The Role of Activin A in The Human Osteoblast Cell Cycle: A Preliminary Experimental in Vitro Study

Abstract

Animal studies have previously shown that activin A enhances osteoblast proliferation in vitro and increases bone formation and bone mechanical strength in vivo. For the further understanding of its action in human osteoblast, we studied the pattern of a cell cycle response to the treatment with activin A. We hypothesize that activin A alters the cell cycle pattern of human osteoblast. Primary cultures of human osteoblast-like cells were treated by activin A in a biologically effective concentration (100 ng/mL). The cells in cultured samples were counted, assayed for cellular alkaline phosphatase activity and calcitonin expression, LDH activity in the medium, cellular BrdU incorporation, cell cycle cytometry and compared to untreated controls. The treated by activin A cells responded by a significant shift toward the G1 phase of the cell cycle with parallel decrease in cell death rate (lower LDH activity and less necrotic cells in cytometric analysis). The treated cells also showed a lower alkaline phosphatase activity and calcitonin expression, indicating their undifferentiated state, and didn’t change their proliferation rate. The number of cells in culture increased following treatment with activin A. We show that activin A increases the net osteoblast number in culture by reducing the cell death rate without affecting the cell proliferation. These findings should be part of cellular pathways that are involved in the initial stages of bone tissue generation.

Key words

activin A - cell cycle - cell death - osteoblast

References

• 1 Bessey OA, Lowry OH, Brock MJ. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum.  J Biol Chem. 1946;  164 321-329
• 2 Centrella M, McCarthy TL, Canalis E. Activin A binding and biochemical effects in osteoblast-enriched cultures from fetal-rat parietal bone.  Molecular Cellular Biol. 1991;  11 250-258
• 3 Chen D, Ji X, Harris MA. et al . Differential Roles for Bone Morphogenic Protein (BMP) Receptor Type 1B and 1A IN Differentiation and Specification of Mesenchymal Precursor Cells to Osteoblast and Adipocite Lineages.  J Cell Biol. 1998;  142 295-305
• 4 Chen D, Zhao M, Mundy GR. Bone morphogenic proteins.  Growth Factors. 2004;  22 233-241
• 5 Chen YG, Lui HM, Lin SL. et al . Regulation of cell proliferation, apoptosis, and carcinogenesis by activin.  Exp Biol Med. 2002;  227 75-87
• 6 Cheng J, Huo D, Kuang D. et al . Human macrophages promote the motility and invasiveness of osteopontin – knockdown tumor cells.  Cancer Res. 2007;  67 5141-5147
• 7 Eghbali-Fatourechi GZ, Lamsam J, Fraser D. et al . Circulating osteoblast-lineage cells in humans.  N Engl J Med. 2005;  352 1959-1966
• 8 Eijken M, Swagemakers S, Koedam M. et al . The activin A – follistatin system:potent regulator of human extracellular matrix mineralization.  FASEB. 2007;  21 2949-2960
• 9 Funaba M, Ogawa K, Abe M. Expression and localization of activin receptors during endochondral bone development.  Eur J Endocrin. 2001;  144 63-71
• 10 Gaddy-Kurten D, Coker JK, Abe E. et al . Inhibin suppresses and activin stimulates osteoblastigenesis and osteoclastogenesis in murine bone marrow cultures.  Endocrinology. 2002;  143 74-83
• 11 Gay RJ, McComb RB, Bowers GN. Optimum reaction conditions for human lactate dehydogenase isoenzyms as they affect total lactate dehydrogenase activity.  Clin Chem. 1968;  14 740-753
• 12 Gericke A, Qin C, Spevak L. et al . Importance of phosphorylation for osteopontin regulation of biomineralization.  Calcif Tissue Int. 2005;  77 45-54
• 13 Gratzner HG. Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication.  Science. 1982;  218 474-475
• 14 Gundle R, Stewart K, Screen J. et al .Isolation and culture of human bone-derived cells.. In: Beresford N, Owen ME (eds). Marrow stromal cell culture. Cambridge: Cambridge university press; 1998: 43-66
• 15 Harris SE, Bonewald LF, Harris MA. et al . Effects of transforming growth factor beta on bone nodule formation and expression of bone morphogenic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type 1 collagen mRNA in long term cultures of fetal rat calvarial osteoblasts.  J Bone Miner Res. 1994;  9 855-863
• 16 Hashimoto M, Shoda A, Inoue S. et al . Functional regulation of osteoblastic cells by the interaction of activin A with follistatin.  J Biol Chem. 1992;  267 4999-5004
• 17 Ikenoue T, Jingushi S, Urabe K. et al . Inhibitary effects of activin A on osteoblast differentiation during cultures of fetal rat calvarial cells.  J Cel Biochem. 1999;  75 206-214
• 18 Sakai R, Miwa K, Eto Y. Local administration of actvin promotes fracture healing in the rat fibula fracture model.  Bone. 1999;  25 191-196
• 19 Shuto T, Sarkar G, Bronk JT. et al . Osteoblasts express type I and II activin receptors during early intramembranous and endochondral bone formation.  J Bone Miner Res. 1997;  12 403-411
• 20 Yamanouchi K, Satomura K, Gotoh Y. et al . Bone formation by transplanted human osteoblasts cultured within collagen sponge with dexamethasone in vitro.  J Bone Mineral Res. 2001;  16 857-867

Correspondence