The extracellular calcium-sensing receptor (CaSR) is a critical modulator of skeletal development

Abstract

The extracellular Ca(2+)-sensing receptor (CaSR) plays a nonredundant role in the functions of the parathyroid gland (PTG) and the kidney. Severe hyperparathyroidism, premature death, and incomplete gene excision in Casr(-/-) mice have precluded the assessment of CaSR function in other tissues. We generated mice with tissue-specific deletion of Casr in the PTG, bone, or cartilage. Deletion of Casr in the PTG or bone resulted in profound bone defects, whereas deletion of Casr in chondrocytes (cartilage-producing cells) resulted in death before embryonic day 13 (E13). Mice in which chondrocyte-specific deletion of Casr was induced between E16 and E18 were viable but showed delayed growth plate development. Our data show a critical role for the CaSR in early embryogenesis and skeletal development.

Fig. 1

Generation of floxed Casr mice. (A) The gene-targeting strategy used to introduce loxP sequences to flank exon 7 of Casr required a construct containing three loxP sites flanking exon 7 of the Casr gene and the cytidine deaminase (CD)–neomycin (NEO) gene cassette. This construct was trans-fected into 129/SvJae ES cells to allow homologous recombination with endogenous Casr alleles. The resulting floxed Casr-containing ES cells were injected into C57/BL6 blastocysts to produce chimeric mice that were bred to obtain heterozygous (Casr^wt/flox) and homozygous (Casr^flox/flox) mice. (B) PCR analyses of genomic DNAs from floxed Casr-containing ES cells before blastocyst injection and from the tails of Casr^wt/wt (WT), heterozygous Casr^wt/flox (Het), and homozygous Casr^flox/flox (Hom) mice were performed with specific primers (see Materials and Methods) to confirm integration of the targeting sequences. (C) PCR analyses of genomic DNAs from (B) after incubation with (+Cre) or without (−Cre) bacteriophage P1 Cre recombinase in vitro for 30 min. The 284-bp band represents the DNA fragment due to excision of exon 7 (ΔExon7).

Fig. 2

Heterozygous and homozygous deletion of the Casr gene in parathyroid cells (PTCs) produces mild and severe hyperparathyroidism (HPT), respectively. (A) Two-week-old PT-KO (^PTCaSR^{Δflox/Δflox}) mice showed severe growth retardation, with body weights about 45% of that of their control and PT-Het (^PTCaSR^WT/Δflox) littermates. (B) PCR analyses of genomic DNAs confirmed the expression of the Cre trans-gene and floxed Casr alleles in PT-Het and PT-KO mice. The control mice used in this experiment were CaSR^flox/flox, which do not express Cre. (C) PCR analyses of genomic DNAs from tissues of PT-KO mice confirmed the deletion of exon 7 only in PTGs. (D) Western blotting of PTG lysates showed the decreased abundance of full-length CaSR protein (WT, 120 to 250 kD) by ∼70% and >95% in the PT-Het and PT-KO mice, respectively, compared with control littermates. PTG ly-sates from PT-Het and PT-KO mice contained the ΔExon7-CaSR (ΔExon7) protein (about 90 kD), which was encoded by exons 1 to 6. (E) qPCR analyses with primers flanking the junctions of either exons 6 and 7 (E6-7) or exons 2 and 3 (E2-3) of Casr and primers specific for PTH mRNA were presented as the percentage of expression of the gene encoding the mitochondrial ribosomal protein L19 (**P < 0.01, n = 4 to 6 mice).

