Nutritional rickets is not only a sequel of vitamin D deficiency, but can also be caused by deficits in phosphate [27] or calcium [28]. There is evidence that in Nigeria, South Africa, Northern India, and Bangladesh a form of rickets occurs in older toddlers and children, which is only partially amenable to vitamin D, but can be fully resolved by calcium supplements alone [29,30]. The partial ineffectiveness of vitamin D could be due to the fact that two-thirds of the children with rickets had serum 25-(OH)D concentrations well above the rachitic range [30]. It has been suggested that, in this case, rickets is attributable to low dietary calcium intake, which is characteristic of cereal-based diets with limited variety and little access to dairy products [28]. According to De Lucia et al. [31], low dietary calcium intake after weaning is another reason for rickets with normal circulating 25-(OH)D, which is occasionally seen in the United States.

In 1998, Riggs et al. [32] had proposed a unitary model for involutional osteoporosis, in which, apart from hormonal imbalances, calcium malnutrition and calcium malabsorption are considered to be of great significance in the development of the disease in both genders. Consequently, nutrient and supplemental calcium has been widely used for the prevention and treatment of osteoporosis (e.g., [33,34,35,36,37,38,39]). A meta-analysis of randomized controlled trials by Boonen et al. [40] emphasized the need to co-administer calcium with vitamin D to significantly (p < 0.025) reduce the risk of hip fractures. Likewise, a patient level pooled analysis of seven major trials conducted in the USA and Europe, with fractures as endpoint [41] found that vitamin D alone is not effective in preventing fractures, but that calcium and vitamin D together significantly (p < 0.025) reduce the rate of total fractures, including hip and probably vertebral fractures as well. Tang and colleagues [42] provided further evidence that in people aged 50 years or older, daily doses of 1200 mg calcium in combination with 800 IU vitamin D have the best therapeutic effect to prevent fractures and osteoporotic bone loss. It is interesting to note that adult, though not elderly, African Americans experience less fragility fractures despite lower serum 25-(OH)D concentrations than whites in the same age range. Aloia [43] suggested that this seeming paradox can be explained by, among others, differences between African Americans and other populations in “calcium economy”. In other words, increased calcium absorption and superior calcium conservation might compensate, in African Americans, for the negative effect of vitamin D insufficiency on bone health.

A lower incidence of fractures could be partially due to the positive effect of calcium and vitamin D supplementation on neuro-musculoskeletal functions [44] leading to a reduction in the number of falls in the elderly [45]. Taken together, calcium in combination with vitamin D is the “gold standard” for treating older persons with decreased bone mineral density, increased propensity to falls, and osteoporotic fractures (for review, see [46]).

Already in 1985, Garland et al. [48] had reported results of a 19 year prospective trial, which allowed the identification of low dietary calcium as an independent risk factor for colon cancer. Additional findings by Garland et al. [49] on an inverse association between nutritional calcium levels and colon cancer risk seem to indicate that the incidence of colon cancer can be reduced by approximately 70% if daily calcium intake is raised from 800 to 1400 mg. Since then, many other observational studies reported a strong association between incidence of colorectal cancer and low calcium intake: for example, Huncharek et al. [50] performed a meta-analysis of 60 epidemiological studies, and showed that high calcium intake, regardless of whether it is dietary or supplemental, had a significant protective effect against tumor development in the distal colon and rectum. Wu et al. [51] analyzed data from two large prospective trials, which had involved more than 80,000 women and 40,000 men. They reported a significant risk reduction for distal colon cancer for subjects whose dietary calcium intake was ≥1250 mg compared to those who ingested 500 mg or less. Cho et al. [52] analyzed pooled primary data from 10 cohort studies, in which more than half a million individuals were followed up for six to 16 years. They found a significant (p < 0.001 for trend) inverse relation between total calcium intake and colorectal cancer incidence. Comparable findings were reported by Jenab et al. [53], who had conducted a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort of more than 520,000 participants from 10 western European countries.

It should be noted that the efficiency of calcium in reducing the risk of colorectal cancer depends very much on the vitamin D status of an individual, so that optimal prevention of the disease necessitates high intake levels of both vitamin D and calcium [52,53,54,55]. Notably, Fedirko et al. [56] reported a significant interaction (p < 0.01) between dietary calcium and vitamin D on reduction of colorectal cancer-related mortality in a nested case–control study within the EPIC cohort.

From their studies on fat-induced carcinogenesis in mice, Lipkin and Newmark suggested that low dietary calcium could also promote breast cancer in humans [57].This notion gained strong support from multiple observations of an inverse relation between calcium intake and disease incidence in both pre-menopausal and post-menopausal women: Lin et al. [58] studied the effects of calcium intake from nutrient sources and supplements on breast cancer risk in a large cohort of premenopausal women. They found that higher intakes of total calcium were associated with a lower risk of premenopausal breast cancer (RR = 0.61; 95% CI: 0.40–0.92). Shin et al. [59] reported that in premenopausal women, dairy calcium (>800 mg/day versus ≤200 mg/day; RR = 0.69) had inverse associations with premenopausal breast cancer risk. McCullough et al. [60] analyzed data from nearly 70,000 postmenopausal women participating in the Cancer Prevention Study II Nutrition Cohort and found a moderately lower risk of breast cancer with intake of dietary calcium >1250 mg/day compared to <500 mg/day ((RR = 0.80, p [trend] = 0.02). This association was even stronger (RR = 0.67) in women with estrogen receptor-positive tumors. Consumption of dairy products was also inversely associated with risk (RR = 0.81; 95% CI, 0.69–0.95; p [trend] = 0.002) in this study. In a recent case-control study conducted among Chinese women by Zhang et al. [61], no significant association was found between dairy product intake and breast cancer risk, but a statistically significant inverse association of dietary calcium intake with breast cancer risk was observed with the adjusted OR (95% CI) of 0.35 (0.22–0.56) comparing the highest with the lowest quartile. Chen et al. [62] performed a meta-analysis of 15 studies on calcium intake and breast cancer risk: an inter-quantile comparison showing that high calcium consumption was associated with overall relative risk of 0.81 (95% CI = 0.72–0.90) adds to the evidence that calcium has a chemopreventive effect against breast cancer.

Bérubé et al. [63] found that combined intake of calcium and vitamin D by pre-menopausal women was superior to separate intakes in reducing mammographic breast density, a surrogate marker of breast cancer incidence. This notion is valid also for breast cancer incidence as shown by Abbas et al. [64] in a population-based case-control study in Germany (for discussion, see [62]).

Rather conflicting data have been reported with respect to the effect of calcium intake on the incidence of prostate cancer. In 1998, a study by Giovannucci et al. [65] found a positive association between calcium intake from food sources and supplements and risk of prostate cancer. Later, Gao et al. [66] analyzed 12 prospective studies published from 1966 to 2005. Dose-response analyses suggested that dairy product and calcium intakes were each positively associated with the risk of prostate cancer (p [trend] = 0.029 and 0.014, respectively). It was, however, not clear whether calcium intake itself was an independent risk factor for prostate cancer. This issue seemed to be clarified by Allen et al. [67], who analyzed data from almost the entire EPIC study cohort. They found that calcium from dairy products was positively associated with risk (p [trend] = 0.02), but not calcium from other foods. The situation elsewhere in the world appears to be less clear. Huncharek et al. [68] performed a meta-analysis of 45 observational studies, the majority of which were conducted in the United States. Results from 23 cohort studies and 26 case-control studies did not support an association between dairy product use and an increased risk of prostate cancer. While calcium data from cohort studies were heterogeneous and could not be used for the analyses, and respective data from case-control studies demonstrated no association with increased risk of prostate cancer. The results of this meta-analysis [68] should be seen with some caution because, as pointed out by the authors, “much of the reviewed literature predated the widespread use of PSA screening in the United States, and only few studies provided information on proportion of cases (controls) screened for prostate cancer and the stage distribution of cancer cases included in individual analyses”. When this information was available, somewhat different results were obtained: For example, Kristal et al. [69] examined nutritional risk factors for prostate cancer among nearly 10,000 participants in the Prostate Cancer Prevention Trial (United States and Canada, 1994–2003) and found that dietary calcium was positively associated (p [trend] = 0.165) with low-grade cancer but inversely associated (p [trend] = 0.034) with high-grade cancer. A preventive effect of high calcium intake against high-grade cancers was also found in a recent case-control study conducted by Williams et al. [70] among US veterans.

Obesity: A clinical study of calcium intake with a skeletal endpoint showed a significant correlation between low calcium intake and increased body weight in women aged 30–80 years [73]. Findings from two studies that were conducted in the USA [73,74] suggested that high calcium intake significantly reduces the risk of obesity. In contrast, Puntus et al. [75] found no association between calcium intake and body mass index in a well controlled, large cohort study on healthy adult Austrians.

Dyslipidemia and glucose intolerance are, apart from hypertension and obesity, constituents of the so-called metabolic syndrome and of non-insulin-dependent diabetes mellitus. A link between low calcium and the metabolic syndrome and type 2 diabetes mellitus has been reported in observational studies (reviewed by [76]). The available evidence is limited because most observational studies were cross-sectional and did not adjust for important confounders.

Hypertension: Evidence from a large number of observational studies and randomized clinical trials indicates that low dietary calcium constitutes a significant risk factor for primary hypertension (for review, see [77]). Calcium appears to be particularly effective in reducing the age-related increase in blood pressure. Dobnig et al. [78] conducted a randomized, double-blind, multi-center study on the effect of daily high-dose calcium supplements in healthy, elderly adults and observed a substantial reduction of systolic and diastolic blood pressure after one year of treatment in individuals who were in the upper third of pre-study blood pressure values. No further improvement was seen with calcium plus vitamin D supplementation. It seems that, apart from hypertension, none of the classical risk factors of cardiovascular disease are significantly associated with low calcium intake.

In 1999, Bostick et al. [79] had published results from a prospective cohort study of more than 30,000 Iowa women, suggesting that a higher intake of calcium is associated with reduced ischemic heart disease mortality in postmenopausal women. However, as mentioned previously, it is still a matter of debate whether higher intake of calcium also has a benefit on incident cardiovascular symptoms such as ischemic heart disease, myocardial infarction, and stroke [71,72]. For example, in a large cohort study of Japanese women and men, Umesawa et al. [80] found a significant inverse association of calcium intake with risk of stroke, particularly of the ischemic subtype, but did not detect any correlation with the risk of ischemic heart disease. The results of a 12 year follow-up of approximately 40,000 male health professionals in USA by Al-Delaimy et al. [81] suggested that neither dietary nor supplemental intakes of calcium were appreciably associated with the risk of ischemic heart disease among men. Lewis et al. [82] conducted a randomized placebo-controlled trial on the effect of calcium supplementation on the risk of atherosclerotic vascular disease in postmenopausal women. Patient-level analysis after a five year follow up period indicated that in patients with pre-existing atherosclerotic cardiovascular disease, calcium therapy reduced the risk of hospitalization and was associated with significantly fewer heart failure death events.

Lewis et al. [82] interpreted their study that it “supplies compelling evidence that calcium supplementation does not have any adverse effect on myocardial function.” This statement refers to studies by Pentti et al. [83] and Bolland et al. [84,85], who had claimed that high calcium intake promotes calcification of coronary arteries and subsequent myocardial infarction. This notion, that was heavily debated in recent years, and particularly the studies by Bolland et al. [84,85] were heavily criticized (cf. [86,87]). In addition, Samelson et al. [88] most recently published the results of their prospective observational study on a cohort within the Framingham offspring study: The authors found no evidence that calcium intake from dietary sources, or from supplements, had any effect on coronary artery calcification. They also concluded from their study that there is no need “to modify current recommendations for calcium intake to protect skeletal health with respect to vascular calcification risk”. It should be noted that vascular calcifications develop secondary to vascular damage, which frequently results from vitamin D insufficiency and associated secondary hyperparathyroidism [89,90,91,92], whereas high calcium has been shown to prevent vascular damage and calcification through activation of the CaSR on vascular smooth muscle cells (for details, see Section 3.3.3).

