The roles of insulin-like growth factors (IGFs) in regulating growth and their modulation by six IGF binding proteins (IGFBP) are well established. IGFBP-5, the most abundant IGFBP stored in bone, is an important regulator of bone formation via IGF-dependent and -independent mechanisms. Two new proteins, four and a half lim (FHL)-2, a transcription modulator that interacts with IGFBP-5, and a disintegrin and metalloprotease (ADAM)-9, an IGFBP-5 protease, have been identified as potential regulators of IGFBP-5 action in bone. We tested the hypothesis that agents which modulate bone formation by regulating IGFBP-5 expression would also regulate FHL-2 and ADAM-9 expression in a coordinated manner. We evaluated the expression of IGFBP-5, FHL-2, and ADAM-9 by real-time reverse transcriptase (RT)-PCR during differentiation of mouse bone marrow stromal cells into osteoblasts and in response to treatment with bone formation modulators in the LSaOS human osteosarcoma cell line. IGFBP-5 and FHL-2 increased 4.3- and 3.0-fold (P < or = 0.01), respectively, during osteoblast differentiation. Dexamethasone (Dex), an inhibitor of bone formation, decreased IGFBP-5 and FHL-2 and increased ADAM-9 in LSaOS cells (P < or = 0.05). Bone morphogenic protein (BMP)-7, a stimulator of bone formation, increased IGFBP-5 and decreased ADAM-9 (P<0.01). To determine if BMP-7 would eliminate Dex inhibition of IGFBP-5, cells were treated with Dex+BMP-7. The BMP-7-induced increase in IGFBP-5 was reduced, but not eliminated, in the presence of Dex (P < or = 0.01), indicating that BMP-7 and Dex may regulate IGFBP-5 via different mechanisms. Transforming growth factor (TGF)-beta, a stimulator of bone formation, increased IGFBP-5 and FHL-2 expression (P < or = 0.01). IGF-I and TNF-alpha decreased expression of ADAM-9 (P<0.05). In conclusion, our findings are consistent with the hypothesis that FHL-2 and ADAM-9 are important modulators of IGFBP-5 actions and are, in part, regulated in a coordinated manner in bone.