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-Subunits Leads to a Phenotype Resembling Germline Knockout of the Extracellular Ca2+-Sensing Receptor
Pharmakologisches Institut (N.W., S.O.), Universität Heidelberg, 69120 Heidelberg, Germany; and Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research (E.L., P.G.R.), Surgery Branch, Center for Cancer Research, National Cancer Institute (S.K.L.), and Molecular Pathophysiology Section, National Institute on Deafness and Other Communication Disorders (A.M.S.), National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Nina Wettschureck, Pharmakologisches Institut der Universität Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. E-mail: nina.wettschureck{at}pharma.uni-heidelberg.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunits of Gq and G11 selectively in parathyroid cells. Mice that were PTH-Cre+/–; Gnaqflox/flox; Gna11–/– (PTH-Gq/G11-double knockouts) were viable, but showed all the features of germline knockout of the CaR except hypocalcuria. Our results demonstrate the critical role of both Gq and G11 in mediating inhibition of PTH secretion by extracellular Ca2+. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The CaR is a member of family 3 of the G protein-coupled receptor superfamily (4). This suggests that heterotrimeric G proteins might be critical for Ca2+ inhibition of PTH secretion, but to date there is no clear evidence supporting this hypothesis. Furthermore, the specific G protein(s) to which the CaR couples to mediate inhibition of PTH secretion is unclear (5). There is considerable evidence that the CaR couples to members of the Gq/G11 family (6, 7) but also evidence for coupling to Gi family members (8) as well as G12/G13 (9). There is evidence for expression of members of each of these G protein families in bovine parathyroid glands (10). It is also theoretically possible that the activated CaR inhibits PTH secretion via a G protein-independent mechanism, given recent evidence that not all of the actions of typical seven-transmembrane receptors involve G protein coupling (11).
To test the importance of the Gq/G11 proteins in inhibition of PTH secretion, we employed cre/lox technology to delete the -subunits of Gq and G11, Gq and G11, selectively in the parathyroid gland. Previous work in which homozygous germline deletions of either Gq or G11 had been performed did not produce mice with a phenotype resembling that of CaR knockout mice (12), but here we show that parathyroid-selective deletion of both copies of Gq and G11 leads to a phenotype closely resembling that of the CaR knockout, thus demonstrating the critical role of these G proteins in the inhibition of PTH secretion by Ca2+.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunits of Gq and G11, Gq and G11, selectively in the parathyroid gland. To do so, we mated mice in which the gene coding for Gq, Gnaq, is flanked with loxP sites (Gnaqflox; Ref. 13) to the constitutively G11-deficient mouse line (Gna11–/–; Ref. 14) and to mice which express the recombinase Cre under control of the human PTH promoter (PTH-Cre+/–; Ref. 15).
To test whether Cre expression indeed leads to parathyroid-specific loss of Gq protein, we performed Western blots with extracts from parathyroid glands, thyroid, kidney, heart, and other organs from 9-d-old PTH-Cre+/–; Gnaqflox/flox; Gna11–/– mice and wild-type animals (Fig. 1A
). We found that Gq/G11 protein was almost undetectable in parathyroid glands of PTH-Cre+/–; Gnaqflox/flox; Gna11–/– mice [PTH-Gq/G11-double knockouts (DKOs)], whereas Gq/G11 protein levels were not significantly altered in any other organ tested (Fig. 1A).
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q/G11-DKO pups were born at the expected ratio and were initially indistinguishable from their PTH-Cre–/–; Gnaqflox/flox; Gna11wt/wt littermates. However, during the first days after birth, double-deficient mice failed to thrive and usually died within the first 3 wk after birth, most of them before postnatal d 14 (Fig. 1B
). Body length and body weight of PTH-Gq/G11-DKO pups was massively reduced at postnatal d 9 (Fig. 1, C and D). The body weight of littermates with only one intact allele Gq or G11 was also reduced (Fig. 1D), but did not result in impaired survival (data not shown).
