Abstract
The ubiquitously expressed calpains-1 and -2 belong to a family of calcium-dependent intracellular cysteine proteases. Both calpains are heterodimers consisting of a large catalytic subunit and a small regulatory subunit encoded by the gene Capn4. Ablation of the calpain small subunit eliminates calpain activity and leads to embryonic lethality. We previously created osteoblast-specific Capn4 knockout mice to investigate a physiological role for the calpain small subunit in cells of the osteoblast lineage. Deletion of Capn4 reduced trabecular and cortical bone, mainly due to impaired proliferation and differentiation of cells of the osteoblast lineage. To further investigate an underlining mechanism by which osteoblast-specific Capn4 knockout mice develop an osteoporotic bone phenotype, we established osteoblastic cell lines stably expressing either control or Capn4 RNA interference for this study. Capn4 knockdown cells showed reduced cell proliferation, accumulation of total and phosphorylated cyclin-dependent kinase inhibitor 1B (p27Kip1) on serine 10, and reduced phosphorylation of retinoblastoma protein on threonine 821. Moreover, ablation of Capn4 increased 27 Kip1 mRNA levels, likely due to stabilized binding of Akt to protein phosphatase 2A, which presumably results in reduced phosphorylation of Akt on S473 and forkhead Box O (FoxO) 3A on T32. Collectively, calpain regulates cell proliferative function by modulating both transcription and degradation of p27Kip1 in osteoblasts. In conclusion, calpain is a critical modulator for regulation of p27Kip1 in cells of the osteoblast lineage.
Similar content being viewed by others
References
WHO (1994) Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. Report of a WHO Study Group. World Health Organ Tech Rep Ser 843:1–129
Goll DE, Thompson VF, Li H, Wei W, Cong J (2003) The calpain system. Physiol Rev 83:731–801
Arthur JS, Elce JS, Hegadorn C, Williams K, Greer PA (2000) Disruption of the murine calpain small subunit gene, Capn4: calpain is essential for embryonic development but not for cell growth and division. Mol Cell Biol 20:4474–4481
Tram KK, Murray SS, Lee DB, Murray EJ (1993) PTH-induced osteoblast contraction is mediated by cysteine proteases. Kidney Int 43:693–699
Tram KK, Spencer MJ, Murray SS, Lee DB, Tidball JG, Murray EJ (1993) Identification of calcium-activated neutral protease activity and regulation by parathyroid hormone in mouse osteoblastic cells. Biochem Mol Biol Int 29:981–987
Murray EJ, Tram KK, Murray SS, Lee DB (1995) Parathyroid hormone–induced retraction of MC3T3–E1 osteoblastic cells is attenuated by the calpain inhibitor N-Ac-Leu-Leu-norleucinal. Metabolism 44:141–144
Murray EJ, Grisanti MS, Bentley GV, Murray SS (1997) E64d, a membrane-permeable cysteine protease inhibitor, attenuates the effects of parathyroid hormone on osteoblasts in vitro. Metabolism 46:1090–1094
Murray SS, Grisanti MS, Bentley GV, Kahn AJ, Urist MR, Murray EJ (1997) The calpain-calpastatin system and cellular proliferation and differentiation in rodent osteoblastic cells. Exp Cell Res 233:297–309
Shimada M, Mahon MJ, Greer PA, Segre GV (2005) The receptor for parathyroid hormone and parathyroid hormone–related peptide is hydrolyzed and its signaling properties are altered by directly binding the calpain small subunit. Endocrinology 146:2336–2344
Shimada M, Greer PA, McMahon AP, Bouxsein ML, Schipani E (2008) In vivo targeted deletion of calpain small subunit, capn4, in cells of the osteoblast lineage impairs cell proliferation, differentiation, and bone formation. J Biol Chem 283:21002–21010
Kashiwagi A, Schipani E, Fein MJ, Greer PA, Shimada M (2010) Targeted deletion of Capn4 in cells of the chondrocyte lineage impairs chondrocyte proliferation and differentiation. Mol Cell Biol 30:2799–2810
