In Vivo Targeted Deletion of Calpain Small Subunit, Capn4, in Cells of the Osteoblast Lineage Impairs Cell Proliferation, Differentiation, and Bone Formation

Calpains are intracellular cysteine proteases, which include widely expressed μ- and m-calpains (1Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2378) Google Scholar). Both μ-calpains and m-calpains are heterodimers consisting of a large catalytic subunit and a small regulatory subunit. The calpain small subunit encoded by the gene Capn4 directly binds to the intracellular C-terminal tail (C-tail) of the receptor for parathyroid hormone and parathyroid hormone-related peptide and modulates its cellular functions in osteoblasts in vitro (2Shimada M. Mahon M.J. Greer P.A. Segre G.V. Endocrinology. 2005; 146: 2336-2344Crossref PubMed Scopus (17) Google Scholar). To investigate a potential role of the calpain small subunit in osteoblasts in vivo, we generated osteoblast-specific Capn4 knock-out mice using the Cre-LoxP system (3Sternberg N. Hamilton D. J. Mol. Biol. 1981; 150: 467-486Crossref PubMed Scopus (511) Google Scholar). Mutant mice had smaller bodies with shorter limbs, reduced trabecular bone with thinner cortices, and decreased osteoblast number. In vitro analysis confirmed that deletion of Capn4 in osteoblasts severely affected multiple osteoblast functions including proliferation, differentiation, and matrix mineralization. Collectively, our findings provide the first in vivo demonstration that the calpain small subunit is essential for proper osteoblast activity and bone remodeling. Calpains are intracellular cysteine proteases, which include widely expressed μ- and m-calpains (1Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2378) Google Scholar). Both μ-calpains and m-calpains are heterodimers consisting of a large catalytic subunit and a small regulatory subunit. The calpain small subunit encoded by the gene Capn4 directly binds to the intracellular C-terminal tail (C-tail) of the receptor for parathyroid hormone and parathyroid hormone-related peptide and modulates its cellular functions in osteoblasts in vitro (2Shimada M. Mahon M.J. Greer P.A. Segre G.V. Endocrinology. 2005; 146: 2336-2344Crossref PubMed Scopus (17) Google Scholar). To investigate a potential role of the calpain small subunit in osteoblasts in vivo, we generated osteoblast-specific Capn4 knock-out mice using the Cre-LoxP system (3Sternberg N. Hamilton D. J. Mol. Biol. 1981; 150: 467-486Crossref PubMed Scopus (511) Google Scholar). Mutant mice had smaller bodies with shorter limbs, reduced trabecular bone with thinner cortices, and decreased osteoblast number. In vitro analysis confirmed that deletion of Capn4 in osteoblasts severely affected multiple osteoblast functions including proliferation, differentiation, and matrix mineralization. Collectively, our findings provide the first in vivo demonstration that the calpain small subunit is essential for proper osteoblast activity and bone remodeling. Calpains are a family of Ca2+-dependent intracellular cysteine proteases that include ubiquitously expressed μ- and m-calpains (1Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2378) Google Scholar, 4Cong J. Goll D.E. Peterson A.M. Kapprell H.P. J. Biol. Chem. 1989; 264: 10096-10103Abstract Full Text PDF PubMed Google Scholar). Both μ-calpains and m-calpains form heterodimers consisting of a large catalytic subunit (80 kDa) encoded by the genes Capn1 and Capn2, respectively, and a small regulatory subunit (28 kDa) encoded by the gene Capn4 (1Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2378) Google Scholar). Disruption of Capn4 eliminates both μ-calpains and m-calpain activities in embryonic fibroblasts (5Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (296) Google Scholar), suggesting that the calpain small subunit is essential for maintenance of calpain stability and activity. Notably, genetic ablation of Capn4 results in early embryonic lethality, which demonstrates an essential role of the calpain small subunit during development (5Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (296) Google Scholar, 6Zimmerman U.J. Boring L. Pak J.H. Mukerjee N. Wang K.K. IUBMB