The concept of an important role of the CaSR in regulation of bone formation and mineralization as developed earlier [112,120], is fully supported by recent findings from a study in a CaSR knock-out mouse model [115]. As osteoblastic lineage cells are endowed with a functional CaSR (e.g., [121,122,123]), changes in [Ca²⁺]_o can be directly transduced into control of osteoblast proliferation, differentiation and function during any phase of mineralized bone formation (Figure 2). This process, in which the canonical Wnt/β-catenin signaling cascade plays a key role, [124] is coordinated by separate receptor-mediated effects of [Ca²⁺]_o and 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) [112,115,125]: As illustrated in Figure 2, activation of the Wnt/β-catenin pathway initiates “osteogenic differentiation” of pluripotent mesenchymal stem cells into osteoprogenitor cells, which characteristically express the osteoblast-specific transcription factor, Core-binding factor-1 (Cbfa1). Signaling from the CaSR in conjunction with the non-canonical Wnt5a/Ca²⁺ pathway [126] increases the expression of Cbfa. This causes osteoprogenitor cells to proliferate and to produce collagen [122]. Subsequently, signaling from 1,25(OH)₂D₃/VDR inhibits proliferation and matrix deposition and at the same time promotes osteoblast differentiation through phases of matrix maturation and mineralization [127]. Independent of vitamin D, activation of the CaSR increases the expression of osteoblast-specific differentiation markers, such as alkaline phosphatase (ALP), osteocalcin (OC) and osteopontin (OP). Finally, the CaSR plays an important role in orderly bone formation by regulating crystal growth and by preventing excessive mineralization [112].

Colorectal cancer: The CaSR is expressed in normal human colonic epithelium, mainly in the upper half of the crypt, and is absent from the crypt base [128]. Thus, expression of the CaSR correlates with a [Ca²⁺]_o gradient that, according to Whitfield [129], decreases in the direction from top to base of the crypt. Pluripotent stem cells at the bottom of the crypt can thus proliferate without restraints from anti-mitotic signaling from the CaSR. Daughter cells, however, while migrating to the crypt top undergo differentiation under the influence of increasing CaSR expression and activity [130]. The assumption of an important role of the CaSR, in control of crypt cell homeostasis, is supported by findings in an intestinal-specific CaSR knockout mouse, which shows changes in crypt structure, expansion of the proliferative zone, and hyperproliferation of colonic epithelial cells [131]. There is suggestive evidence that the CaSR, on the luminal aspect of human colonocytes, reacts to ingestion of calcium-rich food by rapid activation of anti-mitogenic intracellular pathways [98].

The CaSR on human colon carcinoma cells has high sequence homology with the parathyroid CaSR [98,132], and is expressed as long as the cancer cells retain a certain degree of differentiation [128,133]. Epigenetic silencing of the CaSR could be one reason for reduced expression of the receptor in more advanced cancers and in nearly all lymph node metastases [134]. From the evidence that expression of the CaSR is highest in well differentiated cancers and declines during tumor progression [128,133], it can be implied that calcium acting via the CaSR is a chemopreventive rather than a chemotherapeutic agent.

The sequence of events downstream of CaSR activation that actually link CaSR to cell cycle control in colon carcinoma cells starts with inhibition of phospholipase A₂ activity [135] (Figure 1 and Figure 3), which would reduce the amount of arachidonic acid available for the synthesis of proliferation-stimulating prostaglandins. Subsequent down-regulation of c-myc proto-oncogene expression [98], activation of the cyclin-dependent kinase inhibitor p21 [136] and inhibition of cyclin D1 finally leads to cell cycle arrest at the G₁/S-phase transition. CaSR-activated pro-differentiating signaling in colonocytes involves inhibition of the Wnt/β-catenin pathway by down-regulation of the T-cell transcription factor (TCF)-4 with subsequent induction of E-cadherin expression [137,138,139]. Interestingly, part of the anti-proliferative action of 1,25(OH)₂D₃ has been traced to a VDR-mediated, negative effect on TCF-4 [137,140] (Figure 3). These findings illustrate that Ca²⁺ and vitamin D independently inhibit colorectal cancer cell proliferation. Therefore, a combination of both agents is well suited to reduce the risk of colorectal cancer.