Internal organs of PTH-Gq/G11-DKO pups were macroscopically and microscopically normal (data not shown), but reduced in size according to the general growth retardation of these animals. Also, the larynx and its associated structures like thyroid gland were massively retarded in growth (Fig. 2, A and B), whereas the size of the parathyroid glands did not differ significantly from that of control mice (Fig. 2, A and B). To investigate whether this relative increase of parathyroid gland size was due to enhanced proliferation of mutant cells, we treated 6-d-old wild types and PTH-Gq/G11-DKOs with 5-bromo-2'-deoxyuridine (BrdU). We found that the number of BrdU-positive cells was significantly enhanced in mutant parathyroid cells (Fig. 2, C and D).
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). Double-deficient pups showed strongly increased PTH levels, and also mice with only one intact Gq or G11 allele in the parathyroid gland had elevated PTH concentrations. Serum calcium levels were strongly increased in PTH-Gq/G11-DKOs, as well as, although less prominent, in animals with one intact Gq or G11 allele (Fig. 3B). Urine calcium excretion was also strongly increased in PTH-Gq/G11-DKOs, and animals with one intact Gq or G11 allele showed intermediate values (Fig. 3C). To test whether hypercalciuria might be partially due to a down-regulation of PTH receptor subtype 1 (PTH-R1) in response to chronically elevated PTH levels, we performed RT-PCR and Western blotting for the parathyroid receptor subtype 1. We found that expression of PTH-R1 at the mRNA level was not altered in PTH-Gq/G11-DKOs (Fig. 3D), and also in Western blotting no marked differences were detected (data not shown)
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q/G11 would cause compensatory changes in the expression of the parathyroid calcium-sensing receptor. Western blotting of parathyroid glands as well as immunofluorescence staining of laryngeal sections revealed decreased expression of the parathyroid gland calcium-sensing receptor (Fig. 4, A and B).
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q/G11-DKO mice exhibited severe dwarfing, and an axial and appendicular skeletal phenotype compared with wild-type mice. Radiographically, the DKO mice were severely osteopenic throughout the skeleton, with barely detectable mineralization in the bones of the paws and caudal vertebrae (Fig. 5A). By staining with alcian blue/alizarin red, a delay in endochondral bone formation was noted in the cervical and caudal vertebrae, bones of the paws, and all long bones (Figs. 5B and 6, F and J). Mineralization of the ventral ribs was lacking, as well as epiphyseal ossification of the femur and tibia (Fig. 6, D and H). All growth plates displayed rachitic changes, as highlighted by the presence of bulging of the costochondral junction (rachitic rosary) in the ribs (Fig. 6D). In addition to the profound defects in the axial and appendicular skeleton, abnormalities were also noted in the craniofacial complex, which develop by three different mechanisms: intramembranous bone formation, endochondral bone formation, and a third mechanism that requires remodeling of unmineralized cartilage before bone formation (16). PTH-Gq/G11-DKO mice exhibited a delay in all three of these processes, resulting in an anterior-posterior shortening, most pronounced in the snout, and doming of the cranial vault (Fig. 6B). The delay in intramembranous bone formation in the cranial vault resulted in formation of extremely wide sutures, coupled with evidence of bone resorption.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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q/G11-DKO mice, markedly increased serum calcium and PTH, relative increase in parathyroid gland size, severe delay in bone formation and evidence of bone resorption, decreased body size, and early postnatal lethality, all closely resemble the phenotype seen in CaR-null mice. An important phenotypic difference is the increased urinary calcium excretion we observed in PTH-Gq/G11-DKO mice, which contrasts with the relative reduction in urinary calcium excretion seen in CaR-null mice (3). This difference results from the loss in CaR germline null mice of the normal function of the CaR in kidney to promote urinary calcium excretion as serum calcium is elevated, whereas the kidney CaR is intact in PTH-Gq/G11-DKO mice. Increased urinary calcium excretion in the latter is clearly insufficient in preventing severe hypercalcemia. Our results clearly indicate that loss of CaR function exclusively in the parathyroid is sufficient to cause the NSPHT phenotype.
