Closing in on Vitamin D Action in Skeletal Muscle: Early Activity in Muscle Stem Cells?

It is now widely known that the vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) exerts its actions in multiple cell types not only to integrate regulatory control of mineral homeostasis through its effects on intestine, kidney and bone, but to modulate key functional activities in cells of the immune system, the skin, the cardiovascular system, and certainly other tissues as well (1, 2). These actions are mediated by the vitamin D receptor (VDR), which is a member of the steroid receptor transcription factor family that controls the expression of genes in a temporally sensitive and cell type-specific manner. Although early studies defined key components of this activity at single genes, more recent studies have both confirmed many of these fundamental findings on a genomewide scale and provided a wealth of new functional insight (3, 4). A primary discovery, consistent with that of other transcription factors, is the finding that the VDR frequently acts at multiple sites within a gene locus and that these sites can be located many kilobases distal to the gene’s transcriptional start site. This complexity has significant negative implications for the use of traditional methods that have been used to study transcription where gene segments are evaluated out of endogenous gene context as well as in the absence of organized chromatin architecture (5). Vitamin D status has long been linked to definable biological activities in individual tissues. Thus, vitamin D deficiency can result in rickets and in a myriad of other distinct abnormalities that can arise as a result of changes in blood calcium and phosphorus levels (6). The need to distinguish between indirect activities that are secondary to the direct actions of the VDR in well-established target tissues is extremely important, because mechanisms responsible for pathophysiological alterations drive the selection of drugs for therapeutic treatment. As a consequence, it is not surprising that the presence of the VDR in tissues has become an appropriate hallmark determinant for distinguishing between direct and indirect actions. Interestingly, although the biological actions of vitamin D and its mechanisms in a number of tissues fall into this category, no tissues have been the topic of as much investigation as those related to cardiac and skeletal muscle (7). This is because as summarized in multiple reviews, there seems to be no doubt that normal vitamin D levels are necessary for the appropriate functioning of both cardiac and skeletal muscle tissue and that vitamin D deficiency can lead to distinct pathologies (8). There have been a number of recent attempts to demonstrate the presence of the VDR in muscle and other tissues not considered to be traditional targets. The problem here is that these attempts are largely based upon detection of VDR transcripts by RT-PCR analysis, an exquisitely sensitive methodology that defines specific RNA transcript levels but leaves open the question of VDR protein product level. These analyses have established, however, that transcripts for the VDR in some tissues are strikingly lower than those seen in traditional vitamin D target cells. For example, VDR transcript levels in muscle in vivo are 3–4 logs lower than levels measured in the intestine and kidney, and relative concentrations in other tissues such as liver and brain are similarly low (9). These results almost ensure that the detection of the VDR protein by immunological means will be difficult, because the latter methodology is far less sensitive than RT-PCR analysis and in many cases may be conceptually unable to detect existing VDR protein levels. This prediction has proven correct as both earlier studies of VDR expression in muscle tissue as well as more recent studies using either Western