Hormonal regulation of calcium absorption
Vitamin D: 1,25(OH)2D3, the active metabolite of vitamin D, is the main regulating hormone of intestinal Ca2+ absorption. It induces structural and functional modifications in enterocytes and helps to enhance both transcellular and paracellular pathways, either by genomic or nongenomic actions[46,47]. 1,25(OH)2D3 can reach the intestinal target coming from two different sources: Either from the plasma, once its synthesis has been completed by 25(OH)D3 1α-hydroxylase (CYP27B1) in renal proximal tube (endocrine source) or from de novo synthesis in the cytoplasm of the enterocyte, performed by a duodenal 1α-hydroxylase (intracrine source).
This calciotropic lipophilic vitamin passes through the plasma membrane and binds to vitamin D receptor (VDR), its nuclear receptor. Once bound to the ligand, VDR forms a heterodimer with retinoid X receptor (RXR) and the new 1,25(OH)2D3-VDR- RXR complex functions as a transcription factor which binds to different vitamin D response elements in various target genes. This determines a significant increase in the expression of all Ca2+ transporting proteins in the enterocyte: TRPV 6, CB9K, PMCA1b and NCX1, as has been demonstrated in animal models and humans[2,4,49,51-54]. Vitamin D-mediated Ca2+ absorption has mainly been studied in the proximal intestine, where Ca2+ is more efficiently absorbed. However, Christakos et al have recently studied this process in mice with transgenic expression of VDR exclusively in the ileum, cecum and colon of VDR KO mice. Interestingly, these animals did not present the abnormalities in Ca2+ homeostasis and bone mineralization usually seen in VDR KO mice. These findings emphasize the importance of 1,25(OH)2D3-mediated Ca2+ absorption in the distal intestine.
Apart from these Ca2+-transporting proteins, 1,25(OH)2D3 can regulate other important genes in Ca2+ metabolism, such as the one of 24-hydroxylase (CYP24A1) which converts 1,25(OH)2D3 into 1,24,25(OH)3D3 and 25(OH)D3 into 24,25(OH)2D3, and CYP27B1, involved in the renal synthesis of 1,25(OH)2D3, but also expressed in the intestine and parathyroid gland.
In addition to the genomic action described, there is some evidence that 1,25(OH)2D3 also binds to a plasma membrane receptor (MARRS: Membrane-associated, rapid response steroid-binding protein), which, in turn, activates other second messenger systems such as phospholipase A2 and protein kinase C[59-61]. Details of the underlying molecular mechanism of 1,25(OH)2D3-MARRS and its rapid minute-to-minute regulatory capacity remain to be elucidated.
Even though the transcellular pathway has been the focus of most studies concerning the effect of calcitriol on intestinal Ca2+ absorption, vitamin D has proved to exert a positive effect on the paracellular absorptive route as well. 1,25(OH)2D3 is able to change the permeability and selectivity of the tight junctions by altering certain crucial proteins such as CLDNs 2 and 12. This would help to enhance passive diffusion of Ca2+.
Rexhepaj et al have observed that 1,25(OH)2D3 could also stimulate Na+/Ca2+-ATPase and SGLT, and consequently increase water-movement through the junction, thus carrying more Ca2+ inwardly with the flow. Tudpor et al have demonstrated a dose dependent increase in solvent drag-induced Ca2+ movement one hour after direct exposure of rats to 10-100 nmol/L 1,25(OH)2D3. This rapid effect, abolished by inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and MEK, would be mediated by nongenomic mechanisms involving 1,25(OH)2D3-MARRS.
It has also been reported that 1,25(OH)2D3 downregulates intestinal cadherin-17 (involved in cell-to-cell contact) and aquaporin-8 (associated with epithelial selectivity towards cations), which might also affect the Ca2+ absorption[20,65].
PTH: PTH, a hypercalcemic hormone secreted by parathyroid glands, is the other classical hormone known to exert a positive regulatory effect on intestinal Ca2+ absorption. However, this stimulatory effect is achieved indirectly after increasing CYP27B1 transcription for 1α-hydroxylase, the renal enzyme that completes the synthesis of 1,25(OH)2D3 in the kidney. As a result, 1,25(OH)2D3 production augments. In addition, PTH also suppresses the transcription of CYP24A1 that codifies for 24-hydroxylase, a renal enzyme which degrades 1,25(OH)2D3 by converting it into 1,24,25(OH)3D3. Both actions lead to an increase in plasmatic 1,25(OH)2D3, which in turn enhances Ca2+ absorption as we have already revised.