Breast cancer: A role for a functional CaSR in breast cancer can be inferred from the fact that in premenopausal women serum calcium levels vary inversely with breast cancer risk in a concentration-dependent manner [141]. Both normal and malignant mammary gland epithelial cells are endowed with the CaSR [99]. However, little is known of the role played by the CaSR in mediating changes in ambient Ca²⁺ to regulate cellular growth in mammary gland cells. In MCF-7 breast cancer cells, activation of the CaSR is transduced via the PI-PLC pathway into generation of IP₃, which not only causes the release of Ca²⁺ from endoplasmic reticulum, but also evokes Ca²⁺ influx across the plasma membrane through non-selective cation channels (Figure 1; for details, [108,142]). The rise in intracellular Ca²⁺ may conceivably activate pro-apoptotic intracellular signaling [143], particularly through involvement of different plasma membrane calcium ATPase isoforms [144]. In addition, the CaSR can inhibit breast cancer cell growth through its functional linkage with the tumor-suppressor BRCA1: Promkan et al. [145] reported that BRCA1 up-regulates the expression of the CaSR and, at the same time, functions through the receptor in suppressing the expression of survivin, an anti-apoptotic protein that is present in most cancer cells.

An apparent cross-talk between Ca²⁺/CaSR and 1,25(OH)₂D₃/VDR signaling [146] on cytosolic Ca²⁺ may explain, at least in part, how vitamin D and calcium together efficiently inhibit mammary gland cell growth in vivo (cf. Section 2.2.2).

Activation of the CaSR could have dual effects on disease outcome: On the one hand, activation of the CaSR in human MCF-7 breast cancer cells by extracellular calcium concentrations that are detected in bone at tumor metastasis, enhances expression of the estrogen receptor-alpha [147]. An improved estrogen receptor status is considered a good prognostic factor. On the other hand, the CaSR has also been shown to stimulate synthesis and secretion of parathyroid hormone-related peptide, which is thought to play a key role in osteolytic metastases [148].

Prostate cancer: The CaSR is expressed on human prostate cancer cell lines [101,149,150]. Unlike colorectal and breast cancer cells, prostate cancer cells respond to high calcium with increased proliferation. This may be due to transactivation by the CaSR of the epidermal growth factor receptor (EGFR) and subsequent activation of ERK1/2 (cf. Figure 1) and release of PTHrP [101,149]. This can explain why prostate cancers expressing high levels of the CaSR are more likely to metastasize to bone than those poorly expressing the CaSR. Stimulation of PTHrP release from bone metastatic prostate cancer cells induces osteoclastic bone resorption, whereby the Ca²⁺ concentration in the resorptive lacunae of osteoclasts rises to eight to 40 mM [65]. High calcium, acting through the CaSR, causes malignant prostate cells to proliferate and to release even more PTHrP, leading to increased osteolytic activity and enhanced survival of metastatic cells in the bony microenvironment [65]. The relevance of CaSR expression for metastases development, and for disease outcome, is underscored by the recent findings of Shui et al. [151] that common genetic variants of the CaSR are significantly associated with prostate cancer mortality.

Because current literature on expression of the CaSR in human arterial and cardiac cells is rather limited, inference on the function of the receptor in the human cardiovascular system has to be made from the wealth of information on the expression and function of the CaSR in the vasculature (heart and blood vessels and associated structures) of rodents and other experimental animals. In addition, a substantial role of the CaSR in maintaining normal blood pressure and cardiovascular functions is supported by studies on the effect of so-called calcimimetics, i.e., synthetic allosteric activators of the CaSR, in various animal models [152] and patients with chronic kidney disease [153]. It is now very clear that the CaSR is part of an intricate network of calcium channels, pumps, and exchangers, which is involved in the control of intracellular calcium concentration ([Ca²⁺]_i) and thereby in modulation of cardiovascular functions [154,155].