Life. 2000; 50: 63-68Crossref PubMed Scopus (99) Google Scholar). Several lines of evidence suggest that calpains are necessary for proper osteoblast function in vitro (7Tram K.K. Spencer M.J. Murray S.S. Lee D.B. Tidball J.G. Murray E.J. Biochem. Mol. Biol. Int. 1993; 29: 981-987PubMed Google Scholar, 8Tram K.K. Murray S.S. Lee D.B. Murray E.J. Kidney Int. 1993; 43: 693-699Abstract Full Text PDF PubMed Scopus (16) Google Scholar, 9Murray E.J. Murray S.S. Tram K.K. Lee D.B. Exp. Cell Res. 1994; 215: 241-248Crossref PubMed Scopus (2) Google Scholar, 10Murray E.J. Grisanti M.S. Bentley G.V. Murray S.S. Metab. Clin. Exp. 1997; 46: 1090-1094Abstract Full Text PDF PubMed Scopus (27) Google Scholar, 11Murray S.S. Grisanti M.S. Bentley G.V. Kahn A.J. Urist M.R. Murray E.J. Exp. Cell Res. 1997; 233: 297-309Crossref PubMed Scopus (45) Google Scholar). However, a physiological role of calpains in osteoblasts in vivo remains to be established. Chemical inhibition of calpain activity reduces osteoblast proliferation and differentiation in the MC3T3-E1 osteoblastic cell line (11Murray S.S. Grisanti M.S. Bentley G.V. Kahn A.J. Urist M.R. Murray E.J. Exp. Cell Res. 1997; 233: 297-309Crossref PubMed Scopus (45) Google Scholar). Moreover, we have previously reported that the calpain small subunit directly binds to the intracellular C-tail of the receptor for PTH 2The abbreviations used are: PTH, parathyroid hormone; μCT, microcomputed tomography; BrdUrd, bromodeoxyuridine; αMEM, α-minimum essential medium; ALP, alkaline phosphatase; qPCR, quantitative PCR; siRNA, small interfering RNA; UMR, UMR106-01; kbp, kilobase pairs. and PTH-related peptide and modulates its ligand-mediated cellular functions (2Shimada M. Mahon M.J. Greer P.A. Segre G.V. Endocrinology. 2005; 146: 2336-2344Crossref PubMed Scopus (17) Google Scholar). Both ligands are known regulators of bone homeostasis in vivo through their direct actions on cells of the osteoblast lineage (12Reeve J. Hesp R. Williams D. Hulme P. Klenerman L. Zanelli J.M. Darby A.J. Tregear G.W. Parsons J.A. Lancet. 1976; 1: 1035-1038Abstract PubMed Scopus (168) Google Scholar, 13Kronenberg H.M. Lanske B. Kovacs C.S. Chung U.I. Lee K. Segre G.V. Schipani E. Juppner H. Recent Prog. Horm. Res. 1998; 53: 283-303PubMed Google Scholar, 14Schipani E. Provot S. Birth Defects Res. Part C. Embryo Today Rev. 2003; 69: 352-362Crossref PubMed Scopus (75) Google Scholar). Taken together, these findings suggest that the calpain small subunit could play a critical but yet unknown role in osteoblast biology in vivo. To test this hypothesis, we have conditionally ablated Capn4 in cells of the osteoblast lineage in vivo by using the Cre-LoxP system. Lack of the calpain small subunit in osteoblasts caused a significant decrease of both trabecular and cortical bone, which was associated with a severe impairment of osteoblast proliferation and differentiation. These findings are the first in vivo evidence that the calpain small subunit plays a crucial role in osteoblast function and bone homeostasis. Generation of Osx-Cre+/- Capn4flox/flox Mice—Mice homozygous for floxed Capn4 alleles (Capn4flox/flox) were crossed with those expressing Cre under the control of osterix promoter (Osx-Cre+/-) to generate Osx-Cre+/-Capn4flox/+ mice (15Tan Y. Dourdin N. Wu C. Veyra T.De Elce J.S. Greer P.A. Genes. J. Genet. Dev. 2006; 44: 297-303Google Scholar, 16Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2835) Google Scholar, 17Rodda S.J. McMahon A.P. Development (Camb.). 2006; 133: 3231-3244Crossref PubMed Scopus (832) Google Scholar). These mice were then crossed with either Capn4flox/flox or Capn4flox/+ mice to generate Osx-Cre+/-Capn4flox/flox mutant mice. All experiments were performed in compliance with the guiding principles of the Guide for the Care and Use of Laboratory Animals and approved by the subcommittee on Research Animal Care of the Massachusetts General Hospital (MGH). Genotype Analysis—Genomic DNA was isolated from tail biopsies as described previously (18Shimada M. Shimano H. Gotoda T. Yamamoto K. Kawamura M. Inaba T. Yazaki Y. Yamada N. J. Biol. Chem. 1993; 268: 17924-17929Abstract Full Text PDF PubMed Google