Fig. 3

Knockout of Casr in PTCs impedes skeletal development. (A) Whole-mount AR and AB staining showed a smaller skeleton with fractures in the ribs and tibiae (see insets) of 14-day-old PT-KO (^PTCasr^{Δflox/Δflox}) mice, but not in PT-Het (^PTCaSR^WT/Δflox) orcontrol mice. (B) Reconstructed 3D μCT images of femurs from 14-day-old PT-KO mice showed severely undermineralized matrix compared with those of control mice. (C) Histological analyses of femurs from PT-KO mice showed reduced mineralization and osteoid accumulation. Consecutive plastic-embedded femur sections from 14-day-old control and PT-KO mice stained with VK reagents and counterstained with SO or Goldner reagents were visualized at 5× magnification. Boxes in the right panels are enlarged regions of interest (reproduced at 20×). Scale bars: 800 μm at 5×; 200 μm at 20×. (D) Gene expression studies indicated the delayed differentiation of osteoblasts in bones from PT-KO mice compared with that of control mice. qPCR analyses of genes encoding osteoblast markers were performed on RNA isolated from humeral cortices (no marrow) of 14-day-old mice. All measurements of gene expression are presented as the percentage of L19 expression (*P < 0.05, **P < 0.01; n = 6 to 8 mice). Expression of OSX, Col(I), ALP, DMP1, OCN, and SOST in PT-KO mice was reduced by 36%, 48%, 48%, 43%, 91%, and 74%, respectively, compared with that in control mice. PCR analyses with primers flanking the junctions of exons 6 and 7 (E6-7) or exons 2 and 3 (E2-3) of Casr showed reductions in both transcripts in bone in which the PTH-Cre transgene was not expressed. (E) Reconstructed 3D μCT images of Tb bone in distal femurs and Ct bone at the TFJ of 6-month-old control and PT-Het mice. Scale bars: 1 mm for all panels. (F) μCT parameters assessed were BV, BV/TV, Th, N, CD, and Sp for Tb bone and TV, BV, and Th for Ct bone (defined in Results; *P < 0.05, **P < 0.01; n = 6 to 8 mice).

Fig. 4

Knockout of Casr in osteoblasts, driven by 2.3Col(I)-Cre, blocked growth and skeletal development. (A) Body weights of control (n = 8), heterozygous (^Col-BoneCasr^WT/Δflox, COL-Het, n = 12), and homozygous KO (^Col-BoneCasr^{Δflox/Δflox}, COL-KO, n = 9) mice from birth until postnatal day 20. Because the weights of control and Het mice were indistinguishable, they were combined and are presented as Cont+Het. COL-KO mice exhibited growth retardation, which was evident by postnatal day 3. (B) Whole-mount AR and AB staining showed that the skeletons of COL-KO mice were smaller than those of control and heterozygous mice at postnatal day 5. (C) Reconstructed 3D μCT images of skeletons from 20-day-old mice showed severe undermineralization in COL-KO mice, evident in the skull (arrowheads), vertebrae (double arrows), and long bones (arrows). (D) μCT parameters of Tb bone in the distal femur (Tb.BV, Tb.BV/TV, Tb.Th, Tb.N, Tb.CD, and Tb.Sp) and Ct bone at the TFJ (Ct.TV, Ct.BV, and Ct.Th) of 20-day-old control and COL-KO mice showed statistically significant reductions in all parameters except Tb. Sp, which was markedly increased—all of which were confirmatory of reduced bone mass and microarchitecture (**P < 0.01; n = 6 mice). (E) Histological analyses of femurs from COL-KO mice showed poor mineralization and osteoid accumulation compared with those of control mice. VK+SO and Goldner staining were performed on consecutive plastic-embedded sections from 7-day-old control and COL-KO femurs and visualized at 5× magnification. Boxes in the right panels are enlarged regions of interest (reproduced at 20×). Scale bars: 500 μm (5×); 125 μm (20×). (F) The expression of genes encoding osteoblast markers indicated delayed differentiation in COL-KO mice compared with that in control mice. qPCR analyses of samples isolated from humeral cortices (no marrow) from 14-day-old mice with primers specific for OSX, Col(I), ALP, DMP1, OCN, SOST, IGF-1, CCND1, Bcl-2, Bcl-2L1, and IL-10 were performed. Results are presented as the percentage of expression of L19 or glyceraldehyde 3-phosphate dehydrogenase(GAPDH) (**P < 0.01; n = 6 to 9 mice). Reductions (percent decrease in COL-KO mice compared with control mice) of 54%, 35%, 47%, 77%, 79%, and 22% were seen in the expression of Col(I), ALP, DMP1, OCN, SOST, and IGF-1, respectively. (G) TUNEL staining of femoral cortex from 7-day-old control and KO mice with hematoxylin counter-staining. TUNEL-positive cells are indicated by brown DAB stain (arrowheads) (n = 8 sections from four mice).

