Differentiation of human osteoblastic cells in culture: modulation of proteases by extracellular matrix and tumor necrosis factor-alpha.

We have followed the synthesis and secretion of urokinase-type plasminogen activator (u-PA) and its inhibitor, PAI-1, and matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP-1) during differentiation of a human osteoblastic cell line, HOS TE85, and the effect of TNF-alpha on this process. Our results show that the ratio of u-PA/PAI-1 associated with the cell-matrix components increases during differentiation of these cells over a 14-day period. Although TNF-alpha suppresses the induced increase in steady-state mRNA levels of u-PA and PAI-1 during maturation of extracellular matrix (ECM), the u-PA/PAI-1 ratio is altered in such a way that PA activity associated with the ECM is higher than control cells. The expression of MMP-1 is low and remains essentially invariant over a culture period of 14 days. TNF-alpha enhances MMP-1 transcription nearly 12-fold initially, after which mRNA levels drop off but remain significantly higher than the controls. Activities and steady-state mRNA levels of MMP-2 and MMP-9 increase nearly 15-fold during maturation of the ECM, but the level of TIMP-1 mRNA is not appreciably altered. The presence of TNF-alpha suppresses maturation-induced transcription of MMP-2, enhances TIMP-1 transcription, but has little effect on MMP-9 mRNA levels. The data show that chronic exposure to TNF-alpha alters the balance between u-PA/PAI-1 and MMPs/TIMP-1, which favors higher activity of proteinases. Accordingly, the presence of TNF-alpha in chronic inflammatory episodes would be expected to alter bone remodeling by inhibiting maturation of ECM and formation of bone.