IN-VITRO MODEL OF PARATHYROID HORMONE-RELATED PROTEIN SECRETION FROM MAMMARY CELLS ISOLATED FROM LACTATING COWS

Parathyroid hormone-related protein (PTHrP) is produced by the lactati ng mammary gland and is present in milk in a biologically active form. The goal of this investigation was to determine if cells cultured fro m the lactating mammary glands of cows would secrete PTHrP in vitro. M ammary acini were isolated from lactating cows at 1-6 wk after calving , and fresh or cryopreserved mammary acini were cultured for 14 d on T ype I collagen. Cultures on thick layers of collagen (2.5 mm) were det ached and allowed to contract on Day 6. PTHrP production was measured by N-terminal radioimmunoassay and bioassay (increased cAMP levels in ROS 17/2.8 osteoblast-like cells). The mammary cells reached confluenc e at Day 6. PTHrP production was low at Day 2 (<0.5 ng/ml) but increas ed to peak production (2-4 ng/ml) at approximately Day 6 and remained constant until Day 14. Immunoreactive and bioactive PTHrP levels in th e culture medium correlated well. The cultures produced lactoferrin (2 ,000-2,300 ng/ml) and alpha(s1)-casein (14-19 ng/ml). Prolactin stimul ated PTHrP production approximately 50% on Days 6-14. PTHrP production was increased approximately 100% by treatment with epidermal growth f actor (10 ng/ml) for 2 d. Morphologic evaluation of cultures on thick. contracted collagen at Day 14 revealed an inner layer of mammary epit helial cells overlying myoepithelial cells and an outer layer of colla gen containing stromal cells. Immunohistochemistry demonstrated positi ve staining for PTHrP and cytokeratin in both mammary epithelial and m yoepithelial cells and alpha-smooth muscle actin in myoepithelial cell s. These data demonstrated that cryopreserved mammary tissue from lact ating cows could be cultured in vitro and secreted PTHrP in a regulate d manner. This in vitro model will be useful to investigate the functi on and regulation of PTHrP in the lactating mammary gland.