We found no evidence for a compensatory increase in expression of CaR in parathyroids (17) of PTH-Gq/G11-DKO mice, but rather a relative reduction in expression. This may occur as a function of the apparent increase in parathyroid growth, because reduced CaR expression has been noted in other conditions characterized by increased parathyroid cell proliferation (17). A prominent change in CaR expression might affect calcium homeostasis by itself; however, given the small magnitude of CaSR down-regulation, a major contribution to the observed phenotype seems unlikely.
Characterization of the skeletal phenotype in the initial report of germline CaR knockout (KO) mice was limited to a skeletal radiograph showing decreased radiodensity, kyphoscoliosis, and bowing of long bones (3). Subsequently, Garner et al. (18) performed more detailed skeletal analysis and reported that rickets was the predominant skeletal abnormality in germline CaR KO mice. They speculated that aspects of the skeletal phenotype might reflect CaR deficiency in bone cells rather than simply the skeletal consequences of severe hyperparathyroidism. Although we did not perform histologic analyses, our radiographic and skeletal staining results are generally consistent with those of Garner et al., showing not only severe osteopenia but also rachitic changes. Garner et al., however, noted normal craniofacial development in germline CaR KO mice, whereas PTH-Gq/G11-DKO mice show a domed cranial vault, very wide sutures, and other dysmorphic features. A more detailed histological analysis will be required to define fully the basis for abnormal skeletal development in the present model.
Our results show that both Gq and G11 are necessary for normal regulation of PTH secretion by Ca2+. This requirement presumably reflects the coupling of both G proteins to the parathyroid CaR. Both Gq and G11 are expressed in virtually all cells, and they may be functionally redundant in coupling to a variety of G protein-coupled receptors (12). For the parathyroid CaR, there appears to be no functional consequence of deletion of both copies of either Gq or G11. If three of the four alleles of these G proteins were deleted in the parathyroid, however, biochemical abnormalities, albeit less severe than those in DKO mice, were observed. The physiological role, if any, of other G proteins including Gi and G12/13 in coupling to the CaR in parathyroid cells is unclear, but our results do not exclude a role for such proteins in the actions of the CaR in other cells such as in the kidney.
Hypermorphic alleles of Gq or G11 were identified in mice with increased skin pigmentation (19). These mice also showed a reduction in body size that was accentuated with increased number of G protein mutant alleles. No data concerning serum PTH or calcium were provided in this report, nor was the mechanism for growth impairment defined. The functional implications, if any, of activating mutations of Gq or G11 in the parathyroids remains to be determined.
CaR coupling to Gq or G11 is presumed to lead to activation of phospholipase C-ß, which in turn leads to generation of diacylglycerol and inositol 1,4,5-triphosphate and thereby increased intracellular Ca2+. These intracellular signals have been correlated with inhibition of PTH secretion (1, 5). Our results in PTH-Gq/G11-DKO mice do not prove that these signals are critical for inhibition of PTH secretion, but we speculate that this is likely to be the case. Further studies will be needed to verify this hypothesis, and to clarify why such signals, which generally lead to increased protein and hormone secretion, in the parathyroid have the opposite effect.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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q/G11-Double-Deficient Miceq/G11-double-deficient mice (PTH-Gq/G11-DKO) were generated by crossing the parathyroid-specific Cre-expressing line PTH-Cre (PTH-Cre+/–) (15) to mice carrying floxed Gq alleles (Gnaqflox/flox) (13) and constitutively G11-deficient mice (Gna11–/–) (14). Intercrosses between PTH-Cre+/–; Gnaqflox/wt; Gna11–/wt mice resulted in the generation of parathyroid-specific Gq/G11-double-deficient mice (PTH-Cre+/–; Gnaqflox/flox; Gna11–/–), of mice with one intact Gq or G11 allele in the parathyroid gland (PTH-Cre+/–; Gnaqwt/flox; Gna11–/– and PTH-Cre+/–; Gnaqflox/flox; Gna11–/wt, respectively), and control littermates (PTH-Cre–/–; Gnaqflox/flox; Gna11wt/wt). Genotyping for Gnaq and Gna11 alleles was performed as described previously (13). For genotyping of the PTH-Cre transgene, the primers 5'-TAAGCCCCTTGTCAAGCCAAA-3' and 5'-GCAGCGATCGCTATTTTCCAT-3' were used. Animals were on a mixed genetic background with a predominant contribution of the C57BL6/N strain (fourth-generation backcross). In some experiments, also age-matched nonlittermate C57BL6/N wild types were used as controls. Animal experiments were in accordance with Institutional Animal Care and Use Committee regulations.
