Effects of 1,25(OH)2 D3 and 25(OH)D3 on C2C12 Myoblast Proliferation, Differentiation, and Myotube Hypertrophy.

An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2 D by 1α-hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2 D3 and 25(OH)D3 affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . We show that myoblasts not only responded to 1,25(OH)2 D3 , but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2 D3 as well as 25(OH)D3 stimulated VDR mRNA expression and in myotubes 1,25(OH)2 D3 also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α-hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2 D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2 D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . J. Cell. Physiol. 231: 2517-2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Regulation of CYP27B1 mRNA Expression in Primary Human Osteoblasts.

The enzyme 1α-hydroxylase (gene CYP27B1) catalyzes the synthesis of 1,25(OH)2D in both renal and bone cells. While renal 1α-hydroxylase is tightly regulated by hormones and 1,25(OH)2D itself, the regulation of 1α-hydroxylase in bone cells is poorly understood. The aim of this study was to investigate in a primary human osteoblast culture whether parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), calcitonin, calcium, phosphate, or MEPE affect mRNA levels of CYP27B1. Our results show that primary human osteoblasts in the presence of high calcium concentrations increase their CYP27B1 mRNA levels by 1.3-fold. CYP27B1 mRNA levels were not affected by PTH1-34, rhFGF23, calcitonin, phosphate, and rhMEPE. Our results suggest that the regulation of bone 1α-hydroxylase is different from renal 1α-hydroxylase. High calcium concentrations in bone may result in an increased local synthesis of 1,25(OH)2D leading to an enhanced matrix mineralization. In this way, the local synthesis of 1,25(OH)2D may contribute to the stimulatory effect of calcium on matrix mineralization.

Long-term vitamin D deficiency in older adult C57BL/6 mice does not affect bone structure, remodeling and mineralization.

Animal models show that vitamin D deficiency may have severe consequences for skeletal health. However, most studies have been performed in young rodents for a relatively short period, while in older adult rodents the effects of long-term vitamin D deficiency on skeletal health have not been extensively studied. Therefore, the first aim of this study was to determine the effects of long-term vitamin D deficiency on bone structure, remodeling and mineralization in bones from older adult mice. The second aim was to determine the effects of long-term vitamin D deficiency on mRNA levels of genes involved in vitamin D metabolism in bones from older adult mice. Ten months old male C57BL/6 mice were fed a diet containing 0.5% calcium, 0.2% phosphate and 0 (n=8) or 1 (n=9) IU vitamin D3/gram for 14 months. At an age of 24 months, mice were sacrificed for histomorphometric and micro-computed tomography (micro-CT) analysis of humeri as well as analysis of CYP27B1, CYP24 and VDR mRNA levels in tibiae and kidneys using RT-qPCR. Plasma samples, obtained at 17 and 24 months of age, were used for measurements of 25-hydroxyvitamin D (25(OH)D) (all samples), phosphate and parathyroid hormone (PTH) (terminal samples) concentrations. At the age of 17 and 24 months, mean plasma 25(OH)D concentrations were below the detection limit (<4nmol/L) in mice receiving vitamin D deficient diets. Plasma phosphate and PTH concentrations did not differ between both groups. Micro-CT and histomorphometric analysis of bone mineral density, structure and remodeling did not reveal differences between control and vitamin D deficient mice. Long-term vitamin D deficiency did also not affect CYP27B1 mRNA levels in tibiae, while CYP24 mRNA levels in tibiae were below the detection threshold in both groups. VDR mRNA levels in tibiae from vitamin D deficient mice were 0.7 fold lower than those in control mice. In conclusion, long-term vitamin D deficiency in older adult C57BL/6 mice, accompanied by normal plasma PTH and phosphate concentrations, does not affect bone structure, remodeling and mineralization. In bone, expression levels of CYP27B1 are also not affected by long-term vitamin D deficiency in older adult C57BL/6 mice. Our results suggest that mice at old age have a low or absent response to vitamin D deficiency probably due to factors such as a decreased bone formation rate or a reduced response of bone cells to 25(OH)D and 1,25(OH)2D. Older adult mice may therefore be less useful for the study of the effects of vitamin D deficiency on bone health in older people.

Mechanical loading and the synthesis of 1,25(OH)2D in primary human osteoblasts.

The metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D) is synthesized from its precursor 25-hydroxyvitamin D (25(OH)D) by human osteoblasts leading to stimulation of osteoblast differentiation in an autocrine or paracrine way. Osteoblast differentiation is also stimulated by mechanical loading through activation of various responses in bone cells such as nitric oxide signaling. Whether mechanical loading affects osteoblast differentiation through an enhanced synthesis of 1,25(OH)2D by human osteoblasts is still unknown. We hypothesized that mechanical loading stimulates the synthesis of 1,25(OH)2D from 25(OH)D in primary human osteoblasts. Since the responsiveness of bone to mechanical stimuli can be altered by various endocrine factors, we also investigated whether 1,25(OH)2D or 25(OH)D affect the response of primary human osteoblasts to mechanical loading. Primary human osteoblasts were pre-incubated in medium with/without 25(OH)D3 (400 nM) or 1,25(OH)2D3 (100 nM) for 24h and subjected to mechanical loading by pulsatile fluid flow (PFF). The response of osteoblasts to PFF was quantified by measuring nitric oxide, and by PCR analysis. The effect of PFF on the synthesis of 1,25(OH)2D3 was determined by subjecting osteoblasts to PFF followed by 24h post-incubation in medium with/without 25(OH)D3 (400 nM). We showed that 1,25(OH)2D3 reduced the PFF-induced NO response in primary human osteoblasts. 25(OH)D3 did not significantly alter the NO response of primary human osteoblasts to PFF, but 25(OH)D3 increased osteocalcin and RANKL mRNA levels, similar to 1,25(OH)2D3. PFF did not increase 1,25(OH)2D3 amounts in our model, even though PFF did increase CYP27B1 mRNA levels and reduced VDR mRNA levels. CYP24 mRNA levels were not affected by PFF, but were strongly increased by both 25(OH)D3 and 1,25(OH)2D3. In conclusion, 1,25(OH)2D3 may affect the response of primary human osteoblasts to mechanical stimuli, at least with respect to NO production. Mechanical stimuli may affect local vitamin D metabolism in primary human osteoblasts. Our results suggest that 1,25(OH)2D3 and mechanical loading, both stimuli of the differentiation of osteoblasts, interact at the cellular level.