A Potential Role of MMP2 and MMP9 in Regulation of Hepsin and Protein Disulfide Isomerase from Granulosa Cells.

The matrix metalloproteinase (MMP) family of proteolytic enzymes is induced by the preovulatory LH surge and postulated to play a role in the breakdown and reorganization of the ovarian extracellular matrix (ECM) during ovulation. In addition to acting on ECM, the MMPs are also able to function in a variety of non-classical pathways, i.e. the release of cell surface proteins and activation/inactivation of growth factors. However, little is known about the targets of MMP action during the ovulatory process. We have attempted to understand the role of the gelatinases, MMP2 and MMP9, during the periovulatory period by identifying their substrates in granulosa cells using proteomics. In previous experiments, rat granulosa cells were treated with a MMP2/9 Inhibitor, stimulated with hCG for 24 h and the conditioned media subjected to 2D gel electrophoresis, spot identification, trypsinization, and LC/MS and MALDI-TOF analysis. In the current study, we have explored two of the peptide fragments identified by this proteomic approach. One of the upregulated peptides was tentatively identified as serine protease hepsin, a type II membrane-associated protein that is necessary for cell growth in vitro. To confirm our proteomic identification of hepsin in granulosa cells, we determined that Hpn mRNA was present in granulosa cells collected at 0, 6, and 12 h after hCG administration by microarray analysis. To further confirm our proteomic results that the peptide fragment is hepsin, we determined if hepsin is present in granulosa cells using Western blot analysis. In rat granulosa cells cultured for 8, 12, 16 or 24 h with or without hCG, two bands were observed in the cell lysates, a 45 kDa and a 29 kDa peptide, whose expressions were unchanged by hCG treatment. To determine whether hepsin was a target for MMP2/9 action, hepsin was analyzed in granulosa cells cultured with 2 IU of hCG/106 cells/well in the absence or presence of a specific MMP2/9 inhibitor for 24 hours (n=3). Inhibition of the gelatinase activity revealed no change in the 45kDa or 29kDa hepsin peptides in the cell lysates, but an increase in the 29kDa band in the supernatants was observed. This increase from the supernatant samples validates the results seen with the proteomic/mass spectrometry analysis that hepsin is a target for MMP2/9 action. Currently, we are investigating hepsin in the human and have observed that hepsin is present in the normal human ovary as well as in ovarian cancer cells. A second peptide which was downregulated after inhibition of gelationlytic activity was tentatively identified as protein disulfide isomerase (PDI). PDI plays a role in post-transcriptional processing of disulfide bonded proteins catalyzing both formation and rearrangement of intra- and interchain bonds in secreted proteins. To determine if PDI was a target for MMP2/9 action, PDI was incubated with increasing concentrations of MMP2 (0.1- 1.5 ug) for 24 h. There was a dose dependent degradation of PDI with the higher concentrations of MMP2 completely digesting PDI whereas the lower concentrations only partially degraded PDI. In summary, we have identified unique targets for the hCG induced MMPs in rat granulosa cells. Specifically, MMP2 and MMP9 play a role in the regulation of the hepsin at the cell surface and the cleavage of PDI. The function of hepsin and PDI in the ovulatory process or subsequent formation of the CL remains to be elucidated. (Supported by NIH P20RR15592 to TEC)