Electrolyte and Hormone Levels
Serum samples in 9-d-old pups were prepared from blood obtained from the abdominal vena cava under deep anesthesia with 100 mg/kg pentobarbital, ip. Urine was collected by aspiration from the urinary bladder. In PTH-Gq/G11-DKOs mice that survived to an age of 3 wk, blood was collected from the retroorbital plexus. Serum calcium levels were determined with the calcium-o-cresolphthalein complexone method (20). Blood and urine phosphate levels were determined with the Liqui-UV Phosphorus kit (Stanbio, Boernen, TX). Serum PTH levels were determined using a mouse intact PTH ELISA kit (Immutopics, San Clemente, CA).
Histology
Mice were killed at postnatal d 9, and tracheal blocks and other organs were dissected, dehydrated, embedded in paraffin, and sectioned (5 µm) on a rotating microtome (Microm, Walldorf, Germany). Hematoxylin and eosin staining was performed according to standard procedures. For morphometric analysis, sections were digitalized and thyroid and parathyroid cross-sectional area in the section with maximal parathyroid diameter was determined by computerized pixel counting. For immunofluorescence staining, paraffin sections were incubated with polyclonal rabbit calcium-sensing receptor antibody ADD, raised against a synthetic peptide corresponding to residues 214–235 of the human CaR (1:2000; Ref. 21) at 4 C overnight and tetramethylrhodamine isothiocyanate-labeled goat antirabbit antibody (1:200; Jackson ImmunoResearch, West Grove, PA) for 2 h. For BrdU labeling, 6-d-old mice were injected ip with 10 mg/kg body weight BrdU and killed after 4 h. Paraffin sections of the thyroid gland/parathyroid gland were stained with anti-BrdU antibody (BD Biosciences, San Diego, CA; 1:100) and FITC-labeled goat antirabbit antibody (1:200; Jackson ImmunoResearch) for 2 h.
Western Blotting
The thyroid gland was dissected from tracheal blocks, and parathyroid glands were prepared from the lateral lobes of the thyroid gland under a dissection microscope. Parathyroid glands and other tissues were homogenized in hypotonic buffer [10 mM dithiothreitol, 5 mM EDTA, 20 mM HEPES (pH 7.5)] in the presence of protease inhibitors, and after centrifugation the pellet was extracted for 60 min in 20 µl of hypotonic buffer containing 1% deoxycholate. Five micrograms of protein extract were loaded on a 10% SDS-PAGE gel, electrophoresed, blotted, and sequentially probed with polyclonal rabbit antibodies against Gq/G11 (sc-392; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit antibodies directed against the calcium-sensing receptor, ADD (21), or mouse monoclonal -tubulin antibodies (clone DM1a; 1:1000; Sigma-Aldrich, St. Louis, MO). For parathyroid glands, the complete extract from one gland was loaded per lane. Signals were detected by incubation with horseradish peroxidase-conjugated secondary antibodies (1:2000; Sigma-Aldrich). Chemiluminescence was developed using the ECLplus kit (Amersham Biosciences, Freiburg, Germany).
Analysis of the Skeletal Phenotype
Mice were euthanized at postnatal d 9 and radiographic images were obtained using a Faxitron MX-20 Specimen Radiography System (Faxitron X-Ray Corp., Buffalo Grove, IL) at an energy of 30 kV for 90 sec with X-OMAT TL film and processed by an automated x-ray film developer (Eastman Kodak, Rochester, NY). In some cases, mice were skinned and eviscerated, and fixed and stored in 95% ethanol. Cartilage was stained with alcian blue (0.03% in 80% ethanol/20% acetic acid) for 3 d, washed with 95% ethanol for 6 h. After soaking in 2% potassium hydroxide for 24 h, bone was stained with alizarin red (0.03% in aqueous 1% potassium hydroxide) for 24 h, cleared (1% potassium hydroxide/20% glycerol), and stored in 1:1 glycerol/95% ethanol.