Thyroid hormones: Thyroxine (T4) and triiodothyronine (T3) are known to regulate metabolism in general. Overproduction of T4 or T3 in the context of hyperthyroidism can lead to hypercalcemia due to an excessive bone turnover and consequently lead to bone demineralization[67,68]. However, there is some evidence that thyroid hormones would cooperate with vitamin D by increasing the genomic actions of 1,25(OH)2D3 in the intestine. Cross et al have shown that calcitriol added to cultures of 20-day-old embryonic chick small intestine, stimulated Na+ uptake. The calcitriol-mediated increase in Na+ uptake appeared to be related to increased tight-junctional or paracellular permeability. It can be speculated that this effect could favor the paracelullar entry of Ca2+ as well. More recently, Kumar et al have shown that Ca2+ influx in BBM vesicles was higher in enterocytes from hyperthyroid rats as compared to those of hypothyroid ones. The authors have proposed that this could be related to a change in membrane fluidity induced by thyroid hormones. Similarly, they have also observed that efflux of calcium across BLM was also higher in hyperthyroid rats. This difference was associated with a higher NCX1 activity triggered by thyroid hormones, possibly through the cAMP-mediated pathway. cAMP is a potent activator of Na+/Ca2+ exchanger and it was significantly higher in intestinal mucosa of hyperthyroid rats as compared to euthyroid animals. In addition to these actions, thyroid treatment increases serum PTH and 1,25(OH)2D3 levels, which contributes to enhancing Ca2+ absorption indirectly through vitamin D.
Growth hormone: Growth hormone (GH) has a central role in longitudinal bone growth and mineralization during childhood and adolescence. However, this metabolic hormone has receptors in most tissues and exerts various actions apart from skeletal growth. There is evidence that GH has proliferative effects on intestinal mucosa. GH has been used to treat inflammatory bowel disease in pediatric and adult patients. Interestingly, FDA has approved the use of recombinant human GH to treat short bowel syndrome, where it improves absorption of carbohydrates, amino acids and fats[75,76]. GH can also stimulate intestinal Ca2+ absorption, which would occur indirectly by activating renal CYP27B1 and consequently increasing serum 1,25(OH)2D3 levels. It has been demonstrated that GH can prevent the loss of intestinal VDR in ovariectomized (OVX) rats, which would suggest that it could increase intestinal sensitivity to 1,25(OH)2D3 by regulating tissue VDR levels. However, the positive effect of GH on Ca2+ absorption would not be exclusively dependent on vitamin D. Fleet et al have shown that GH increases intestinal Ca2+ absorption and duodenal CB9k levels in aged rats without increasing serum 1,25(OH)2D3 levels. Analogous results have been found in humans. In adult men, Ca2+ absorption has a positively correlation with IGF-1 and age-related decline in IGF-1 has a negative impact on Ca2+ absorption that could not be justified by a decrease in serum 1,25(OH)2D3.
Estrogens: There is evidence that post-menopausal women experiment an increase in bone resorption together with a reduction in Ca2+ absorption and an increase in urinary Ca2+ excretion as a consequence of estrogen loss[82,83]. Post-menopausal low estrogen levels have been associated with reduced serum 1,25(OH)2D3. However, OVX rats have no reduction in serum 1,25(OH)2D3 levels, which would suggest the implication of vitamin D independent mechanisms. Thus, O’Loughlin et al observed that estradiol replacement in OVX rats increases intestinal Ca2+ absorption without stimulation of circulating 1,25(OH)2D3 levels. In the same line, van Abel et al found increased duodenal gene expression of TRPV5, TRPV6, CB9k, and PMCA1b in OVX rats treated with estradiol. In order to determine whether this stimulatory effect on Ca2+ transporting proteins was calcitriol-dependent, they used CYP27B1 KO mice and found that estradiol treatment increased mRNA levels of duodenal TRPV6. In contrast, Gennari et al found that oophorectomy in young women reduces the intestinal Ca2+absorption induced by vitamin D, which was reversed by estrogen repletion. Other studies suggest the possibility of a deficient intestinal responsiveness to vitamin D due to a reduction of VDR levels[78,89,90]. However, the loss of intestinal VDR levels following estrogen reduction could not be confirmed in all studies.
Cell-culture experiments suggest that estrogen is able to reverse the decline in the efficiency of intestinal Ca2+ absorption at menopause onset, but the mechanisms that underlie this effect remain to be elucidated. Estrogen receptor alpha (ERα) KO mice showed a decrease in duodenal TRPV6 mRNA expression, without modification in CB9k, PMCA1b and VDR levels. Therefore, it seems that the genomic effects of estrogen on mice intestinal mucosa are mainly mediated by ERα. However, Nie et al have recently reported that estrogen regulates duodenal Ca2+ absorption through differential effects of ERα and ERβ on TRPV6 and PMCA1b expressions in duodenal epithelial cells, respectively.