Begum N, Ragolia L (1996) cAMP counter-regulates insulin-mediated protein phosphatase-2A inactivation in rat skeletal muscle cells. J Biol Chem 271:31166–31171
Patel YM, Lane MD (2000) Mitotic clonal expansion during preadipocyte differentiation: calpain-mediated turnover of p27. J Biol Chem 275:17653–17660
Choi YH, Lee SJ, Nguyen P, Jang JS, Lee J, Wu ML, Takano E, Maki M, Henkart PA, Trepel JB (1997) Regulation of cyclin D1 by calpain protease. J Biol Chem 272:28479–28484
Delmas C, Aragou N, Poussard S, Cottin P, Darbon JM, Manenti S (2003) MAP kinase–dependent degradation of p27Kip1 by calpains in choroidal melanoma cells. Requirement of p27Kip1 nuclear export. J Biol Chem 278:12443–12451
Bertoli C, Copetti T, Lam EW, Demarchi F, Schneider C (2009) Calpain small-1 modulates Akt/FoxO3A signaling and apoptosis through PP2A. Oncogene 28:721–733
Xu Y, Xing Y, Chen Y, Chao Y, Lin Z, Fan E, Yu JW, Strack S, Jeffrey PD, Shi Y (2006) Structure of the protein phosphatase 2A holoenzyme. Cell 127:1239–1251
Yajima Y, Kawashima S (2002) Calpain function in the differentiation of mesenchymal stem cells. Biol Chem 383:757–764
Partridge NC, Opie AL, Opie RT, Martin TJ (1985) Inhibitory effects of parathyroid hormone on growth of osteogenic sarcoma cells. Calcif Tissue Int 37:519–525
MacDonald BR, Gallagher JA, Russell RG (1986) Parathyroid hormone stimulates the proliferation of cells derived from human bone. Endocrinology 118:2445–2449
Finkelman RD, Mohan S, Linkhart TA, Abraham SM, Boussy JP, Baylink DJ (1992) PTH stimulates the proliferation of TE-85 human osteosarcoma cells by a mechanism not involving either increased cAMP or increased secretion of IGF-I, IGF-II or TGF beta. Bone Miner 16:89–100
Scutt A, Duvos C, Lauber J, Mayer H (1994) Time-dependent effects of parathyroid hormone and prostaglandin E2 on DNA synthesis by periosteal cells from embryonic chick calvaria. Calcif Tissue Int 55:208–215
Tam VK, Clemens TL, Green J (1998) The effect of cell–matrix interaction on parathyroid hormone (PTH) receptor binding and PTH responsiveness in proximal renal tubular cells and osteoblast-like cells. Endocrinology 139:3072–3080
Nasu M, Sugimoto T, Kaji H, Chihara K (2000) Estrogen modulates osteoblast proliferation and function regulated by parathyroid hormone in osteoblastic SaOS-2 cells: role of insulin-like growth factor (IGF)-I and IGF-binding protein-5. J Endocrinol 167:305–313
Swarthout JT, Doggett TA, Lemker JL, Partridge NC (2001) Stimulation of extracellular signal–regulated kinases and proliferation in rat osteoblastic cells by parathyroid hormone is protein kinase C–dependent. J Biol Chem 276:7586–7592
Onishi T, Hruska K (1997) Expression of p27Kip1 in osteoblast-like cells during differentiation with parathyroid hormone. Endocrinology 138:1995–2004
Qin L, Li X, Ko JK, Partridge NC (2005) Parathyroid hormone uses multiple mechanisms to arrest the cell cycle progression of osteoblastic cells from G1 to S phase. J Biol Chem 280:3104–3111
Acknowledgements
We thank Dr. Hanno Hock and Laura B. Prickett-Rice at the Center for Regenerative Medicine, Massachusetts General Hospital (MGH), for consultation and assistance with the flow-cytometric analysis. This work was partially supported by National Institutes of Health grant R01 DK072102, the William F. Milton Fund, and the MGH interim support fund (to M. S.).
Author information
Authors and Affiliations
Corresponding author
Additional information
The authors have stated that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Kashiwagi, A., Fein, M.J. & Shimada, M. Calpain Modulates Cyclin-Dependent Kinase Inhibitor 1B (p27Kip1) in Cells of the Osteoblast Lineage. Calcif Tissue Int 89, 36–42 (2011). https://doi.org/10.1007/s00223-011-9491-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00223-011-9491-3