Journal Facts

Journal
BoneKEy Knowledge Environment
ISSN
1533-4368
Volume
Year
2002

Article Facts

Title
How does vitamin D drive active intestinal calcium absorption?
DOI
10.1138/2002037
Author

Online Date
05/02/2002

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BoneKEy-Osteovision | Commentary

How does vitamin D drive active intestinal calcium absorption?


DOI:10.1138/2002037

The role of gut calcium absorption has been the focus of research on the actions of vitamin D from the earliest days of the vitamin D field. It was appreciated, soon after the recognition of vitamin D being a “vitamin”, that a high dietary calcium intake could overcome much of the adverse effects in animals on a vitamin D deficient diet and deprived of ultraviolet exposure. Calcium absorption in the gut has two major components, active transport through the gut intestinal cell (enterocyte) and passive paracellular transport (beside the enterocyte). The latter may be the more important calcium absorption pathway with high calcium intake, such as with dietary calcium supplements, but is inherently unlikely to be a regulated or regulatable pathway. Hence active calcium absorption has been the focus of considerable interest. The importance of calcium absorption in the small intestine helped focus the search for and eventual cloning of the putative vitamin D receptor from chick intestine in the early 80′s. However, identification of cis-acting vitamin D responsive elements (VDRE) in intestine-related gene promoters has been slow and the VDRE was first identified in the bone-specific gene osteocalcin ().

Active gut calcium absorption has been considered to consist of three steps: apical transport of calcium from the gut lumen into the enterocyte, binding/ transport of calcium within the enterocyte cytoplasm and extrusion of calcium from the basolateral surface into the extracellular fluid (). As the vitamin D endocrine system was responsive to dietary calcium manipulation, the hormonal form of vitamin D, 1,25-dihydroxyvitamin D, theoretically could regulate any of these steps. Hence a protein/pathway that responded to dietary calcium manipulation was a good starting point for identifying a vitamin D responsive pathway. An intracellular calcium binding protein, calbindin, was the first to be shown to change in relation to calcium transport across the intestine (). Two calbindins-D have been described, calbindin-D9K and calbindin-D28K. However the calbindin-D9K will be focused on, as calbindin-D28K may be more relevant to renal than intestinal calcium handling and even in the kidney may be only modestly vitamin D sensitive () or unaffected by a complete VDR knock-out (). Similarly intestinal cell calmodulin may fulfil a protective intracellular calcium binding role but there is little evidence for it being directly vitamin D-regulated (). There has been considerable debate as to whether changes in calbindin-D9K protein precede or follow changes in gut calcium transport (,,,). Some studies have detected a calbindin-D9K protein increase as early as one hour post 1,25-dihydroxyvitamin D, but even more rapid changes in calcium absorption in response to 1,25-dihydroxyvitamin D treatment have been reported (). Such a response would precede the detected calbindin-D9K changes or any other nuclear 1,25-dihydroxyvitamin D-mediated response. These very rapid responses are more consistent with post-translational activation of existing proteins and will need to be evaluated with respect to changes in protein modifications, such as phosphorylation, glycosylation, prenylation, etc. With these questions unanswered, it is not clear whether calbindin-D9K reacts to intracellular calcium levels rather than specifically driving the process. If the former, calbindin-D9K could serve an important “buffer” protecting the cell during the calcium transport process without fulfilling a key rate-limiting step. This unresolved “chicken-and-egg” question has led others to focus on alternate control points, including those at the luminal and contra-luminal (basolateral) cell surfaces, particularly calcium channels and calcium-ATPases.

The next component of the active calcium transport pathway to be defined was the basolateral calcium-ATPases (). These are considered to function in extrusion of calcium from the cell into the extra-cellular fluid. One of these, PMCA1, has been shown to respond to 1,25-dihydroxyvitamin D3 treatment in vitamin D-deficient rats (). In this study, the induction of calbindin-D9K and the intestinal Ca-ATPase peaked at 12-24 hours after a single of 1,25-dihydroxyvitamin D3 dose. Although some increase in expression was apparent at 3 - 6 hours, these data were again not suggestive of a driving process but rather of a response to increased intracellular calcium levels.