Semiquantitative RT-PCR Analysis of PTH-R1 Expression
Total RNA was extracted from mouse kidneys using Trizol according to the manufacturer’s instructions (Invitrogen, Carslbad, CA), and digested with deoxyribonuclease I (Roche Diagnostics, Mannheim, Germany) for 10 min at 37 C. cDNA was synthesized using SuperScript synthesis according to the manufacturer’s instructions (Promega, Madison, WI). A 200-bp sequence spanning exons 8 and 9 of the PTH-R1 gene was amplified in GoTaq green mater mix containing the forward primer, 5'-ATCTTCGTGAAGGACGCTGT-3' and the reverse primer, 5'-CCCTCCACCAGAATCCAGTA-3' (GenBank accession no. NM_011199, bases 709–728, bases 955–974). The target cDNA was amplified during 25 and 30 cycles at 94 C for 30 sec for denaturation, 55 C for 30 sec for annealing, and 72 C for 15 sec for extension. A final extension was at 72 C for 15 sec. For comparison, a 816-bp sequence of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using the forward primer 5'-AGCCGCATCTTCTTTTGCGTC-3' and the reverse primer 5'-TCATATTTGGCAGGTTTTTCT-3' (GenBank accession no. M33197, bases 12–32, bases 807–827) by running the reaction for 15 min at 94 C, and then 25 and 35 cycles at 94 C for 45 sec, 58 C for 45 sec, and 72 C for 1 min, and final extension at 72 C for 7 min. The amplification products were separated by 1.5% TBE-agarose gel.
Statistics
Values were expressed as mean ± SEM. Differences between two groups were statistically analyzed using unpaired Student’s t test. Differences between more than two groups were analyzed using ANOVA. Statistical significance was accepted at P < 0.05.
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FOOTNOTES |
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Present address for A.M.S.: Office of the Dean, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461.
N.W., E.L., S.K.L., and S.O. have nothing to declare. P.G.R. and A.M.S. have equity interests.
First Published Online September 20, 2006
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; CaR, Ca2+-sensing receptor; DKO, double knockout; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout; NSPHT, neonatal severe primary hyperparathyroidism; PTH-R1, PTH receptor subtype 1.
Received for publication March 7, 2006. Accepted for publication September 14, 2006.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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12/13 to activate phospholipase D in Madin-Darby canine kidney cells. Am J Physiol 286:C22–C30-subunits in bovine parathyroid. Endocrinology 136:4390–4396[Abstract]q/G11 in cardiomyocytes. Nat Med 7:1236–1240[CrossRef][Medline]q/G11-mutant mice. EMBO J 17:4304–4312[CrossRef][Medline]This article has been cited by other articles:
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  T. Meir, R. Levi, L. Lieben, S. Libutti, G. Carmeliet, R. Bouillon, J. Silver, and T. Naveh-Many Deletion of the vitamin D receptor specifically in the parathyroid demonstrates a limited role for the receptor in parathyroid physiology Am J Physiol Renal Physiol, November 1, 2009; 297(5): F1192 - F1198. [Abstract] [Full Text] [PDF]
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V. Lavi-Moshayoff, J. Silver, and T. Naveh-Many Human PTH gene regulation in vivo using transgenic mice Am J Physiol Renal Physiol, September 1, 2009; 297(3): F713 - F719. [Abstract] [Full Text] [PDF]
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 N. Ogata, H. Kawaguchi, U.-i. Chung, S. I. Roth, and G. V. Segre Continuous Activation of G{alpha}q in Osteoblasts Results in Osteopenia through Impaired Osteoblast Differentiation J. Biol. Chem., December 7, 2007; 282(49): 35757 - 35764. [Abstract] [Full Text] [PDF]
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