Scholar). PCR-based strategies were used to genotype these mice. The Cre transgene was detected by PCR using the primers P1 (5′-CGCGGTCTGGCAGTAAAAACTATC-3′) and P2 (5′-CCCACCGTCAGTACGTGAGAT ATC-3′) to generate an internal sequence of Cre. Taking an advantage of a floxed construct, in which an intron 9 was shortened by 1.6 kilobase pairs (kbp), the Capn4 floxed and wild-type alleles were determined using two sets of PCR reactions (15Tan Y. Dourdin N. Wu C. Veyra T.De Elce J.S. Greer P.A. Genes. J. Genet. Dev. 2006; 44: 297-303Google Scholar, 19Arthur J.S. Greer P.A. Elce J.S. Biochim. Biophys. Acta. 1998; 1388: 247-252Crossref PubMed Scopus (17) Google Scholar). The floxed and wild-type alleles were determined as 0-bp (no amplified product) and 1.0-kbp PCR products, respectively, using primers P3 (5′-GTCAGGCTAGATGCCATGTTCC-3′) in exon 9 and P4 (5′-CGACTATCCGAGCGCTGCC-3′) within a sequence deleted in floxed allele in intron 9 and as 0.4-kbp and 2.0-kbp PCR products, respectively, using primers P3 (5′-GTCAGGCTAGATGCCATGTTCC-3′) and P5 (5′-GTTCACTTGGATCTGTCCGGTGCC-3′) corresponding to sequences in exons E9 and E10. Serology—Ionized calcium was measured with the Ciba-Corning 634 Ca2+/pH analyzer (Ciba-Corning Diagnostics Corp., Medfield, MA). Serum levels of osteocalcin (Biomedical Technologies Inc., Stoughton, MA) and tartrate-resistant acid phosphatase (TRACP) 5b (Immunodiagnostic Systems, Tyne and Wear, UK) were determined using enzyme-linked immunosorbent assays as described by the manufacturers' instructions. Whole Mount Skeletal Staining—The whole mount skeletal staining was performed as described previously (20McLeod M.J. Teratology. 1980; 22: 299-301Crossref PubMed Scopus (546) Google Scholar). Briefly, newborn mice were fixed in ethanol for 5 days and then in acetone for 2 days. Staining with Alizarin red S and Alcian blue was performed for 3 days at 37 °C. After washing with distilled water, the skeleton was cleared in 1% KOH and taken through graded steps into 100% glycerol. Sample Preparation and Histological Analysis—For histological analysis, Osx-Cre+/-Capn4flox/flox and sex-matched control littermates (Osx-Cre+/-Capn4flox/+ and Capn4flox/flox) were sacrificed at birth and at 2, 4, 9, and 12 weeks of age. Tissues from Osx-Cre+/-Capn4flox/flox, and control littermates were fixed and stored as described previously (21Calvi L.M. Sims N.A. Hunzelman J.L. Knight M.C. Giovannetti A. Saxton J.M. Kronenberg H.M. Baron R. Schipani E. J. Clin. Investig. 2001; 107: 277-286Crossref PubMed Scopus (310) Google Scholar). In selected cases, hind limbs were decalcified, and paraffin blocks were prepared by standard histological procedures. To detect osteoclast-like cells, tartrate-resistant acid phosphatase (TRAP) staining was performed using an acid phosphatase detection kit (Sigma-Aldrich) for some selected samples. In Situ Hybridization—In situ hybridization analysis was performed as described previously (22Lee K. Lanske B. Karaplis A.C. Deeds J.D. Kohno H. Nissenson R.A. Kronenberg H.M. Segre G.V. Endocrinology. 1996; 137: 5109-5118Crossref PubMed Scopus (235) Google Scholar). Complementary 35S-labeled riboprobes were transcribed from the plasmids encoding mouse type I collagen (Col.1), mouse matrix metalloproteinase (MMP)13, mouse osteocalcin (OC), mouse osteopontin (OP), and rat TRAP using Riboprobe from were with and of 3 and days were fixed and in as described previously (21Calvi L.M. Sims N.A. Hunzelman J.L. Knight M.C. Giovannetti A. Saxton J.M. Kronenberg H.M. Baron R. Schipani E. J. Clin. Investig. 2001; 107: 277-286Crossref PubMed Scopus (310) Google Scholar). were with or and analysis was performed with the system Inc., using standard procedures. were measured in the the of the and were by using a system as described previously R. J. Res. 2005; 20: PubMed Scopus Google Scholar, J. Res. 2005; 20: PubMed Scopus Google Scholar, E. L. J. Res. 22: PubMed Scopus Google Scholar). trabecular bone in the and the and cortical bone at the 12 trabecular bone, we measured bone trabecular and trabecular cortical bone, we the cortical bone cortical and the of of mice were with of and 12 of of 2 After hind limbs were decalcified, and in and the