Mechanism of Mitogenic Action of Aluminum Ion on Bone Cells : Potential Involvement of the Insulin-Like Growth Factor Regulatory System

Abstract

Micromolar concentrations of aluminum ion (oxidation state of 3 +) consistently stimulated [3H]thymidine incorporation into cell DNA and increased cellular alkaline phosphatase activity (an osteoblastic differentiation marker) in osteoblast-like cells of chicken and human origin. Although biphasic, dose-dependent stimulations were highly reproducible, the maximal stimulatory dose varied among different experiments. Mitogenic doses of aluminum ion also stimulated collagen synthesis in cultured human osteosarcoma TE85 cells. In addition, the same mitogenic concentrations of aluminum ion enhanced the 1,25 dihydroxy vitamin D3-dependent stimulation of osteocalcin secretion. Together, these findings indicate that aluminum at micromolar concentrations, stimulates the proliferation, differentiation, and activity of human osteoblastic-line cells in vitro. With respect to the mechanism of its mitogenic action, the mitogenic concentrations of aluminum ion did not stimulate cAMP production in human osteosarcoma TE85 cells, indicating that the mechanism of aluminum ion does not involve cAMP. Additional studies have also revealed that the mitogenic activity of aluminum ion is different from that of fluoride because (a) unlike fluoride, the mitogenic action of aluminum was unaffected by culture medium changes (/. e., it was not dependent on the presence of essential factors in the cell-conditioned media); (b) unlike fluoride, the mitogenic action of aluminum was not specific for bone cells; and (c) interaction studies with fluoride revealed that aluminum and fluoride did not share the same rate-limiting step(s) for their effects in stimulating the proliferation of osteoblastic-like cells. To test the additional hypothesis that the mitogenic action of aluminum ion is mediated through the insulin-like growth factor (IGF) regulatory system, the effects of mitogenic concentrations of aluminum on IGF-I and IGF-II production were evaluated. TE85 cells treated with aluminum ion for 48 hours showed biphasic stimulation of the synthesis/secretion of IGF-I and IGF-II into the conditioned medium (CM). The addition of IGFBP-4, an inhibitory IGF binding protein, significantly reduced the effect of aluminum to stimulate [3H]thymidine incorporation, supporting the hypothesis that the mitogenic action of aluminum was mediated by an increase in IGF production. Western ligand blot analysis revealed that mitogenic concentrations of aluminum ion also inhibited the secretion of IGF binding proteins, particularly the inhibitory IGFBP-4, an action which could lead to the potentiation of the overall activity of IGFs. Based on these findings, it is concluded that aluminum ion, at micromolar concentrations, acts directly on human bone cells to stimulate their proliferation and differentiation by a mechanism that involves increase in the production and activity of IGFs in bone cells