In vitro model of parathyroid hormone-related protein secretion from mammary cells isolated from lactating cows |
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| Institution: | 1. Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University; Columbus, OH, USA;7. Department of Veterinary Pathology, Rakuno Gakuen University, Ebetsu, Hokkaido 069, Japan;71. Department of Dairy Science, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH, USA;2. Department of Periodontics/Prevention & Geriatrics, The University of Michigan, Ann Arbor, Ml 48109, USA;1. CAS Key Laboratory of Genome Sciences and Information, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China;2. School of Life Science and Technology, ShanghaiTech University, Shanghai, China;3. University of Chinese Academy of Sciences, Beijing, China;4. Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China;5. Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China;6. MOE Key Laboratory of Bioinformatics, Center for Stem Cell Biology and Regenerative Medicine, THU-PKU Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China;1. Center for Personalized Cancer Therapy, UCSD Moores Cancer Center, University of California San Diego, La Jolla, CA, USA;2. Division of Hematology-Oncology, University of California San Diego, La Jolla, CA, USA;1. Howard Hughes Medical Institute Research Laboratories, Department of Embryology, Carnegie Institution for Science, Baltimore, MD 21218, USA |
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| Abstract: | Parathyroid hormone-related protein (PTHrP) is produced by the lactating mammary gland and is present in milk in a biologically active form. The goal of this investigation was to determine if cells cultured from the lactating mammary glands of cows would secrete PTHrP in vitro. Mammary acini were isolated from lactating cows at 1–6 wk after calving, and fresh or cryopreserved mammary acini were cultured for 14 d on Type I collagen. Cultures on thick layers of collagen (2.5 mm) were detached 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 confluence at Day 6. PTHrP production was low at Day 2 (<0.5 ng/ml) but increased to peak production (2–4 ng/ml) at approximately Day 6 and remained constant until Day 14. Immunoreactive and bioactive PTHrP levels in the culture medium correlated well. The cultures produced lactoferrin (2,000–2,300 ng/ml) and αs1-casein (14–19 ng/ml). Prolactin stimulated PTHrP production approximately 50% on Days 6–14. PTHrP production was increased approximately 100% by treatment with epidermal growth factor (10 ng/ml) for 2 d. Morphologic evaluation of cultures on thick, contracted collagen at Day 14 revealed an inner layer of mammary epithelial cells overlying myoepithelial cells and an outer layer of collagen containing stromal cells. Immunohistochemistry demonstrated positive staining for PTHrP and cytokeratin in both mammary epithelial and myoepithelial cells and a-smooth muscle actin in myoepithelial cells. These data demonstrated that cryopreserved mammary tissue from lactating cows could be cultured in vitro and secreted PTHrP in a regulated manner. This in vitro model will be useful to investigate the function and regulation of PTHrP in the lactating mammary gland. |
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