and were To cells, that had were detected using a kit Inc., of the and the were All and in these were or were for of two or Osx-Cre+/- and Osx-Cre+/-Capn4flox/flox at of in osteoblasts were detected in of of Osx-Cre+/-Capn4flox/+ and Osx-Cre+/-Capn4flox/flox littermates by a using from the in situ cell detection The were with and were isolated from of newborn mice by in α-minimum essential type I and type were for at 37 with The was with and were with αMEM, and in with 1% for was Capn4flox/flox osteoblasts were with either control or at a of of for experiments D. T. Knight M.C. E. A.J. Schipani E. Development (Camb.). PubMed Scopus Google Scholar). were DNA and were from osteoblasts for of of activity was measured in cell using a (Sigma-Aldrich) as described previously P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). were in 1% for and were in with and 1% with acid and (Sigma-Aldrich) for and were then fixed with and were with either Alizarin red S or were using a In some Alizarin red S was and as described P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). PCR of by the osterix promoter or was using on DNA isolated from of Osx-Cre+/-Capn4flox/flox and Capn4flox/flox mice and from osteoblasts from Capn4flox/flox mice with either or In of Osx-Cre+/-Capn4flox/flox and Capn4flox/flox mice were in and in I and at 37 for to cells and The were then and with in DNA was isolated using standard was then performed at for in the by using a PCR kit and primers P3 and To of was from cells by using the kit was using the kit for by were in and the results were to D. T. Knight M.C. E. A.J. Schipani E. Development (Camb.). PubMed Scopus Google Scholar). sequences were as ALP, and and and and and and and and and and and J.S. Greer P.A. Elce J.S. Biochim. Biophys. Acta. 1998; 1388: 247-252Crossref PubMed Scopus (17) Google Scholar, X. J. R. M.C. C. Williams S. A. PubMed Scopus Google Scholar, S. K. X. D. E. Exp. Cell Res. 2005; PubMed Scopus Google Scholar, N. Inaba H. T. M. K. S. A. PubMed Scopus (75) Google Scholar). in osteoblasts were in at a of were with and using kit and by Inc., by rat cells were at a of 5 in Capn4 and control were into cells using as by the were Capn4 sequences are as and and of Capn4 was by calpain activity as described previously (2Shimada M. Mahon M.J. Greer P.A. Segre G.V. Endocrinology. 2005; 146: 2336-2344Crossref PubMed Scopus (17) Google Scholar). were from 3 to 5 experiments and expressed as the of either or analysis was performed using the analysis of or the was determined using significant and were as Generation of Capn4 in of the and of expressing Cre under the control of the osterix promoter (Osx-Cre+/-) were crossed with floxed Capn4 mice (Capn4flox/flox) to Osx-Cre+/-Capn4flox/+ which were then with either Capn4flox/flox or Capn4flox/+ to generate osteoblast-specific Capn4 knock-out and Capn4flox/flox were used in the as control of Cre activity by of either DNA or isolated from mutant was Osx-Cre+/-Capn4flox/flox mice were at the and were into and in both was reduced from weeks of in with and control at weeks of of Osx-Cre+/-Capn4flox/+ mice was that of the Capn4flox/flox and calcium levels of mice were mutant and control The newborn skeleton of Osx-Cre+/-Capn4flox/flox mice was smaller that of Capn4flox/flox and Osx-Cre+/- Capn4flox/+ littermates but Notably, of the was in Osx-Cre+/-Capn4flox/flox mice Osx-Cre+/-Capn4flox/flox and of histological and in situ hybridization were then performed on hind limbs isolated from and respectively, to investigate a role of Capn4 in bone and remodeling. of Osx-Cre+/- Capn4flox/flox mice had reduced trabecular bone with both Osx-Cre+/-Capn4flox/+ and Capn4flox/flox control littermates Moreover, of osteoblast as and was severely in mutant and was in and mutant mice of either 2, and and at these Osx-Cre+/- mice a of trabecular bone, which was from that of Osx-Cre+/- Capn4flox/flox as by and detection of by in situ hybridization analysis 2, and mice bone at or age. analysis significant in and Capn4flox/flox with as bone trabecular and trabecular for These of the Cre transgene on bone homeostasis. that of a Capn4 allele in cells of the osteoblast lineage is to bone