Previous studies have shown that the actions of
insulin-like growth factor-II (
IGF-II) in bone are determined by both its concentration and the concentrations of the
IGF-binding proteins (IGFBPs). As
IGFBP concentrations may be regulated not only at the level of production, but also at the level of degradation,
IGFBP proteases may be an important component of the
IGF-II regulatory system. In this study, we have identified
IGFBP-4 and
IGFBP-5 protease activity in the
conditioned medium (CM) of the human
osteosarcoma U2 cell line (U2OS) and untransformed normal human bone cell (HBC) derived from skull. Proteolysis of the 29-kilodalton (kDa) [125I]
IGFBP-5 produced an 18- to 20-kDa fragment of
IGFBP-5, and 25 kDa [125I]
IGFBP-4 yielded two lower mol wt fragments in the presence of
IGF-II. CM from
IGF-II-treated U2OS and normal HBC cultures exhibited decreased
IGFBP-5 proteolytic activity compared to control cultures. In contrast, CM from
IGF-II-treated HBC cultures had increased proteolytic activity against
IGFBP-4. To determine the mechanisms by which
IGF-II modulates
IGFBP-4 and -5 proteolytic activity, CM from control U2OS cell culture was incubated with [125I]
IGFBP-4 or -5 in the presence of various concentrations of
IGF-II and IGF analogs under cell-free conditions. It was found that exogenous
IGF-II stimulated
IGFBP-4 proteolysis, but IGF analogs that had no or extremely low affinity to
IGFBP-4 failed to induce
IGFBP-4 proteolysis. On the contrary, exogenous
IGF-II had no effect on
IGFBP-5 proteolysis in cell-free U2OS CM. Both
IGFBP-4 and
IGFBP-5 proteolytic activities were inhibited by
aprotinin,
zinc chloride, and
EDTA and eluted as a single major peak between mol wt markers of 160 and 67 kDa upon gel filtration. Based on the findings that HBCs in culture produce a
protease(s) capable of cleaving both
IGFBP-4 and
IGFBP-5 and that
IGF-II can promote or inhibit proteolytic degradation of
IGFBP-4 and
IGFBP-5, respectively, it is proposed that
IGFBP protease(s) may be an important modulator of IGF activity in bone.