John L. Fowlkes

Matrix metalloproteinases as insulin-like growth factor binding protein-degrading proteinases

Insulin-like growth factor (IGF) bioavailability is modulated by specific IGFBPs, six of which are known (IGFBPs 1-6). Since IGFBPs have equal or higher affinity for IGFs than do IGF receptors, it is believed that degradation of IGFBPs by IGFBP-degrading proteinases is an important step in regulating IGF bioactivity. Recent studies from our laboratory have demonstrated that at least two IGFBPs, i.e. IGFBP-3 and -5, are degraded under physiologic conditions by matrix metalloproteinases (MMPs). In vitro, we have demonstrated that IGFBP-3 is degraded in human dermal fibroblast cultures by MMPs using a variety of techniques, including proteinase inhibition with a specific inhibitor of MMPs, i.e. tissue inhibitor of metalloproteinases (TIMP-1), immunoabsorption with specific antisera to human MMPs and a unique method developed in our laboratory, IGFBP-3 substrate zymography. Using similar methods, we have also demonstrated that MMPs, along with an unidentified 97-kDa proteinase, degrade IGFBP-5 in murine osteoblast cultures. In rat pregnancy serum, we have shown that degradation of IGFBP-3 is associated with MMP activity present in the serum, which likely arises from the placental compartment. Analysis of the cleavage products of IGFBP-3 produced by MMPs 1, 2 and 3 reveals that MMPs cleave exclusively within the non-homologous, mid-region of the molecule. Together, these studies suggest that MMPs, beyond their previously described roles as extracellular matrix degrading enzymes, may also exert effects on cellular growth and differentiation via degradation of IGFBPs.

Differentiation of MC3T3-E1 osteoblasts is associated with temporal changes in the expression of IGF-I and IGFBPs

We examined the relationship between osteoblast maturation and temporal changes in the secretion of IGF-I and the IGF-binding proteins (IGFBPs) in the MC3T3-E1 model of osteoblast development. IGF-I was present at low levels in conditioned media in proliferating preosteoblasts (3.7 +/- 1.7 ng/micrograms DNA and 3.9 +/- 0.6 at culture (days 3 and 9) and increased progressively in postmitotic differentiating osteoblasts, reaching a maximal concentration of 13.1 +/- 1.5 ng/micrograms DNA by day 25 of culture. We also observed an increase in IGF-I mRNA expression. Using Western ligand blot and immunoblot techniques, we found that IGFBP-2, -4, and -5 also displayed temporal differences in expression during MC3T3-E1 development. We observed a sustained increase in IGFBP-2, -4, and -5 mRNA expression between days 10-14, coincident with the onset of differentiation. IGFBP-2 and IGFBP-4 protein concentrations increased in parallel with IGFBP mRNA expression, but IGFBP-5 levels peaked between days 8-14 of culture, and declined thereafter in spite of persistent IGFBP-5 mRNA levels. These findings suggest complex transcriptional and post-transcriptional regulation of IGFBP metabolism during osteoblast development. Thus, IGF-I and IGFBP production are regulated during osteoblast development. In turn, time-dependent changes in IGF-I and modulation of IGF-I bioavailability by IGFBPs may regulate the osteoblastic developmental sequence.