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Cilia-like Structures and Polycystin-1 in Osteoblasts/Osteocytes and Associated Abnormalities in Skeletogenesis and Runx2 Expression

2006; Elsevier BV; Volume: 281; Issue: 41 Linguagem: Inglês

10.1074/jbc.m604772200

ISSN

1083-351X

Autores

Zhousheng Xiao, Shiqin Zhang, Josh Mahlios, Gan Zhou, Brenda S. Magenheimer, Dayong Guo, Sarah L. Dallas, Robin L. Maser, James P. Calvet, Lynda F. Bonewald, L. Darryl Quarles,

Tópico(s)

Microtubule and mitosis dynamics

Resumo

We examined the osteoblast/osteocyte expression and function of polycystin-1 (PC1), a transmembrane protein that is a component of the polycystin-2 (PC2)-ciliary mechano-sensor complex in renal epithelial cells. We found that MC3T3-E1 osteoblasts and MLO-Y4 osteocytes express transcripts for PC1, PC2, and the ciliary proteins Tg737 and Kif3a. Immunohistochemical analysis detected cilia-like structures in MC3T3-E1 osteoblastic and MLO-Y4 osteocyte-like cell lines as well as primary osteocytes and osteoblasts from calvaria. Pkd1m1Bei mice have inactivating missense mutations of Pkd1 gene that encode PC1. Pkd1m1Bei homozygous mutant mice demonstrated delayed endochondral and intramembranous bone formation, whereas heterozygous Pkd1m1Bei mutant mice had osteopenia caused by reduced osteoblastic function. Heterozygous and homozygous Pkd1m1Bei mutant mice displayed a gene dose-dependent decrease in the expression of Runx2 and osteoblast-related genes. In addition, overexpression of constitutively active PC1 C-terminal constructs in MC3T3-E1 osteoblasts resulted in an increase in Runx2 P1 promoter activity and endogenous Runx2 expression as well as an increase in osteoblast differentiation markers. Conversely, osteoblasts derived from Pkd1m1Bei homozygous mutant mice had significant reductions in endogenous Runx2 expression, osteoblastic markers, and differentiation capacity ex vivo. Co-expression of constitutively active PC1 C-terminal construct into Pkd1m1Bei homozygous osteoblasts was sufficient to normalize Runx2 P1 promoter activity. These findings are consistent with a possible functional role of cilia and PC1 in anabolic signaling in osteoblasts/osteocytes. We examined the osteoblast/osteocyte expression and function of polycystin-1 (PC1), a transmembrane protein that is a component of the polycystin-2 (PC2)-ciliary mechano-sensor complex in renal epithelial cells. We found that MC3T3-E1 osteoblasts and MLO-Y4 osteocytes express transcripts for PC1, PC2, and the ciliary proteins Tg737 and Kif3a. Immunohistochemical analysis detected cilia-like structures in MC3T3-E1 osteoblastic and MLO-Y4 osteocyte-like cell lines as well as primary osteocytes and osteoblasts from calvaria. Pkd1m1Bei mice have inactivating missense mutations of Pkd1 gene that encode PC1. Pkd1m1Bei homozygous mutant mice demonstrated delayed endochondral and intramembranous bone formation, whereas heterozygous Pkd1m1Bei mutant mice had osteopenia caused by reduced osteoblastic function. Heterozygous and homozygous Pkd1m1Bei mutant mice displayed a gene dose-dependent decrease in the expression of Runx2 and osteoblast-related genes. In addition, overexpression of constitutively active PC1 C-terminal constructs in MC3T3-E1 osteoblasts resulted in an increase in Runx2 P1 promoter activity and endogenous Runx2 expression as well as an increase in osteoblast differentiation markers. Conversely, osteoblasts derived from Pkd1m1Bei homozygous mutant mice had significant reductions in endogenous Runx2 expression, osteoblastic markers, and differentiation capacity ex vivo. Co-expression of constitutively active PC1 C-terminal construct into Pkd1m1Bei homozygous osteoblasts was sufficient to normalize Runx2 P1 promoter activity. These findings are consistent with a possible functional role of cilia and PC1 in anabolic signaling in osteoblasts/osteocytes. Correction: Peter Reichard and the reduction of ribonucleosides.Journal of Biological ChemistryVol. 