Cardiomyocytes: The CaSR is functionally expressed on rat neonatal and adult cardiomyocytes [154,156,157]. Upon activation by [Ca²⁺]_o the receptor transduces signals preferentially along the PLC pathway causing intracellular accumulation of IP₃ (cf. Figure 1) and, in turn, a rise in [Ca²⁺]_i, the classic “second messenger” in cardiac physiology. Thus, the CaSR has been implicated in regulating cardiac development, function, and homeostasis [154]. Apart from regulating [Ca²⁺]_i, the CaSR may be involved in excitation-contraction coupling through a unique feedback mechanism. Hofer et al. [158] observed that intracellular signaling events can produce changes in [Ca²⁺]_o that are detected by the CaSR on cells in close proximity. According to Tfelt-Hansen et al. [154], the CaSR on cardiomyocytes may therefore change its activity with every heart contraction.

Endothelial and smooth muscle cells of human arteries express a functional CaSR [105]. The receptor was detected also in endothelial cells from rat mesenteric and porcine coronary arteries [155], and in rat aortic smooth muscle cells [152]. Indeed, the CaSR is functionally expressed on all cells of the vascular wall-adventitia, fibroblast, vascular smooth muscle cells (VSMC), and endothelium. It is closely involved in the regulation of vascular tone and arterial blood pressure (for review, see [159]). In a hypothetical model proposed by Weston et al. [155] the CaSR plays a key role in myo-endothelial coupling in the vasculature. Activation of the CaSR induces endothelium-dependent hyperpolarization in neighboring myocytes. In short, activation of the endothelial CaSR is associated with the opening of a Ca²⁺-sensitive K⁺ conductance channel. K⁺ released into the intercellular space is taken up by the myocyte Na⁺/K⁺-ATPase. This contributes to a reversal of spasminogen-induced depolarization of vascular myocytes and, consequently, to vascular relaxation. This can explain, at least in part, the well documented hypotensive effect of dietary Ca²⁺ (see section 2.3.1).

The CaSR has also been shown to be involved in vascular repair and maintenance of vascular integrity [159]. Normal expression of the receptor on VSMC seems to be important for prevention of vascular calcification, since calcification is associated with reduced expression of the CaSR in blood vessels [160]: large and small arteries of normal subjects and of patients with advanced chronic kidney disease express the CaSR, although the expression is lower in epigastric arteries of patients with advanced renal impairment, compared with healthy transplant donors. Analysis of epigastric arteries, taken from chronic kidney disease patients, showed that a decrease in CaSR expression was accompanied by progressive calcification of VSMC areas [105]. Moreover, there is increasing evidence from in vitro and in vivo studies that calcimimetic compounds upregulate the CaSR in vascular cells and thereby attenuate vascular mineralization associated with reduced kidney function (for review, see [153]).

Juxtaglomerular cells: According to Atchison and Beierwaltes [161], signaling from Ca²⁺/CaSR in juxtaglomerular cells inhibits a calcium-inhibitable isoform of adenylate cyclase (cf. Figure 1), which is associated with formation of renin. Reduced activity of the renin-angiotensin II-aldosterone system could partially account for a CaSR mediated hypotensive effect of dietary and supplemental calcium.

Renal epithelial cells: Activation of the basolateral CaSR in the thick ascending limb of Henle’s (TAL) mediates the anti-hypertensive effect of elevated extracellular calcium in two ways: (i) CaSR signaling via PKC results in up-regulation of COX-2 and enhanced synthesis of the natriuretic prostaglandin E₂ [162,163]; (ii) by activation of PLA₂, CaSR signaling targets apical electrolyte transporters and channels participating in the control of electrolyte reabsorption from the lumen [164]. The importance of the CaSR in this process has been proven by identification of activating mutations in the CaSR gene, which cause Bartter syndrome type 5, a human disease characterized by excessive wasting of NaCl and other electrolytes [165]. Taken together, signaling from the basolateral CaSR in the TAL induces natriuresis and thereby causes reduction of plasma volume. This can partially explain the blood pressure-lowering effect of elevated extracellular calcium