VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: Potential role in suppression of myoblast proliferation
Center for Muscle Biology, Department of Physiology, College of Medicine, University of Kentucky, Lexington, USA.AJP Cell Physiology (Impact Factor: 3.78). 05/2012; 303(4):C396-405. DOI: 10.1152/ajpcell.00014.2012
1α,25(OH)(2)D(3), the active form of vitamin D(3), has been reported to regulate the cell biology of skeletal muscle. However, there has been some controversy about the expression of the vitamin D receptor (VDR) and thus the potential role of vitamin D(3) in skeletal muscle. In this study, we isolated and sequenced the full-length Vdr and Cyp27b1 transcripts in C2C12 myoblasts and myotubes. Western blots and immunocytochemistry confirmed protein expression in both myoblasts and myotubes clearly demonstrating that C2C12 cells express VDR and CYP27B1. To determine the vitamin D(3) action, we found that C2C12 myoblasts treated with either 1α,25(OH)(2)D(3) or 25(OH)D(3) inhibited cell proliferation and this was associated with increased Vdr expression. The observation that treatment of C2C12 myoblasts with the inactive form of vitamin D(3), [25(OH)D(3)], inhibited proliferation suggested that CYP27B1 was functionally active. We used small interfering RNA to knock down Cyp27b1 in myoblasts, and cells were treated with 25(OH)D(3). The growth-suppressive effects of 25(OH)D(3) were abolished, suggesting that CYP27B1 in myoblasts is necessary for the ability of 25(OH)D(3) to affect cell proliferation. Finally, we analyzed expression of VDR and CYP27B1 in regenerating skeletal muscle in vivo. We found that expression of VDR and CYP27B1 increased significantly at day 7 of regeneration, and these results confirm the expression of Vdr and Cyp27b1 in vivo and suggest a potential role for vitamin D(3) in skeletal muscle regeneration following injury.
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- "with 10% FBS was in addition reported by Srikuea et al. (2012), who also demonstrated VDR expression in C2C12 cells. The authors used 20 nM of 1,25D for 48 h and under these conditions, they observed an increase in VDR expression and a significant decrease in the number of cells (Srikuea et al. 2012). We also demonstrated that p38 participates in 1,25D-dependent G0/G1-phase arrest in C2C12 cells. "
ABSTRACT: Previously, we have reported that 1,25(OH)2-vitamin D3 (1,25D) activates p38 MAPK (p38) in a vitamin D receptor (VDR)-dependent manner in proliferative C2C12 myoblast cells. It was also demonstrated that 1,25D promotes muscle cell proliferation and differentiation. However, we did not study these hormone actions in depth. In this study we have investigated whether the VDR and p38 participate in the signaling mechanism triggered by 1,25D. In C2C12 cells, the VDR was knocked down by a shRNA, and p38 was specifically inhibited using SB-203580. Results from cell cycle studies indicated that hormone stimulation prompts a peak of S-phase followed by an arrest in the G0/G1-phase, events which were dependent on VDR and p38. Moreover, 1,25D increases the expression of cyclin D3 and the cyclin-dependent kinase inhibitors, p21(Waf1/Cip1) and p27(Kip1), while cyclin D1 protein levels did not change during G0/G1 arrest. In all these events, p38 and VDR were required. At the same time, a 1,25D-dependent acute increase in myogenin expression was observed, indicating that the G0/G1 arrest of cells is a pro-differentiative event. Immunocytochemical assays revealed co-localization of VDR and cyclin D3, promoted by 1,25D in a p38-dependent manner. When cyclin D3 expression was silenced, VDR and myogenin levels were downregulated, indicating that cyclin D3 was required for 1,25D-induced VDR expression and the concomitant entrance into the differentiation process. In conclusion, the VDR and p38 are involved in control of the cellular cycle by 1,25D in skeletal muscle cells, providing key information on the mechanisms underlying hormone regulation of myogenesis.
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- "Srikuea et al.  confirmed that C2C12 cells express the full-length CYP27B1 mRNA transcript and CYP27B1 protein could be detected in the cytoplasm of myoblasts, exhibiting partially overlapping with the mitochondria to which CYP27B1 has been reported to be typically localized . Furthermore, they showed that siRNA-mediated knockdown of CYP27B1 could alleviate inhibitory effects of 25(OH)D 3 on cell proliferation . These observations provide direct evidence that CYP27B1 is biologically active in myogenic cells and mediates to convert 25(OH)D 3 to 1í µí»¼,25(OH) 2 D 3 . "
ABSTRACT: Muscle mass and strength progressively decrease with age, which results in a condition known as sarcopenia. Sarcopenia would lead to physical disability, poor quality of life, and death. Therefore, much is expected of an effective intervention for sarcopenia. Epidemiologic, clinical, and laboratory evidence suggest an effect of vitamin D on muscle function. However, the precise molecular and cellular mechanisms remain to be elucidated. Recent studies suggest that vitamin D receptor (VDR) might be expressed in muscle fibers and vitamin D signaling via VDR plays a role in the regulation of myoblast proliferation and differentiation. Understanding how vitamin D signaling contributes to myogenesis will provide a valuable insight into an effective nutritional strategy to moderate sarcopenia. Here we will summarize the current knowledge about the effect of vitamin D on skeletal muscle and myogenic cells and discuss the potential for treatment of sarcopenia.
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- "Additionally, the mutant mice had a total 33% body weight reduction compared to controls at maturity; implying a post-natal role for VDR in maintaining weight (Song et al., 2003). Increased VDR expression is also correlated with regeneration (Srikuea et al., 2012), but levels of VDR appear to decrease with increasing age, which has been proposed as a potential mechanism contributing to reduced muscle strength in the Bischoff-Ferrari et al. (2004). In the context of muscle biology, VDR mediates both nongenomic and genomic effects of vitamin D (Buitrago et al., 2001; Capiati et al., 2002). "
ABSTRACT: Myopathy is a feature of many inflammatory syndromes. Chronic inflammation has been linked to pathophysiological mechanisms which implicate 1,25 dihydroxyvitamin D3 (1,25-(OH)2D3)-mediated signaling pathways with emerging evidence supporting a role for the vitamin D receptor (VDR) in contractile and metabolic function of both skeletal and cardiac muscle. Altered VDR expression in skeletal and cardiac muscle has been reported to result in significant effects on metabolism, calcium signaling and fibrosis in these tissues. Elevated levels of serum inflammatory cytokines, such as IL-6, TNF-α and IFNγ, have been shown to impact myogenic and nuclear receptor signaling pathways in cancer-induced cachexia. The dysregulation of nuclear receptors, such as VDR and RXRα in muscle cells, has also been postulated to result in myopathy via their effects on muscle structural integrity and metabolism. Future research directions include generating transcriptome-wide information incorporating VDR and its gene targets and using systems biology approaches to identify altered molecular networks in human tissues such as muscle. These approaches will aid in the development of novel therapeutic targeting strategies for inflammation-induced myopathies.
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