Activating calcium-sensing receptor mutation in the mouse is associated with cataracts and ectopic calcification

Abstract

The extracellular calcium-sensing receptor (CaSR) plays a pivotal role in the regulation of extracellular calcium such that abnormalities, which result in a loss or gain of function, lead to hypercalcemia or hypocalcemia, respectively, in patients. Mice carrying CaSR knockout alleles develop hypercalcemia that mimics the disorders observed in humans. To date, there is no mouse model for an activating CaSR mutation. Here, we describe such a mouse model, named Nuf, originally identified for having opaque flecks in the nucleus of the lens in a screen for eye mutants. Nuf mice also display ectopic calcification, hypocalcemia, hyperphosphatemia, and inappropriately reduced levels of plasma parathyroid hormone. These features are similar to those observed in patients with autosomal dominant hypocalcemia. Inheritance studies of Nuf mice revealed that the trait was transmitted in an autosomal-dominant manner, and mapping studies located the locus to chromosome 16, in the vicinity of the CaSR gene (Mouse Genome Database symbol Gprc2a). DNA sequence analysis revealed the presence of a Gprc2a missense mutation, Leu723Gln. Transient expression of wild-type and mutant CaSRs in human embryonic kidney 293 cells demonstrated that the mutation resulted in a gain of function of the CaSR, which had a significantly lower EC(50). Thus, our results have identified a mouse model for an activating CaSR mutation, and the development of ectopic calcification and cataract formation, which tended to be milder in the heterozygote Nuf mice, indicates that an evaluation for such abnormalities in autosomal dominant hypocalcemia patients who have activating CaSR mutations is required.

Fig. 1.

Ectopic calcification and cataract formation in Nuf mice. (A–C) Cataracts in lenses (arrowheads). (A) Normal control 102/H mice. (B) Nuf/+ mice with few nuclear flecks. (C) Nuf/Nuf mice with more and larger flecks. (D–J) Soft tissue mineralization in Nuf/Nuf mutants. Arrows indicate sites of mineralization. (D) Faxitron x-ray image of a fixed tongue. (E–J) Hematoxylin/eosin-stained sections original magnification ×200 of the tongue (E), colon (F), skin vibrissa dermal sheath (G), testis artery wall (H), and blood vessels (I) in kidney cortex. (J) Positive Von Kossa staining confirms a calcium component of the mineralized deposits in the renal blood vessels.

Fig. 2.

Missense mutation in exon 7 of the CaSR (Gprc2a) of the Nuf mouse. (A) Analysis of the DNA sequence revealed an A-to-T transversion at codon 723 in the Nuf/Nuf mice. (B) At codon 723, the wild-type (WT) sequence is CTG encoding an evolutionarily conserved (arrowed) Leu (L) residue, whereas the mutant (M) sequence is CAG encoding a Gln (Q) residue. (C) The missense mutation resulted in the loss of a PstI restriction enzyme site (CTGCA/G) and this mutation facilitated its confirmation. Amplification with PCR and digestion with PstI resulted in two products of 179 and 172 bp, respectively, from the 102/H control sequence, but a larger product of 351 bp from the mutant sequence. S, DNA size marker (100-bp ladder).

Fig. 3.

Functional expression in HEK293 cells of the wild-type (WT) (L723) and mutant (Q723) CaSRs. HEK293 cells were transiently transfected with wild-type or the mutant CaSR-EGFP construct or with empty EGFP vector. Fluorescence microscopy was used to confirm successful transfection. (A and B) HEK293 cells transfected with wild-type (A) or mutant (B) CaSR showed similar expression patterns with fluorescence in the cytoplasm and at the plasma membrane, but not in the nucleus. (C) Cells transfected with pEGFP alone showed a uniform, nonspecific fluorescence pattern, whereas untransfected cells had no fluorescence (data not shown). The cells were counterstained with 4′,6-diamidino-2-phenylindole to show the nuclei (blue). (D) Western blot analysis of total cell protein extracts from HEK293 cells (transfected with either wild-type or the mutant CaSR by using an anti-GFP antibody) confirmed the expression of EGFP-tagged CaSRs (167 kDa), which was not present in untransfected (UT) cells. (E) Single, live cells loaded with indo-1 acetoxymethylester, emitting fluorescence at 525 nm, and hence containing transfected CaSR were selected by fluorescence-activated cell sorting and the [Ca²⁺]_o-evoked increases in [Ca²⁺]_i were measured. The increments in [Ca²⁺]_o from 0 to 10 mM are shown on the x axis, and the [Ca²⁺]_i response, which was measured as a percentage of the maximum normalized response, is shown on the y axis (mean ± SD of six estimations). The EC₅₀ of the mutant CaSR was significantly lower (P < 0.01) than that of the wild-type CaSR. The lower EC₅₀ and the leftward shift of the concentration–response curve of the mutant CaSR indicate that this mutation confers a gain of function of the mutant CaSR.