PRL: The main lactogenic hormone, PRL, is elevated during pregnancy and lactation. Apparently, this pituitary hormone is able to enhance Ca2+ absorption in order to supply calcium for milk production. It has been shown that PRL enhances CYP27B1 protein expression and increases levels of 1,25(OH)2D3 during lactation, a moment when there is an increased Ca2+ requirement for the neonate. However, its calciotropic action is not only achieved via vitamin D. It has been shown that PRL stimulates active intestinal Ca2+ transport in vitamin D-deficient rats. Charoenphandhu et al demonstrated that PRL directly stimulates active duodenal Ca2+ transport. Wongdee et al observed that lactating rats exhibit some adaptive changes in their intestinal mucosa tending to increase the absorptive surface area. These rats have larger duodenal, jejunal and ileal villous as well as deeper cecal crypts than age-matched nulliparous rats. These histological modifications were diminished by bromocriptine, an inhibitor of pituitary PRL release. PRL also upregulated TRPV6 and PMCA1b in the duodenum of lactating rats. These changes were associated with a compensatory increase in FGF-23 expression, a local negative regulator of Ca2+ absorption, presumably to prevent Ca2+ hyperabsorption. Bromocriptine also manages to abolish FGF-23 increment, confirming it was induced by PRL. In addition, it has been suggested that PRL has also a stimulating effect on paracellular pathway by upregulating CLDN 15 in the tight junctions.
FGF-23: It is a glycoprotein secreted by osteocytes and osteoclasts and regulated by plasma levels of 1,25(OH)2D3 and Pi. The enhancement of these regulators leads to the serum increase in FGF-23, which in turn reduces the concentration of 1,25(OH)2D3 by inhibiting 1α-hydroxylase and stimulating 24 α-hydroxylase. As for Pi, FGF-23 increases its renal excretion.
FGF-23 has been indicated as a vitamin D antagonist in intestinal absorption of Ca2+. Khuituan et al have demonstrated that intravenous administration of FGF-23 to male rats abolished the increase in intestinal absorption of Ca2+ caused by the injection of 1,25(OH)2D3. However, the inhibitory effect of FGF-23 could not be observed in the absence of the previous supply of 1,25(OH)2D3. The mechanisms underlying the effect of FGF-23 would be related to the decrease in the gene expression of TRPV5, TRPV6 and CB9k caused by this phosphaturic hormone. In this same work, the presence of FGFR1-4 in the BLM of rat enterocytes was confirmed. However, their functions are unclear since the direct exposure of the intestinal epithelium to FGF-23 did not cause alterations.
FGF-23 also blocks the stimulatory effect of 1,25(OH)2D3 on the paracellular pathway of intestinal Ca2+ absorption. Since vitamin D favors this process by increasing water flow across paracellular space and consequently dragging solutes as Ca2+, it has been proposed that FGF-23 could decrease the water flow and the dragging of this cation.
The activation of the mechanisms mediated by FGF-23 would be crucial to avoid the Ca2+ hyperabsorption. Therefore, it was thinkable that a molecule that senses extracellular Ca2+ as the calcium sensing receptor (CaSR) would play an important role. In fact, Rodrat et al have demonstrated that CaSR was involved in the inhibition of intestinal Ca2+ absorption mediated by FGF-23. According to their findings in a cell monolayer, the use of allosteric inhibitors of CaSR could reverse the inhibitory effect of FGF-23 on the stimulation of Ca2+ transport triggered by 1,25(OH)2D3.
Glucocorticoids: The negative side effects of glucocorticoid (GC) treatment on bone health are well known. Impaired function and number of osteoblasts and osteoclasts, high resorption rate, deficiency in mineralization are some of the effects of chronic treatment that lead to the development of GC-induced osteoporosis (GIO). GIO is also partially due to the alterations that GC produces in intestinal Ca2+ absorption. Van Cromphaut et al evaluated the effect of dexamethasone treatment on the gene expression of proteins involved in the intestinal absorption of the cation. They did not find alterations in gene expression or Ca2+ absorption in the treated mice, justifying the absence of effects with the short treatment duration. Kim et al determined that a single dose of dexamethasone increased the gene expression of TRPV6 and CB9k, while when it was given for 5 days, it led to a reduction in the expression of both genes. In concordance with these results, mRNA levels for duodenal VDR increased on day one, while they were reduced after 5 days of treatment. Zhang et al observed reduced protein expression of TRPV6 and CB9k in the intestine of male mice injected with dexamethasone 3 times a week, for 12 weeks, effect that was accompanied by hypercalciuria and reduction in serum Ca2+ levels. Although the role of GC in intestinal Ca2+ absorption is not clear, the results presented would allow infer a certain negative effect of GC on cation transfer from the lumen to the interstitium.
