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3 The Calcium-Sensing Receptor and Its Implication in the Control of PTH Secretion
M.E. Rodríguez-Ortiz et al.
Although Ca is the main agonist of the CaR, other ligands are also capable of
activate it. They are classified as a) type I agonists, including other divalent (Mg2+,
Sr2+, Ba2+) and trivalent (La3+, Gd3+) cations, and b) type II agonists, as polyamines
(putrescine, spermine, spermidine) and amino acids . The ability of these molecules to activate the CaR has prompted the development of calcimimetics, drugs
capable of inhibiting the secretion of PTH upon the activation of the receptor.
The human CaR gene is located in the long arm of chromosome 3, at position 13
(3q13) whereas in the rat and mouse it resides on chromosomes 11 and 16, respectively. The protein of CaR has a molecular weight of 120 KDa. It possesses a large
N-terminal extracellular domain, a cysteine-rich domain linking the extracellular
domain to the first transmembrane helix, a seven transmembrane domain, and an
intracellular C-terminal domain. Although it also has been reported a binding site
for Ca in the transmembrane region of the receptor, the site located in the extracellular domain is required for the fully activation. The interaction between Ca and the
receptor induces a conformational change in its structure, the activation of transduction signals and the mobilization of intracellular Ca.
Mutations in CaR gene are responsible for several disorders. Inactivating mutations cause familial hypocalciuric hypocalcemia (FHH, OMIM 14598) as well as
neonatal severe hyperparathyroidism (NSHPT, OMIM 239200), whereas autosomal
dominant hypoparathyroidism (ADH, OMIM 601298) is associated to gain-offunction mutations in the CaR gene .
The CaR is not only expressed in organs directly involved in mineral homeostasis, such as parathyroid cells, C cells of the thyroid gland, bone or kidney, but also
in other tissues as gastrointestinal tract (esophagus, stomach, small intestine, and
colon), skin, cardiac and smooth muscle, and nervous system. The presence of the
CaR in the surface of parathyroid cells allows them to respond exquisitely to small
changes in the concentration of extracellular Ca. The activation of the receptor activates several signaling pathways (phospholipase A2 and C, protein kinase C, and
several mitogen-activated protein kinases) that eventually lead to the inhibition of
The activation of the CaR has also been shown to regulate the synthesis of
PTH. Indeed, in a state of hypocalcemia the stability of PTH mRNA is increased by
the binding of proteins to the specific region in the 3ʹ-untranslated region (UTR) that
protects the transcript from degradation. Naveh-Many et al. identified the A+U-rich
element binding factor (AUF1) as a protein that may confer stability to the PTH
mRNA transcript .
The expression of the CaR is modulated by several factors. Vitamin D augments
the expression of the receptor at parathyroid level. In an in vivo study, Brown and
collaborators found that administration of 1,25(OH)2D3 in rats fed with diets deficient in vitamin D stimulated the expression of the parathyroid CaR dosedependently . Of note, the expression of CaR in these vitamin D-deficient
animals was already diminished. The effect of vitamin D, as a modulator of the
parathyroid CaR, was confirmed in a subsequent work by Canaff and Handy. They
identified the transcriptional start sites of promoters P1 and P2 in the CaR and the
existence of vitamin D responsive elements (VDREs) in both sites .
7 Vitamin D and Parathyroid Hormone Regulation in Chronic Kidney Disease
In addition to vitamin D, other modulators of parathyroid function have also
been shown to regulate the CaR expression. For instance, moderately elevated
magnesium (Mg) upregulated parathyroid CaR expression in vitro . Similarly,
the addition of FGF23 to incubated parathyroid glands produced an increase in the
So far, the results obtained concerning the role of Ca itself as a regulator of the
CaR are contradictory. While some authors have not found an independent effect of
Ca , we observed that high Ca significantly increased the expression of CaR in
rat parathyroid glands culture in vitro . On the other hand, several studies have
shown that calcimimetics up-regulate the CaR in parathyroid glands from uremic
rats, where the expression of the receptor is already decreased . Moreover, this
effect appears to be independent of a possible effect on proliferation, as observed in
rats shortly after acute administration of calcimimetic .