is in mice. and were performed on bone of Capn4flox/flox and Osx-Cre+/-Capn4flox/flox mice and confirmed a significant decrease of bone and trabecular with of trabecular in Osx-Cre+/-Capn4flox/flox with Capn4flox/flox control mice at 12 weeks of and Moreover, bone osteoblast and were reduced in Osx-Cre+/- Capn4flox/flox was decreased in as by both and TRAP staining and Taken together, these suggest that bone in Osx-Cre+/- Capn4flox/flox mice is the of decreased osteoblast and activity. with the findings described levels of both osteocalcin and of osteoblast and respectively, were reduced in mutant mice Capn4flox/flox Osx-Cre+/-Capn4flox/flox Capn4flox/flox Capn4flox/flox Lack of Capn4 in of the and bone was by analysis at the and cortical bone and cortical were reduced in Osx-Cre+/-Capn4flox/flox mice suggesting that the bone is smaller and that is in Osx-Cre+/-Capn4flox/flox mice In the of an of was in deletion of Capn4 in cells of the osteoblast lineage bone in both trabecular and cortical and severely that bone Lack of Capn4 of of the reduced osteoblast in Osx-Cre+/-Capn4flox/flox mice could be a of either decreased proliferation or To these two proliferation of cells of the osteoblast lineage was by in and mice. The of cells detected in the was reduced in Osx-Cre+/-Capn4flox/flox with Osx-Cre+/- Capn4flox/+ and Capn4flox/flox littermates proliferation, which as an internal control for proper and was affected significant in the of cells determined by staining was detected in mutant Collectively, these that the reduced osteoblast in Osx-Cre+/-Capn4flox/flox is to reduced proliferation of cells of the osteoblast Lack of Capn4 and in functions were in this osteoblasts were from Osx-Cre+/-Capn4flox/+ and Osx-Cre+/- Capn4flox/flox newborn mice and at a of of Capn4 were reduced by in Osx-Cre+/-Capn4flox/flox osteoblasts in with Cell was Osx-Cre+/-Capn4flox/flox and Osx-Cre+/- Capn4flox/+ cells and in Osx-Cre+/-Capn4flox/flox cells Osx-Cre+/-Capn4flox/+ cells on differentiation and by activity and of osteoblast and of bone by cell at the of respectively, were in Osx-Cre+/-Capn4flox/flox in Osx-Cre+/-Capn4flox/+ cells to investigate a role of Capn4 in osteoblast differentiation, cells isolated from Capn4flox/flox mice were with expressing either or The of was at both DNA and levels using of alleles were in cells, and their levels of Capn4 were reduced to of those in control cells were at the of to to an cell at the of the of proliferation on the differentiation Notably, cell of both mutant and control cells and a cells were at the the of the was determined by significant was detected in cells with the of early and cells with the in vivo cells Capn4 reduced activity as as of genes including and that are early of osteoblast differentiation X. J. R. M.C. C. Williams S. A. PubMed Scopus Google Scholar). of of osteoblast differentiation as ALP, and was in cells cells in differentiation for and respectively, of the bone in control cells and Taken together, these in vitro results that of Capn4 both proliferation and differentiation of cells of the osteoblast lineage as by the in vivo Lack of Capn4 in of the in an of Capn4 ablation on gene in cells of the osteoblast cells were with PTH for and 2 and levels of were using is a gene of PTH, and its and PTH Lee S.J. P. J.S. Lee J. Wu E. M. P.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, D. P. Elce J.S. Greer P.A. M.C. Mol. Cell Biol. 2002; 22: PubMed Scopus Google Scholar, W. 1997; PubMed Scopus Google Scholar). In control cells, PTH for and 2 levels and was reduced by in cells the calpain small subunit in cells by using that Capn4 and reduced calpain activity to and respectively, with control in cells in a significant impairment of of Cell of the receptor for PTH and PTH-related peptide determined by was control and cells Collectively, these results suggest that Capn4 could osteoblast at in by PTH activity in these we the that Capn4 is in bone development and in vivo. the early of the Capn4 knock-out Osx-Cre+/-Capn4flox/flox was an to investigate the role of the calpain small subunit in cells of the osteoblast lineage (5Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (296) Google