CT: The role of CT on intestinal Ca2+absorption is controversial. Some studies have suggested that CT inhibits the process; in contrast, others indicate that has a stimulatory effect. Swaminathan et al have demonstrated that CT may produce an inhibitory effect at low doses, whereas high doses increase the intestinal Ca2+ absorption. CT effect could be mediated by the vitamin D endocrine system, since it has been demonstrated in diabetic rats that CT increases 1,25(OH)2D3 synthesis at renal level. Use of CT has been suggested to treat patients with β-thalassemia because they usually have low plasma levels of this hormone. CT chronic use has benefited osteoporosis associated with thalassemia, not only for its inhibitory effect on osteoclasts but also for the possible role in the 1,25(OH)2D3 synthesis.
Regulation of intestinal Ca2+ absorption by dietary calcium
The main dietary factor that can modify intestinal Ca2+ absorption is calcium itself. Low-calcium uptake could eventually produce hypocalcemia, which would augment PTH secretion leading to stimulate vitamin D endocrine system and demineralize bone. On the other hand, high calcium diets and calcium hyperabsorption could increase cardiovascular risk associated with vascular calcification, nephrolithiasis and dementia, among other conditions[110,111]. Since the gut is the only gate for Ca2+ uptake, it is subjected to both local and systemic regulations, which protect against either insufficient or excessive Ca2+ absorption. Low calcium diets enhance serum levels of vitamin D and, consequently, activate the endocrine actions of this vitamin. Thus, a chronic dietary Ca2+ deficiency increases all transcellular pathway genes and proteins[109,113,114], and increases the activity of the intestinal PMCA1b and NCX1 all along the villus, independently of cell maturation degree. Benn et al have gone further to demonstrate that this adaptive increase in Ca2+ absorption is present even in TRPV6 KO and CB9k KO mice, suggesting that TRPV6, which has been postulated as the rate-limiting factor in transcellular pathway, may not be so or it may be successfully replaced by other factors able to partially compensate its function. In our laboratory, we have observed in animals under low Ca2+ diets that the increment in Ca2+ transport is accompanied by a concomitant increase in the activity of intestinal alkaline phosphatase (IAP), a marker enzyme of enterocytic differentiation that may have a role in intestinal Ca2+ absorption. Brun et al have reported that luminal Ca2+ concentration increases the activity of IAP and simultaneously decreases the percentage of Ca2+ absorption, functioning as a minute-to-minute local regulatory mechanism of Ca2+ entry, independent of vitamin D. This would limit an excessive Ca2+ intake secondary to dietary calcium restriction, thus preventing possible acute toxic effects. This regulatory mechanism may probably be one of the reasons why high Ca2+ intake (1500 mg/d) was not followed by a significant increase in Ca2+ absorption in a clinical trial[116,117], as it would have been expected from the positive effect of stimulated vitamin D endocrine system. Interestingly, L-Phenylalanine, an inhibitor of IAP, prevented this regulatory effect and Ca2+ uptake remained increased. A more recent study showed that IAP activity induced by luminal calcium concentration provoked changes in luminal pH that could modulate intestinal Ca2+ absorption In addition, a recent study revealed that IAP KO mice have higher intestinal Ca2+ uptake, which correlates with better biomechanical properties of trabecular bone.
It has also been suggested that CaSR, abundantly expressed in apical and basolateral membranes of enterocytes in humans, rats and mice[120-122] may also participate in the local regulation of intestinal Ca2+ absorption. Intestinal CaSR -specific KO mice showed an altered intestinal integrity, disbalanced gut microbiota and a pro-inflammatory status[123-125]. Rodrat et al have recently observed that high-dose of 1,25(OH)2D3 or high concentration of luminal calcium reduced Ca2+ transport across a Caco-2 monolayer. The authors proposed that CaSR would sense luminal calcium triggering a local inhibitory feedback mechanism to restrict excessive Ca2+ uptake. This inhibitory loop could possibly involve locally produced FGF-23, which has been observed to counteract the enhanced duodenal Ca2+ transport in mice exposed to 1,25(OH)2D3 for a long term[32,100].