293Issue 48PreviewVOLUME 281 (2006) PAGE e13 Full-Text PDF Open Access The study of the genetics of hereditary cystic diseases of the kidney led to the recognition of a novel mechano-sensing mechanism involving cilia, PKD1, 2The abbreviations used are: PKD1, polycystic kidney disease gene 1; PKD2, polycystic kidney disease gene 2; PC1, polycystin 1; PC2, polycystin 2; Runx2, Runt-related transcription factor 2; OPG, osteoprotegerin; RANKL, Rank ligand; TRAP, tartrate-resistant acid phosphatase; BMD, bone mineral density; μCT, microcomputed tomography; RT, reverse transcription; PBS, phosphate-buffered saline; GFP, green fluorescent protein; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. and PKD2 (1Delmas P. Padilla F. Osorio N. Coste B. Raoux M. Crest M. Biochem. Biophys. Res. 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The initial mechano-sensing event that stimulates the release of osteogenic factors and the activation of second messengers leading to the osteo-inductive response, however, has not been established. The possibility that polycystin 1 may be involved in mechano-sensing in osteoblasts/osteocytes, however, is suggested by the observation that Pkd1 null mice have abnormal skeletal development characterized by spina bifida occulta and osteochondrodysplasia (35Boulter C. Mulroy S. Webb S. Fleming S. Brindle K. Sandford R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12174-12179Crossref PubMed Scopus (252) Google Scholar, 36Lu W. Shen X. Pavlova A. Lakkis M. Ward C.J. Pritchard L. Harris P.C. Genest D.R. Perez-Atayde A.R. Zhou J. Hum. Mol. Genet. 2001; 10: 2385-2396Crossref PubMed Scopus (159) Google Scholar, 37Herron B.J. Lu W. Rao C. Liu S. Peters H. Bronson R.T. Justice M.J. McDonald J.D. Beier D.R. Nat. Genet. 2002; 30: 185-189Crossref PubMed Scopus (164) Google Scholar). 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In the current study, we examined whether osteoblasts/osteocytes express Pkd1 and Pkd2 and other cilia genes, determined whether mono cilia are present in these cells, assessed the skeletal phenotype in Pkd1m1Bei mice that have an inactivating missense mutation in the Pkd1 gene (37Herron B.J. Lu W. Rao C. Liu S. Peters H. Bronson R.T. Justice M.J. McDonald J.D. Beier D.R. Nat. Genet. 2002; 30: 185-189Crossref PubMed Scopus (164) Google Scholar), and evaluated the function of PC1 in osteoblasts ex vivo. We have found evidence for the presence of polycystin complexes and cilia in osteoblasts/osteocytes that are coupled to Runx2-dependent regulation of osteoblast/osteocyte function. Mice—Pkd1m1Bei heterozygous mice, which has an inactivating point mutation in Pkd1 gene caused by ENU mutagenesis leading to substitution of an arginine for methionine in the first transmembrane domain of the PC1 protein (37Herron B.J. Lu W. Rao C. Liu S. Peters H. Bronson R.T. Justice M.J. McDonald J.D. Beier D.R. Nat. Genet. 2002; 30: 185-189Crossref PubMed Scopus (164) Google Scholar), were obtained from mutant Mouse Regional Resource Center (University of North Carolina). These mice were bred and maintained on a C57BL/6J background. The mice were genotyped using SYBR® Green (Bio-Rad) real-time PCR reagents, extracted genomic DNA, and the following primers: wild-type (T allele) forward primer, 5′-CTG GTG ACC TAT GTG GTC AT-3′; mutant (G allele) forward primer, 5′-CTG GTG ACC TAT GTG GTC AG-3′; common reverse primer, 5′-AGC CGG TCT TAA CAA GTA TTT C-3′. The Pkd1 allele discrimination was determined by delayed signal on one mismatch. Animal experiments were performed after review and approval by University of Kansas Medical Center's Animal Care and Use Committee. Cell Cultures and Transient or Stable Transfections—MLO-Y4, osteocyte-like cells, were plated onto culture dishes coated with rat tail type I collagen, whereas the MC3T3-E1 cells were plated directly onto the plastic culture dish. For total RNA isolation, MC3T3-E1 cells were cultured in α-minimal essential