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ARE MATRIX METALLOPROTEINASE 2 (MMP-2) AND TISSUE INHIBITOR OF METALLOPROTEINASES 2 (TIMP-2) COORDINATELY EXPRESSED WITH MEMBRANE TYPE 1 METALLOPROTEINASE (MT1-MMP) IN THE BOVINE CORPUS LUTEUM DURING THE ESTROUS CYCLE?
Zhang, Bo1, Tsang, Paul1, 1
ABSTRACT- Matrix metalloproteinases (MMPs) and their endogenous regulators, tissue inhibitors of metalloproteinases (TIMPs), constitute a family of proteins that may participate in the extracellular matrix (ECM) remodeling that occurs during corpus luteum (CL) development and demise. Most MMPs are secreted as zymogens and then cleaved extracellularly into their active forms by serine proteinases. However, pro-MMP2 is activated instead on the cell surface by membrane-type 1 metalloproteinase (MT1-MMP). It is now believed that this process also involves TIMP-2, which together with MT1-MMP and MMP-2, forms a triplex on the cell surface to facilitate activation. Previously, we cloned the bovine MT1-MMP gene and showed that it was variably expressed in endothelial and large luteal cells of the bovine CL. The aim of the present study was to investigate whether MMP-2 and TIMP-2 are coordinately expressed with MT1-MMP in the bovine CL over the estrous cycle. Luteal tissues were collected from regularly cycling, nonlactating dairy cows at early (4-days old, day 0=estrus, n=3), mid (10-days old, n=3), and late (16-days old, n=3) stages of the estrous cycle. Northern blotting revealed that MMP-2 mRNA (3.1kb) expression did not differ (P>0.05) over the cycle. However, two TIMP-2 transcripts, a major 1kb band and a minor 3.5kb band, were detected. The expression of both was higher (P<0.05) in mid and late cycle CL than in the early stage. Additionally, Western blotting indicated that immunoreactive TIMP-2 protein (~21kD) was also higher (P<0.05) in mid and late CL than in the early stage, while immunoreactive pro-MMP-2 (~69kD) did not vary (P>0.05) between these stages. Furthermore, immunohistochemistry revealed that TIMP-2 and MMP-2 were co-localized in endothelial and large luteal cells. However, MMP-2 staining was more intense in endothelial cells, while TIMP-2 was more strongly expressed in large luteal cells. Taken together with our previous findings of peak MMP-2 activity and the high level of active MT1-MMP protein in mid and late cycle CL, these results suggest that MMP-2, TIMP-2 and MT1-MMP are temporally and spatially available in the bovine CL to coordinate the process of pro-MMP-2 activation in vivo. (Supported by Regional Project NE-161 to PCWT and Sigma Xi GIAR Award to BZ)
KEY WORDS: corpus luteum, MMP-2, TIMP-2, MT1-MMP