Two apical calcium channel proteins have been cloned recently. The first, the renal epithelial calcium channel (ECaC), cloned from rabbit kidney was also expressed in intestinal and placental tissues (). The second, the intestinal calcium channel (CaT1), cloned from rat duodenum and found to be expressed predominantly in intestinal but not renal cells, has 75% homology to ECaC (). A subsequent study mapped these two calcium channel genes to adjacent positions on chromosome 7, suggesting a gene duplication origin (). Both calcium channel proteins conferred renal cell-like calcium influx on Xenopus oocytes (). Expression of the renal epithelial calcium channel (ECaC) appeared to be regulated by changes in calcium intake, which would be expected to change circulating 1,25-dihydroxyvitamin D levels, and by 1,25-dihydroxyvitamin D3 treatment (). By contrast, the intestinal calcium channel protein, CaT1, did not appear to be regulated by changes in calcium intake or by 1,25-dihydroxyvitamin D3 treatment (). The human homologue (ECaC2/CaT1) was cloned from human duodenum, but duodenal mRNA levels did not correlate with serum 1,25-dihydroxyvitamin D levels (). The same group had earlier reported a weak association of duodenal expression of calbindin-D9K, but not intestinal calcium ATPase (PMCA1), with serum 1,25-dihydroxyvitamin D levels (). However, in the later study, ECaC2/CaT1 mRNA levels correlated strongly with the level of the PMCA1 Calcium-ATPase and to a lesser extent with that of calbindin-D9K (). Of interest in earlier studies, 1,25-dihydroxyvitamin D treatment was shown to be associated with rapid changes in expression of the basolateral calcium ATPase (). Thus, as for calbindin-D9K and the calcium ATPase, relationships for either of the putative apical calcium channels have not been clearly shown for vitamin D metabolite levels and calcium absorption.

Recent publications using the VDR-KO mouse models have shed some light on the relative roles of these putative calcium regulatory mechanisms (). The VDR-KO mice very closely resemble the human hereditary vitamin D resistant rickets type II, i.e. that associated with failure of VDR function and high or very high 1,25-dihydroxyvitamin D levels ().

In one study the calbindin-D9K levels were very low in the KO mice and did not respond to 1,25-dihydroxyvitamin D3 treatment but did respond to a diet high in calcium, lactose (and phosphate) that presumably increased enterocyte calcium by passive diffusion (). A high calcium diet also normalised calcium homoeostasis, including bone mineralisation, in VDR knock-out mice (), analogous to the cure of rickets in human hereditary forms of vitamin D resistance (). Although Demay and colleagues concluded that vitamin D regulated the calbindin-D9K expression (), their data would seem to be more consistent with a secondary rather than a direct response. The recent publication by Van Cromphaut and colleagues has taken similar advantage of two additional VDR knock-out strains to clarify some of these questions, using the strain generated in their own group at Leuven and the earlier knock-out developed in Tokyo (). In their study, expression of ECaC2/CaT1 was exquisitely regulated in wild-type control mice in response to exogenous 1,25-dihydroxvitamin D3 administration and to the physiological increase in circulating 1,25-dihydroxyvitamin D level that occurs with low dietary calcium intake. Perhaps more critically, in both VDR-KO lines ECaC2/CaT1 gene expression was markedly decreased in duodenum and, as might be expected for a target gene, did not respond to dietary calcium manipulations or 1,25-dihydroxyvitamin D3 treatment in either KO strain. While calbindin-D9K mRNA and protein were also lower in the KO mouse duodenum, these changes were modest, and surprisingly they were lower on both high or low calcium diet compared with the standard chow in the Leuven KO strain. Due to their greater sensitivity to low calcium intake, this unexpected outcome could not be evaluated in the Tokyo strain. There were some interesting differences in calcium channel gene expression in the kidney of these mice. Specifically, the ECaC2/CaT1 gene was expressed at much lower levels in kidney than in intestine but did not differ consistently between VDR-KO and wild-type strains. Thus, although ECaC2/CaT1 seems clearly involved in gut calcium transport, it equally clearly appears to have little if any role in effects of vitamin D on renal calcium transport. Thus the mechanisms of renal calcium reabsorption and regulation of these pathways by vitamin D, PTH and other hormonal factors are yet to be adequately addressed. Despite these uncertainties, these recent studies have identified the apical calcium channel as a key element in the calcium transport pathway in the intestine. The similar results in two distinct VDR-KO strains strengthens confidence in this work.

Major questions about the initial steps in regulation of intestinal active calcium absorption remain. Thus it is not clear that increased ECaC2/CaT1 gene expression precedes increased gut calcium absorption in response to endogenous or exogenous increases in 1,25-dihydroxyvitamin D levels. The demonstration of increased gut calcium transport with targeted intestinal over-expression of ECaC2/CaT1 would be powerful evidence for a primary role, particularly if this were observed despite a high dietary calcium intake or with VDR-KO mutations. The precise roles of ECaC2/CaT1 and its interactions with other elements of the calcium pathways, particularly calbindin-D9K and the basolateral calcium ATPase, remain to be elucidated. While it is clear that all three of these proteins are co-coordinately involved in the normal active calcium absorption process, it is unclear which (if any) of these drives the process or plays an anchor role in its regulation by the vitamin D endocrine system.

Acknowledgement: The constructive contributions of Dr Edith Gardiner are gratefully acknowledged.

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