medium containing 10% fetal calf serum supplemented with 5 mm β-glycerophosphate and 25 μg/ml ascorbic acid for 4, 7, and 14 days, whereas MLO-Y4 cells were cultured in α-minimal essential medium supplemented with 5% fetal bovine serum and 5% calf serum for 4, 7, and 14 days. For transient transfections, 1 × 106 MC3T3-E1 or MLO-Y4 cells were transfected with either control expression vector, gain-of-function, or loss-of-function PC1 C-tail constructs along with the Runx2-P1 luciferase reporter (p1.4Runx2-P1-Luc) construct by using the electroporation protocol from Amaxas Biosystems as described by the manufacturer (Amaxa, Inc.). A total of 6.6 μg of plasmid DNA was used for each electroporation, with 3.6 μg of PC1 C-tail construct, 2.4 μg of p1.4Runx2-P1-Luc reporter, and 0.6 μg of Renilla luciferase-null (RL-null) as the internal control plasmid. Promoter activity was assessed by measuring luciferase activity 48 h after transfection. The cells were then lysed in Passive lysis buffer (Promega), and 20 μl of cell lysate was used with the dual luciferase assay kit (Promega) using an EG&G Berthold 9507 luminometer. Stable transfection of MC3T3-E1 was performed by a protocol that maintains the differentiation potential of these osteoblasts as previously described (41Quarles L.D. Siddhanti S.R. Medda S. J. Cell. Biochem. 1997; 65: 11-24Crossref PubMed Scopus (19) Google Scholar). Briefly, MC3T3-E1 cells were only transfected with 6.0 μg of PC1 C-tail fusion construct and selected by incubation in media containing 500 μg/ml G418 (Invitrogen) 48 h after transfection. Measurement of Alkaline Phosphatase Activity and Mineralization Assays in Immortalized Osteoblast Cultures—Calvaria from Pkd1m1Bei E15.5 embryos were used for the isolation of osteoblasts by sequential collagenase digestion as previously described (42Xiao Z.S. Hjelmeland A.B. Quarles L.D. J. Biol. Chem. 2004; 279: 20307-20313Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 43Borton A.J. Frederick J.P. Datto M.B. Wang X.F. Weinstein R.S. J. Bone Miner. Res. 2001; 16: 1754-1764Crossref PubMed Scopus (131) Google Scholar). To engineer immortal osteoblast cell lines, isolated primary osteoblasts were infected using a retroviral vector carrying SV40 large and small T antigen. To induce differentiation, immortalized osteoblasts were plated at a density of 1 × 105 cells/well in a 6-well plate and grown for a period of up to 14 days in α-minimal essential medium containing 10% fetal calf serum supplemented with 5 mm β-glycerophosphate and 25 μg/ml ascorbic acid. Alkaline phosphatase activity and alizarin red-S histochemical staining for mineralization were performed as previously described (42Xiao Z.S. Hjelmeland A.B. Quarles L.D. J. Biol. Chem. 2004; 279: 20307-20313Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Total DNA content was measured with a PicoGreen® double-stranded DNA quantitation reagent and kit (Molecular Probes, Eugene, OR). RT-PCR Analysis—RT-PCR was done using the Titan™ One tube RT-PCR kit from Roche Applied Science. DNase I-treated total RNA (1.0 μg) was reverse-transcribed into cDNA with the reverse primer described before (44Xiao Z.S. Simpson L.G. Quarles L.D. J. Cell. Biochem. 2003; 88: 493-505Crossref PubMed Scopus (45) Google Scholar). The reverse transcription reaction was incubated at 50 °C for 30 min. PCR was performed with thermal cycling parameters of 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 30 s for 35 cycles followed by final extension at 68 °C for 7 min. To amplify mouse Pkd1 transcript, we used the forward primer (5′-CAT TGT ACC CCT GGA GGA GA-3′) in combination with the reverse primer (5′-GAT GTC CAG GCT GTT TCG AT-3′). To amplify mouse Pkd2 transcript, we used the forward primer (5′-GGG GCT GCT ACA GTT TCT TG-3′) with the reverse primer (5′-CCG GAG ACT CTC TGA GAT GG-3′). The mouse Tg737 transcript was RT-PCR-amplified using the forward primer (5′-TCC AAC TGA TTC CCA AGC TC-3′) and the reverse primer (5′-TGG CAC TCA GTC GTT CAC TC-3′). Mouse glyceraldehyde-3-phosphate dehydrogenase was amplified as a control for the RT-PCR reactions. Immunofluorescence—MC3T3-E1 osteoblasts and MLO-Y4 osteocytes were grown on collagen-coated coverslips and kept at confluence for at least 3 days. At the end of the culture the cells were washed 3 times with PBS, then fixed with cold 4% paraformaldehyde, 0.2% Triton for 10 min at room temperature and washed with PBS 3 times. The coverslips were incubated for 30 min in 1% bovine serum albumin before incubation with the primary acetylated α-tubulin antibody (Sigma-Aldrich, T6793) for 1 h at room temperature. After washing 3 times in PBS, they were treated with secondary Texas Red-labeled anti-mouse IgG (Jackson ImmunoResearch, 715-076-150) in 1% bovine serum albumin for 1 h at room temperature and washed 3 times in PBS before mounting with ProLong® Gold antifade reagent (Invitrogen, P36935). Nuclei were counterstained with 4,6-diamidino-2-phenylindole. Photographs were taken under a microscope with magnifications of 60×. Whole Mount Calvaria Immunostaining—For whole-mount bone immunostaining for α-tubulin, transgenic mice expressing the topaz variant of enhanced green fluorescent protein (eGFP-tpz) targeted to the osteocyte were used (45Kalajzic I. Braut A. Guo D. Jiang X. Kronenberg M.S. Mina M. Harris M.A. Harris S.E. Rowe D.W. Bone (NY). 2004; 35: 74-82Crossref PubMed Scopus (181) Google Scholar). These mice were kindly provided by Dr. David Rowe (University of Connecticut Health Center, Farmington, CT 06030). The GFP reporter was expressed under control of a 7892-bp fragment of the dentin matrix protein-1 (Dmp1) promoter together with a 4439-bp region containing the first exon, the first intron, and part of exon 2. In these mice GFP is expressed almost exclusively in osteocytes and preosteocytes. This transgenic mouse model, therefore, provides an extremely useful tool for imaging of osteocytes either in situ within the bones or in cell cultures (45Kalajzic I. Braut A. Guo D. Jiang X. Kronenberg M.S. Mina M. Harris M.A. Harris S.E. Rowe D.W. Bone (NY). 2004; 35: 74-82Crossref PubMed Scopus (181) Google Scholar, 46Yang X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li L. Brancorsini S. Sassone-Corsi P. Townes T.M. Hanauer A. Karsenty G. Cell. 2004; 117: 387-398Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 47Xiao G. Jiang D. Ge C. Zhao Z. Lai Y. Boules H. Phimphilai M. Yang X. Karsenty G. Franceschi R.T. J. Biol. Chem. 2005; 280: 30689-30696Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Whole calvaria from 12-day-old neonatal Dmp1-GFP mice were excised, and the periostea was carefully removed under a dissection microscope. The bones were fixed overnight at 4 °C in the dark in neutral-buffered formalin. The bones were then washed twice in PBS for 15 min with shaking. Next, the bones were decalcified for 1 week in 10% EDTA followed by washing 3 times in PBS at 4 °C for 15 min. Background blocking was performed overnight at 4 °C in PBS, 1% normal horse serum, 0.05% sodium azide followed by washing twice with PBS. Because the primary antibody was a mouse monoclonal against α-tubulin (Sigma-Aldrich, T 6793), the Vectastain-Elite kit was used according to manufacturer's instructions to block endogenous mouse immunoglobulin. The bones were incubated for 4 h at room temperature with primary antibody diluted in Vectastain-Elite diluent. After 4 washes in PBS, the bones were then incubated for 2 h with a Cy3-conjugated donkey anti-mouse second antibody diluted 1:250 in Vectastain-Elite diluent (Jackson Immunoresearch Laboratories Inc., West Grove, PA). The bones were then washed four times in PBS and counterstained with 4,6-diamidino-2-phenylindole nuclear stain (4 μg/ml in PBS) for 30 s followed by two final PBS washes. The whole bones were mounted under a coverslip in 2 drops of 9:1 glycerol:PBS containing 5% N-propyl gallate as an anti-fade reagent. The specimens were