Importantly, a novel mechanism that modulates CaR trafficking from cytosol to
the cell membrane has been recently described. The agonist-driven insertional signaling implies that the maturation, trafficking, and insertion of the CaR in the
plasma membrane are mediated by the ligand binding .
The Vitamin D Receptor
The biological effects of 1,25(OH)2D3, the most active vitamin D metabolite, are
mediated by the VDR. The VDR is a ligand-activated transcription factor belonging
to the superfamily of steroid/thyroid hormone receptors nuclear receptor that, when
bound to vitamin D, acts as a transcription factor.
The VDR was discovered in 1969 in the intestine of vitamin D–deficient chicks
. The VDR protein is composed of different structural regions with a certain
degree of variation between species. The primary amino acid sequence of the VDR
consists of six functional domains: the variable regions (A and B domains) which
include an activator region called AF-1, DNA binding (the C domain) is the most
conserved region and responsible for the recognition of the VDRE in the target
genes; this region contains two zinc fingers which are absolutely essential to stabilize the binding VDR-DNA. The hinge region (D domain) which gives to VDR an
ability of rotation enabling adequate link to VDREs, the ligand-binding region
(LBD, E domain) responsible for binding of vitamin D, and transcriptional activation (domain F) is the C-terminal region which contains a site for dimerization and
a AF-2 domain with regulatory function of transcription. To date, N terminal isoforms of the receptor have been identified that differ only in the length of their A/B
VDR is a DNA-binding transcription factor, which generates an active signaling
transduction complex. VDR control of gene transcription is mediated by several
stages including binding of vitamin D in the C-terminal portion of VDR, heterodimerization with retinoid X receptor (RXR) and nuclear translocation, binding of
VDR–RXR to specific DNA sequences (VDRE) in target gene promoter, and
M.E. Rodríguez-Ortiz et al.
recruitment of VDR-interacting nuclear co-regulators or co-factors resulting in activation or inhibition of gene transcription.
At the parathyroid level, the VDR directly down-regulates the transcription of
the gene encoding PTH through binding to VDRE. Negative VDREs have been
mapped both in the human and rat PTH promoters, resulting in a reduction of the
PTH synthesis [24, 25]. Results from VDR activation in other target tissues will not
be commented here.
Regulation of Vitamin D Receptor in Parathyroid Glands
An homologous regulation of VDR expression by 1,25(OH)2D3 amplifies the effect
of circulating 1,25(OH)2D3 on the PTH gene. This effect was described by NavehMany et al. : the administration of 1,25(OH)2D3 increased the levels of the VDR
mRNA in rat parathyroid glands. Other studies have confirmed this finding. Denda
et al.  reported that low serum 1,25(OH)2D3 levels were, at least in part, responsible for the decrease in VDR content in parathyroid glands of uremic rats and that
treatment with 1,25(OH)2D3 prevented this decrease, ameliorating the development
of SHPT. In vitro studies also demonstrated a direct effect of 1,25(OH)2D3 to
increase VDR expression .
Of special importance in the regulation of parathyroid function is the cooperation
of Ca and vitamin D on reducing parathyroid function. Several experimental and
clinical data suggests that the 1,25(OH)2D3 system is relatively ineffective in controlling PTH when serum Ca concentration is low. A study by Naveh-Many et al. 
showed that the ability of exogenous 1,25(OH)2D3 to inhibit PTH mRNA in vitamin
D–deficient rats was largely prevented when serum Ca was low, suggesting that the
inhibition of PTH by 1,25(OH)2D3 was not possible if hypocalcemia was present. In
another study , the administration of calcitriol (CTR) in vitamin D–deficient
chickens for 3–6 days increased parathyroid VDR mRNA, and this effect was
enhanced when serum Ca levels were normal. In this same study it was observed
that 6 days of dietary Ca restriction decreased VDR mRNA and that 1,25(OH)2D3
administration only upregulated VDR expression when chickens were fed a normal
Ca diet. In rats fed for 6 weeks with diets containing different vitamin D, Brown
et al.  did not observe an independent effect of 1,25(OH)2D3, but upregulation
of VDR by 1,25(OH)2D3 appeared as being mediated primarily by an increased
serum Ca concentration.