Scholar, 6Zimmerman U.J. Boring L. Pak J.H. Mukerjee N. Wang K.K. IUBMB Life. 2000; 50: 63-68Crossref PubMed Scopus (99) Google Scholar). Calpains to a family of intracellular cysteine proteases that have to and (1Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2378) Google Scholar). direct evidence that of Capn4 severely proliferation of cells of the osteoblast lineage both in vivo and in with these was previously reported that a calpain proliferation in the MC3T3-E1 cell line E.J. Grisanti M.S. Bentley G.V. Murray S.S. Metab. Clin. Exp. 1997; 46: 1090-1094Abstract Full Text PDF PubMed Scopus (27) Google Scholar, 11Murray S.S. Grisanti M.S. Bentley G.V. Kahn A.J. Urist M.R. Murray E.J. Exp. Cell Res. 1997; 233: 297-309Crossref PubMed Scopus (45) Google Scholar). The role of calpain in the cell embryonic fibroblasts with calpain activity (5Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (296) Google Scholar). However, have that calpain plays a role in the cell at to S by of as and 2, and Lee S.J. P. J.S. Lee J. Wu E. M. P.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, D. P. Elce J.S. Greer P.A. M.C. Mol. Cell Biol. 2002; 22: PubMed Scopus Google Scholar, W. 1997; PubMed Scopus Google Scholar). demonstrates that of cell proliferation is a crucial function of the calpain small at in Lack of Capn4 in osteoblasts reduces activity and of differentiation as ALP, and with our a calpain was reported to osteoblast differentiation in the MC3T3-E1 cell line (11Murray S.S. Grisanti M.S. Bentley G.V. Kahn A.J. Urist M.R. Murray E.J. Exp. Cell Res. 1997; 233: 297-309Crossref PubMed Scopus (45) Google Scholar). The the first in vivo evidence that Capn4 is an essential of cell differentiation in Notably, for μ- and m-calpains performed in the MC3T3-E1 cell line that m-calpain is a is both and which that calpains control gene directly at the (11Murray S.S. Grisanti M.S. Bentley G.V. Kahn A.J. Urist M.R. Murray E.J. Exp. Cell Res. 1997; 233: 297-309Crossref PubMed Scopus (45) Google Scholar). reported that PTH of the early which a critical role in bone by of the at by Biol. Cell. PubMed Scopus Google Scholar, K. Deeds J.D. S. M. Segre G.V. Endocrinology. 1994; PubMed Scopus Google Scholar, B. A.J. Endocrinology. 2002; PubMed Scopus Google Scholar, M.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Endocrinology. PubMed Google Scholar). Moreover, we have previously reported that MC3T3-E1 osteoblastic cells expressing reduced (2Shimada M. Mahon M.J. Greer P.A. Segre G.V. Endocrinology. 2005; 146: 2336-2344Crossref PubMed Scopus (17) Google Scholar). our that of Capn4 reduces of in both and Collectively, these suggest that Capn4 osteoblast at in by PTH activity in these role of calpains in bone homeostasis previously by the analysis of knock-out which to an activity. osteoblast was reported in these mutant mice M. R. L. Baron R. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). their bone from that of the osteoblast-specific Capn4 knock-out which both osteoblast and function and reduced and activity. the of the Capn4 knock-out mice either of m-calpain function or of both m-calpains and μ-calpains function in Several lines of evidence that of a Capn4 allele in cells of the osteoblast lineage a bone Osx-Cre+/-Capn4flox/+ mice to weeks of and their from that of Osx-Cre+/- Capn4flox/flox mice at weeks of age. as an of skeletal the suggest skeletal in Osx-Cre+/-Capn4flox/+ mice. Moreover, histological analysis that Osx-Cre+/-Capn4flox/+ mice an 2 and weeks of age. proliferation of cells of the osteoblast lineage as by at 2 weeks was reduced in Osx-Cre+/-Capn4flox/+ mice with Capn4flox/flox was in Osx-Cre+/-Capn4flox/flox Osx-Cre+/- and as confirmed by analysis, from wild-type which that the bone of Osx-Cre+/- Capn4flox/+ was the of a to the of the Cre transgene in cells of the osteoblast These results are of a in mice allele of Capn4 in cells of the osteoblast In of Capn4 in cell of the osteoblast lineage both trabecular and cortical bone by proliferation and differentiation of osteoblastic that calpains could be in as M. Kronenberg for critical of the and and at and at for

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