viewed on a Nikon E800 microscope under epifluorescent illumination. Digital photographs were acquired from multiple optical planes using an optronics CCD camera driven by the AnalySIS software (Olympus Soft Imaging Solutions Corp, Lakewood, CO). Extended focus images were generated using this software. Scanning Electron Microscopy—Renal tubular Madin-Darby canine kidney cells and MLO-Y4 cells were cultured on Thermanox coverslips and at the end of the culture washed with PBS and fixed with 10% formalin for 20 min, washed again with PBS, dehydrated in graded concentrations of ethanol, and dried using hexamethyldisilazone for 5 min. After dehydration, the coverslips were mounted on a stub, sputtered with gold-palladium, and viewed with the FEI/Philips XL30 Field emission environmental scanning electron microscope (48Nanci A. Zalzal S. Gotoh Y. McKee M.D. Microsc. Res. Tech. 1996; 33: 214-231Crossref PubMed Scopus (76) Google Scholar). Whole Skeletal Mount Alizarin Red/Alcian Blue Staining and Histological Preparation—This procedure provides information regarding developmental abnormalities. Embryos from 13.5 (E13.5) to 15.5 (E15.5) days of gestation were collected and fixed for more than 3 days in 95% ethanol. Samples were defatted for 2-3 days in acetone and stained sequentially with Alcian blue and alizarin red S in 2% KOH. The stained skeleton preparations were cleared with 1% KOH, 20% glycerol and stored in 50% EtOH, 50% glycerol (42Xiao Z.S. Hjelmeland A.B. Quarles L.D. J. Biol. Chem. 2004; 279: 20307-20313Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Femurs from E15.5 embryos were decalcified at 4 °C in 12.5% EDTA and 2.5% paraformaldehyde in phosphate-buffered saline. Longitudinal sections were stained with hematoxylin and eosin to assess the histology of the growth plate and bone marrow cavity in femurs (42Xiao Z.S. Hjelmeland A.B. Quarles L.D. J. Biol. Chem. 2004; 279: 20307-20313Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Microcomputed Tomography Analysis—The distal femoral metaphyses were scanned using a μCT 40 (Scanco Medical AG, Bassersdorf, Switzerland) and 167 slices of the metaphyses under the growth plate, constituting 1.0 mm in length, were selected. The three-dimensional images were generated using the following values for a gauss filter (σ0.8, support 1) and a threshold of 275. A three-dimensional image analysis was done to determine bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Cortical bone was measured on the mid-shaft region of cortical bone. 50 slices of the diaphysis, 0.3 mm in length, were selected. The mean cortical thickness was determined by distance measurements at eight different points on the cortical slice (42Xiao Z.S. Hjelmeland A.B. Quarles L.D. J. Biol. Chem. 2004; 279: 20307-20313Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Real-time RT-PCR and Western Blot—For quantitative realtime RT-PCR, 2.0 μg total RNA isolated from whole E15.5 embryos and femurs of 12-week-old mice was reverse-transcribed as described (42Xiao Z.S. Hjelmeland A.B. Quarles L.D. J. Biol. Chem. 2004; 279: 20307-20313Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). PCR reactions contained a 100-ng template (cDNA or RNA), 300 nm each forward and reverse primer, and 1× iQ™ SYBR® Green Supermix (Bio-Rad) in 50 μl. Samples were amplified for 40 cycles in an iCycler iQ™ real-time PCR detection system with an initial melt at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. PCR product accumulation was monitored at multiple points during each cycle by measuring the increase in fluorescence caused by the binding of SybrGreen I to double-stranded DNA. The threshold cycle (Ct) of tested-gene product from the indicated genotype was normalized to the Ct for 18 S rRNA (49Marino J.H. Cook P. Miller K.S. J. Immunol. Methods. 2003; 283: 291-306Crossref PubMed Scopus (94) Google Scholar). Nuclear extracts were prepared using N

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