Gene Expression, Localization, and Regulation of Activated Leukocyte Cell Adhesion Molecule/CD166 in the Bovine Corpus Luteum.

Abstract

Activated leukocyte adhesion molecule (ALCAM/CD166) is a transmembrane glycoprotein expressed on a variety of epithelial cell types in normal tissues, but it is predominantly relevant to the biology of ovarian, breast, prostate, pancreatic and colorectal cancers. The objectives of the present study were to 1) determine if the ALCAM gene and protein are expressed in the CL, 2) examine if there are temporal changes in expression during the estrous cycle and early pregnancy (day 17), 3) identify the cell types on which it is expressed, and 4) evaluate regulation of expression by paracrine factors. Gene expression was determined by qPCR in luteal tissue collected at early (day 5), midcycle (days 10-12) and late (day 18) stages of the estrous cycle, and 12hr post prostaglandin F2a injection (pPGF2a) administered at midcycle (n= 3-6). In addition to gene expression, protein ALCAM was determined in early (day 4), midcycle and regressing CL (1, 8 and 12hrs pPGF2a) using Western blot analysis (n= 3). The steady-state concentration of ALCAM mRNA was higher in early compared to midcycle and late CL (P<0.05) and lower at midcycle compared to 12hrs pPGF2a (P<0.05). In contrast, the pattern of ALCAM protein expression was inversely proportional to the mRNA (P<0.05). ALCAM protein at 12hrs pPGF2a was lower compared to midcycle, 1 and 8hrs pPGF2a, (P<0.05). Compared to day 17 of the estrous cycle, ALCAM protein was lower at day 17 of pregnancy (P<0.05).To determine the proportion of cells expressing ALCAM in functional and regressing CL, freshly isolated and cultured luteal cells from CL collected at midcycle and 8hrs pPGF2a were labeled with anti-ALCAM. Furthermore, to identify if ALCAM in luteal cells was expressed on monocytes/macrophage and activated leukocytes, cells were colabeled with either anti-CD14 or anti-CD18 antibodies. The cultured luteal cells from midcycle CL were also treated with luteinizing hormone (LH), PGF2a, interferon gamma (IFNg) and phorbolmyristate acetate (PMA) for 48hrs and later labeled with anti-ALCAM antibody. All labeled cells were analyzed by flow cytometry. Both freshly isolated and cultured cells expressed ALCAM. There was no difference in the proportion of ALCAM+ cells between midcycle and regressing luteal cells (P>0.05). The proportion of ALCAM+ cells expressing CD14 and CD18 ranged between 5-20%. The proportion of ALCAM+ cells was increased by LH, IFNg and PMA (P<0.05), but not PGF2a. To further determine the cell types expressing ALCAM, we performed fluorescence microscopy on luteal cells and frozen tissue sections labeled with anti-ALCAM and Bandeira simpilicifolia-1 lectin (labels endothelial cells). In dissociated luteal cells, ALCAM was mainly expressed on steroidogenic-like cells, but not endothelial cells. In tissue sections, ALCAM was observed on steroidogenic-like cells as well as endothelial cells lining blood vessels. In conclusion, this is the first report to show that ALCAM is expressed in the CL. It is predominantly expressed in steroidogenic cells and its abundance changes over time. Because ALCAM has been shown to activate T lymphocytes through the CD6 receptor, it is suggested that luteal cells may activate T cells via ALCAM. This project was supported by National Research Initiative Competitive Grant no.2008-35203-04617 from the USDA National Institute of Food and Agriculture.