Early in primate evolution (about 60 million years ago), the major groups of Hominidae (humans and other apes), OWMs, NWMs and prosimians (lemurs and lorises) evolved independently and the origin and amplification of Alu elements was concomitant with this primate radiation . Examples of Alu elements implicated in gene regulation have been identified [9–11, 21, 22, 26, 33–36]. Identification of convincing examples of evolutionarily-fixed, novel regulatory patterns requires evidence of: 1) a known transposable element (TE) sequence in the region of the gene; 2) the TE sequence affecting regulation of the nearby gene; 3) the gene having some function and 4) the TE having been present long enough to be fixed [10, 34]. For some of the above examples, it was demonstrated that the Alu elements are conserved in humans, apes and/or OWMs, but was not examined in NWMs; therefore, the last requirement was only partially addressed .
Previously, we and others established the first three criteria for the CAMP gene [12, 13]. Here we provide convincing evidence for the last requirement. We demonstrate that the TE (AluSx SINE) acquired a VDRE through a duplication event and has undergone approximately 60 million years of purifying selection during the primate radiation to become fixed in the genomes of present-day humans, apes, OWMs and NWMs (Figure 1). The distribution of this Alu in the different lineages is consistent with current primate phylogeny and the ancient age of this class of Alu . Furthermore, we have shown that, as with humans, the non-human, primate VDREs are functional both in vitro and in vivo (Figure 2). Regulation of the CAMP gene by vitamin D as conferred by the AluSx in its promoter meets each of the above criteria. This study demonstrates that exaptation of vitamin D-mediated gene regulation by an AluSx SINE provided a novel, biologically-important innate immune response that is conserved in humans and non-human primates, but not other mammalian species. It is a convincing example of an evolutionarily-fixed, Alu-mediated divergence in steroid hormone nuclear receptor gene regulation between humans/primates and other mammals.
During the course of evolution, each group of primates has acquired differences in the ability to respond to steroid hormones . Surprisingly, NWM have circulating 1,25(OH)2D3 levels that are up to two orders of magnitude higher than those observed in OWM, apes and humans [30, 38]. The NWMs are naturally resistant to 1,25(OH)2D3 due to the over expression of VDRE-binding proteins (VDRE-BP) that requires NWMs to maintain high levels of 1,25(OH)2D3 to displace it from binding sites [39, 40]. Also, NWM are resistant to estrogen, testosterone and glucocortocoids [31, 38], but remain sensitive to retinoic acid and thyroid hormone . Although these differences in vitamin D physiology exist, we have demonstrated that the VDRE is conserved in all three groups of primates. Subclasses of AluS sequences provide a significant source of potential hormone response elements for retinoic acid and thyroid hormone receptors [36, 41]. Interestingly, in NWMs these elements are well conserved in the CAMP promoter Alu and have acquired additional changes that make them potentially better binding sites for RAR and TR (Figures 3 and 5). On the other hand, these sites were altered during hominid, ape and OWM evolution and did not respond to RA or TH (Figures 3 and 4). Retaining responses to both vitamin D and these other steroid hormones may be necessary for a proper innate immune response in vitamin D-resistant, but RA- and TH-sensitive NWMs. The importance of these binding sites to the expression of the CAMP gene remains to be fully elucidated.
The importance of vitamin D and the active metabolite 1,25(OH)2D3 in immune function became apparent with the discovery of VDR expression in activated inflammatory cells [42, 43]. Also, it was demonstrated that 1,25(OH)2D3 was produced by activated macrophages [44, 45] and 1,25(OH)2D3 inhibited T-cell activation and proliferation [46–49]. Subsequently, it was shown that vitamin D has an inhibitory action on the adaptive immune system with a shift from Th1 to Th2 and T regulatory cells and inhibition of Th17 development [50–54]. Suppression of the adaptive system and the anti-inflammatory effects of vitamin D are probably beneficial for conditions that involve autoimmunity ; however, it could prove detrimental for some infections [56, 57].
The human CAMP gene is not induced consistently by pro-inflammatory stimuli [12, 16, 58–61]. Additionally, infection of macrophages with Mtb and other cell types with pathogens leads to the repression of the CAMP gene [18, 62, 63]. Acquisition of the VDRE by ancestral primates that likely possessed high levels of vitamin D like today's non-human primates  would have provided a pathway for induction of the CAMP gene in cells such as macrophages or epithelial barrier cells that are capable of activating the vitamin D pathway in response to infection or wounding [17, 19]. The activation of the vitamin D pathway provides a way for human macrophages to prevent the suppression of the CAMP gene when activated with TLR2 or TLR4 ligands . Thus, induction of the CAMP gene by 1,25(OH)2D3 provides a possible mechanism for primates to counteract pathogen-mediated suppression and modulate the immune response.