Activation of caspases is required for osteoblastic differentiation.
Previous studies have shown that mouse osteoblastic MC3T3-E1 cells undergo apoptosis when exposed to a mixture of proinflammatory cytokines. Bone morphogenetic protein (BMP)s are important regulators of osteoblast differentiation. Because regulation of osteoblastic differentiation is poorly understood, we sought to determine if BMP-4-induced differentiation of osteoblastic cells depends on the activity of the key apoptotic proteases, i.e. the caspases. BMP-4 induced the growth arrest and differentiation of osteoblastic cell line MC3T3-E1, as evidenced by the appearance of osteoblastic phenotypes such as alkaline phosphatase (ALP) activation and parathyroid hormone (PTH)-dependent production of cAMP. Surprisingly, BMP-4 induced transient and potent activation of caspase-8, caspase-2, and caspase-3, in this order. However, no apoptosis or necrosis in BMP-4-treated cells could be detected by FACS using annexin-V/propodium iodine double staining. Peptide inhibition of caspase activity led to a dramatic reduction in ALP activation and PTH-induced production of cAMP in BMP-4-treated cells. Although BMP-4 treatment resulted in cell-cycle G0/G1 arrest as detected by FACS cell-cycle analysis, caspase inhibitors (caspase-8, caspase-2, and caspase-3 inhibitors) could block the G0/G1 arrest in MC3T3-E1 cells. Taken together, these results confirm a unique and unanticipated role for the caspase-mediated signal cascade in the differentiation of osteoblasts.
Mogi
Makio
M
Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, Nagoya 464-8650, Japan. makio@dpc.aichi-gakuin.ac.jp
Togari
Akifumi
A
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D053143:Caspase 2; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002469:Cell Separation; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D051379:Mice; D010006:Osteoblasts / Q000166:cytology*; D010281:Parathyroid Hormone / Q000378:metabolism; D010641:Phenotype; D013997:Time Factors
C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D010281:Parathyroid Hormone; D000242:Cyclic AMP; D000469:Alkaline Phosphatase; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053143:Caspase 2; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases