Overgaard, Michael T.

Pregnancy-associated plasma protein A (PAPP-A) is an IGF-binding protein-4 (IGFBP-4) metalloproteinase that cleaves inhibitory IGFBP-4 to amplify local IGF-I bioavailability in vitro. Thus it has functional implications in injury/repair responses. In this study we determined PAPP-A expression in healing human skin. Wounds were induced with a scalpel on the forearms of three normal subjects and were allowed to heal by first intention. Biopsies obtained on d 0, 2, 8, and 14 were processed for immunohistochemical detection of PAPP-A, IGF-I, and IGFBP-4. In uninjured skin (d 0), strong staining for PAPP-A was present in the epidermis, sweat and sebaceous gland epithelial cells, hair follicles, and blood vessels; no PAPP-A was detected in dermal fibroblasts or with mature collagen bundles. IGF-I localized strongly to epithelial cells of skin glands was weak to moderate in epidermis and blood vessels, and was absent in dermal cells. Weak focal staining for IGFBP-4 was found within uninjured epidermis. During wound healing, PAPP-A expression was induced in dermal granulation tissue within and adjacent to the injury. PAPP-A was present in dermis on d 2 and was increased in intensity and extent on d 8 and 14. PAPP-A expression also increased in the epidermis. PAPP-A expression in cells of granulation tissue colocalized with α-smooth actin staining of myofibroblasts and new blood vessels as well as with CD68 staining of macrophages and was associated with the compact, newly synthesized collagen of the healing wound. IGF-I staining was enhanced in the epidermis localized to the area of the incision and in granulation tissue associated with lymphoid cells. IGFBP-4 staining of the epidermis remained unchanged during wound healing, but was induced in the fibroblastic cells of granulation tissue over time. These data demonstrate localized and regulated expression of PAPP-A in human skin and suggest that PAPP-A may play an important role in an integrated IGF system in wound healing and tissue remodeling in vivo.

Human pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and -II. Through this mechanism, PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system. PAPP-A belongs to the metzincin superfamily of zinc metalloproteinases, and is the founding member of a fifth metzincin family, the pappalysins. Herein, we first determined that PAPP-A cleaves IGFBP-4 at a single site (Met-135/Lys-136), and we analysed the influence of ionic strength, pH and zinc ion concentration on the cleavage reaction. Secondly, we sought to delineate the role of substrate residues in PAPP-A-mediated cleavage by the construction and analysis of 30 IGFBP-4 mutants in which various residues were replaced by alanine, by the analysis of eight mutants of IGFBP-5 (found recently to be a second PAPP-A substrate), and by cleavage analysis of synthetic peptides derived from IGFBP-4. Our data reveal a complex mode of substrate recognition and/or binding, pointing at important roles for several basic residues located up to 16 residues N-terminal to the scissile bond. An unexpected parallel can be drawn with an intracellular enzyme, the mitochondrial processing peptidase, that may help us to understand properties of the pappalysins. Further, proteinase-resistant variants of IGFBP-4 and −5, presented here, will be useful tools for the study of proteolysis in cell-based systems, and our finding that a synthetic peptide can be cleaved by PAPP-A provides the basis for development of quantitative assays for the investigation of PAPP-A enzyme kinetics.

Proteolytic cleavage of the six known insulin-like growth factor binding proteins (IGFBPs) is a powerful means of rapid structure and function modification of these important growth-regulatory proteins. Intact IGFBP-4 is a potent inhibitor of IGF action in vitro, and cleavage of IGFBP-4 has been shown to abolish its ability to inhibit IGF stimulatory effects in a variety of systems, suggesting that IGFBP-4 proteolysis acts as a positive regulator of IGF bioavailability. Here we report the isolation of an IGF-dependent IGFBP-4-specific protease from human fibroblast-conditioned media and its identification by mass spectrometry microsequencing as pregnancy-associated plasma protein-A (PAPP-A), a protein of unknown function found in high concentrations in the maternal circulation during pregnancy. Antibodies raised against PAPP-A both inhibited and immunodepleted IGFBP-4 protease activity in human fibroblast-conditioned media. Moreover, PAPP-A purified from pregnancy sera had IGF-dependent IGFBP-4 protease activity. PAPP-A mRNA was expressed by the human fibroblasts and osteoblasts, and PAPP-A protein was secreted into the culture medium. In conclusion, we have identified an IGF-dependent IGFBP protease and at the same time assigned a function to PAPP-A. This represents an unanticipated union of two areas of research that were not linked in any way before this report.