Fig. 5

Knockout of floxed Casr in osteoblasts by OSX-Cre results in retarded growth and altered gene expression. (A) PCR analyses of genomic DNAs confirmed that deletion of exon 7 occurred only in bone from homozygous ^Osx-BoneCasr^{Δflox/Δflox} (OSX-KO) mice and not in other tissues. (B) One-month-old OSX-KO mice were growth-retarded compared with control mice. (C) μCT images of trabecular (Tb) and cortical (Ct) bones in the distal femur and TFJ, respectively, showed severely under-mineralized matrix in 4-week-old KO mice compared with that in control mice. Scale bars: 1 mm for all panels. (D) μCT parameters assessed included BV, BV/TV, Th,N,CD, and SpforTbbone and TV, BV, and Th for Ct bone in 4-week-old control and OSX-KO mice. (*P < 0.01; n = 6 for KO and n = 12 for control mice). (E) Analysis of gene expression by qPCR in samples isolated from humeral cortices (no marrow) of newborn mice indicated delayed differentiation of osteoblasts in OSX-KO mice compared with that of control mice, with 55% reduced expression of Col(I) and 35% reduced expression of OCN. Results are presented as the percentage of L19 expression (*P < 0.05, n = 4 to 6 mice).

Fig. 6

Knockout of Casr in chondrocytes blocks embryonic development and cartilage maturation. (A) Whole-mount AR and AB staining shows that the skeletons of Cart-KO (^CartCasr^{Δflox/Δflox}) mice are smaller and undermineralized compared with those of their heterozygous (^CartCasr^WT/Δflox) and control littermates at E12.5. (B to F) Tamoxifen (Tam)-induced knockdown of the Casr in cartilage produces small, undermineralized skeletons in E18-19 ^Tam-CartCasr^{Δflox/Δflox} (Tam-Cart-KO) embryos. (B) PCR analysis of genomic DNAs from embryos with (+) or without (−) exposure to 4OH-Tam for the expression ofthe ^TamCre transgene, homozygous floxed Casr (fl-Casr) alleles, and sequences lacking exon 7 (ΔExon7). (C) Whole-mount AR and AB staining of E19 embryos showed smaller skeletons, including humeri, femurs, and tibiae in Tam-Cart-KO compared with those of control mice. (D) Immunohistochemical staining of CaSR, depicted by brown DAB staining, in the proximal tibial growth plates from E18 to E19 Tam-Cart-KO and control mice. PZ, proliferation zone; MZ, maturation zone; HZ, hypertrophic zone. (E) VK and SO staining of plastic sections of proximal tibial growth plates from E18 to E19 Tam-Cart-KO and control mice. The histogram presents HZ widths in proximal tibial growth plates from control and Tam-Cart-KO mice (*P < 0.01; n = 4 mice). (F) Gene profiling in the epiphyseal growth plates indicates a delay in cell maturation and differentiation in Tam-Cart-KO mice compared with that in control mice. qPCR was performed on epiphyseal growth plate samples from E18 to E19 Tam-Cart-KO and control mice with primers specific for early [AGG and Col(II)] and late [Col(X), OPN, and RUNX2] chondrocyte markers and for IGF-1, IGF-1R, and Casr. Results are presented as the percentage of L19 expression (*P < 0.05, **P < 0.01; n = 6 mice).

Fig. 7

Knockout of Casr in chondrocytes blocks the expression of IGF-1 and IGF-1R and delays cell differentiation. (A) Immunohistochemical staining of IGF-1 and IGF-1R, depicted by brown DAB staining, in the proximal tibial growth plates from E18 to E19 Tam-Cart-KO and control mice. (B) In vitro knockout of IGF-1R was performed by infecting floxed IGF-1R-containing GPCs with Ad-Cre (Cre) or, as a control, Ad-Cont (Cont) viruses (16 PFU/cell). Expression of IGF-1R and Col(X) mRNAs was assessed by qPCR 72 hours postinfection. (C) Western blots were incubated with antisera against the β subunit of IGF-1R to confirm the knockdown of IGF-1R in GPCs infected with Ad-Cre (Cre) compared with that in Ad-Cont (Cont) viruses. (D) AR staining was performed and quantified by absorbance to assess mineralization in cultures infected with Ad-Cre (Cre) or Ad-Cont (Cont) viruses and grown in media containing 2.0 mM Ca²⁺ for 14 days (**P < 0.001; n = 3).