A definitive evidence for an independent effect of Ca on parathyroid VDR
expression came from in vivo experiments by Garfia et al. . By using a 6-h
hypercalcemic or hypocalcemic clamp in rats, it was found that 1,25(OH)2D3 produced an increase in VDR levels only when Ca concentration was elevated; however, 1,25(OH)2D3 did not increase VDR expression in rats maintained with
hypocalcemia during a 6-h period. In addition, the downregulation of VDR by
hypocalcemia resulted in impairment of the inhibitory action of 1,25(OH)2D3 on
parathyroid cell function. 1,25(OH)2D3 administered to rats after the induction of
7 Vitamin D and Parathyroid Hormone Regulation in Chronic Kidney Disease
hypocalcemia (when VDR was already downregulated) was not able to reduce PTH
mRNA, whereas it was downregulated in rats receiving 1,25(OH)2D3 before the
initiation of hypocalcemia. These results were also confirmed in vitro by Cañadillas
et al.  and by Carrillo et al. , who observed in rat parathyroid glands that the
higher the Ca, the higher VDR expression. Thus, beside the direct posttranscriptional effect on PTH mRNA, the low Ca may also stimulate PTH levels indirectly
through lowering the parathyroid VDR expression which makes 1,25(OH)2D3 less
effective. Of note, downregulation of parathyroid VDR caused by low Ca prevents
PTH inhibition by 1,25(OH)2D3. This is an important concept in the pathogenesis of
SHPT because hypocalcemia may not allow a normal inhibition of parathyroid cells
by 1,25(OH)2D3 even when 1,25(OH)2D3 levels are normal.
Finally, Cañadillas et al.  characterized in vitro the signaling system responsible for the stimulation of parathyroid VDR by extracellular Ca, which takes place
through the elevation of the cytosolic Ca level, and the subsequent stimulation of the
PLA2-AA-dependent ERK1/2- pathway. High extracellular Ca also increased the
VDR promoter activity through the activation of the Sp1 transcription factor in
VDR-transfected HEKCaR cells.
Taken together, the above data support that the tight control of PTH secretion and
synthesis by Ca may in part be explained by the fact that high Ca enhances the
inhibitory action of vitamin D on parathyroid glands by augmenting VDR
Besides 1,25(OH)2D3 and Ca, several other factors have been reported to regulate
VDR expression and/or function. Calcimimetics, such as R-568 and cinacalcet HCl,
are allosteric modulators of the CaR, acting by increasing the sensitivity of the parathyroid gland to extracellular Ca. Thus, it was hypothesized that administration of
the calcimimetic R-568 may result in increased VDR expression in parathyroid tissue. In in vitro studies with whole rat parathyroid glands, the calcimimetic elicited
an increase in VDR mRNA similar to the maximum increase detected with 1.5 mM
Ca . Treatment with R-568 also increased VDR protein in normal rat parathyroid glands and, interestingly, in human parathyroid glands with diffuse but not
nodular hyperplasia. These results support the convenience of using vitamin
D-calcimimetic combinations in the clinical settings. Any increase in VDR would
facilitate the inhibitory feedback of vitamin D on the parathyroid glands and would
assist in optimization of the positive action of the pharmacological administration
of 1,25(OH)2D3 or other vitamin D analogs, which in turn may reduce unwanted
side effects as high CaxP products and vascular calcifications.
It is now well-known that FGF23 decreases PTH secretion and PTH mRNA .
Furthermore, in normal rat parathyroid glands in vitro, addition of FGF23 to the
low-Ca medium increased parathyroid VDR and CaR mRNA and protein expression to levels similar to those observed with a high-Ca concentration . The
FGF23-dependent changes in VDR and CaR expression were paralleled by the activation of the ERK1/2 activation. FGF23 also increased VDR and CaR expression
and activated ERK1/2 in the parathyroid glands of normal rats in vivo. This upregulation of CaR and VDR may represent another mechanism whereby FGF23 reduces