, +/+; Crx‐Cre, Tph1 fl/fl and Tph1 pineal −/− mice on 129/SvJ background. (F) Serum melatonin, vertebral histomorphometry and serum osteocalcin levels in WT C57BL/6 mice treated with 0, 10 or 100 mg/kg/d melatonin for 6 wk. n for each group is indicated within each panel. Values are mean±SEM. Following symbols have been used to indicate level of significance: *P<.05; **P<.01; ***P<.001 compared to WT or control samples. ns, not significant. See also Fig. S1
Figure 2
Figure 2
Melatonin promotes bone mass through MT2. (A) Real‐time PCR analysis of MT1 and MT2 expression in different tissues of the WT mice. (B) Real‐time PCR analysis of MT1 and MT2 in calvarial preosteoblast, differentiated osteoblast and long bone of the WT mice. (C) Vertebral histomorphometry and serum levels of osteocalcin (Ocn) and Ctx in MT1+/+,MT1−/−, MT2+/+ and MT2−/− mice. (D) μCT analysis of tibia in MT1+/+,MT1−/−, MT2+/+ and MT2−/− mice. (E) Real‐time PCR analysis of Cyclins and osteoblast marker genes in the tibia of MT1+/+,MT1−/−, MT2+/+ and MT2−/− mice. (F) Vertebral histomorphometry of vehicle or melatonin‐treated +/+ and MT2−/− mice. n for each group is indicated within each panel. Values are mean±SEM. Following symbols have been used to indicate level of significance: *P<.05; **P<.01; ***P<.001 compared to WT or control samples. ns, not significant. See also Fig. S2
Figure 3
Figure 3
Melatonin directly regulates osteoblast functions through MT2 receptor. (A) BrdU incorporation assay in melatonin‐treated WT calvarial osteoblasts. (B) BrdU incorporation assay in MT1+/+,MT1−/−, MT2+/+ and MT2−/− osteoblasts treated with vehicle or melatonin. (C, D) Real‐time PCR analysis of Cyclins in vehicle or melatonin‐treated WT osteoblasts (C) and in MT1+/+,MT1−/−, MT2+/+ and MT2−/− osteoblast cells (D). (E) Alkaline phosphatase activity in WT osteoblasts treated with melatonin at different concentrations. (F) Alkaline phosphatase activity in MT1+/+,MT1−/−, MT2+/+ and MT2−/− osteoblasts treated with vehicle or melatonin. (G) Real‐time PCR analysis of osteoblast marker genes and (H) OPG/RankL expression ratio in melatonin‐treated WT osteoblasts. (I) Alizarin red staining and quantification in WT cells treated with melatonin for 21 d. (J, K) Alizarin red staining (J) and quantification (K) in the calvarial osteoblast cells from MT1+/+,MT1−/−, MT2+/+ and MT2−/− mice differentiated toward mineralizing osteoblasts. n for each group is indicated within each panel. Values are mean±SEM. Following symbols have been used to indicate level of significance: *P<.05; **P<.01; ***P<.001 compared to WT or control samples. ns, not significant. See also Fig. S3
Figure 4
Figure 4
Daily melatonin treatment cures ovariectomy‐induced osteoporosis. (A) Schematic representation of the experimental protocol used and representative images of von Kossa stained vertebral sections of sham/OVX mice treated with vehicle or melatonin at 10 or 100 mg/kg/d dose. (B) Vertebral histomorphometric analysis in sham and OVX mice treated with vehicle or melatonin at 10 or 100 mg/kg/d dose. (C, D) 3D μCT images of proximal tibia (C) and its microarchitectural parameters (D) in sham/OVX mice treated with vehicle or melatonin. (E‐G) Serum melatonin (E), osteocalcin (F) and Ctx levels (G) in sham/OVX mice treated with vehicle or melatonin. (H, I) Femur stiffness (H) and femur maximum force analysis (I) by three‐point bending test in sham/OVX mice treated with vehicle or melatonin. Values are mean±SEM. n for each group is indicated within each panel. Results were considered significant at P<.05. In all the panels, following symbols have been used to indicate different levels of significance *P<.05, **P<.01 and ***P<.001 as compared to OVX (vehicle) and ! P<.05, @ P<.01, # P<.001 as compared to sham (Veh)
Figure 5
Figure 5
Model of melatonin regulation of bone mass. Diagrammatic representation of the proposed pathway through which pineal‐derived melatonin regulates bone mass. Tryptophan regulates through a series of reactions the synthesis of pineal‐derived melatonin (PDM) in a Tph1‐dependent manner. PDM then enters the blood stream and acts on the osteoblasts through MT2 receptors to positively regulate bone mass. Trp, tryptophan; Tph1, tryptophan hydroxylase 1; 5‐HTP, 5‐hydroxytryptophan; AADC, aromatic amino acid decarboxylase; AANAT, arylalkylamine N‐acetyltransferase; ASMT, N‐acetylserotonin O‐methyltransferase

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