1,25-Dihydroxyvitamin D3 modulates calcium transport in goat mammary epithelial cells in a dose-and energy-dependent manner

摘要

Background: Calcium is a vital mineral and an indispensable component of milk for ruminants. The regulation of transcellular calcium transport by 1,25-dihydroxyvitamin D3(1,25-(OH)2D3, the active form of vitamin D) has been confirmed in humans and rodents, and regulators, including vitamin D receptor(VDR), calcium binding protein D9k(calbindin-D9k), plasma membrane Ca2+-ATPase 1b(PMCA1b), PMAC2 b and Orai1, are involved in this process.However, it is still unclear whether 1,25-(OH)2D3could stimulate calcium transport in the ruminant mammary gland.The present trials were conducted to study the effect of 1,25-(OH)2D3supplementation and energy availability on the expression of genes and proteins related to calcium secretion in goat mammary epithelial cells.Methods: An in vitro culture method for goat secreting mammary epithelial cells was successfully established. The cells were treated with different doses of 1,25-(OH)2D3(0, 0.1, 1.0, 10.0 and 100.0 nmol/L) for calcium transport research, followed by a 3-bromopyruvate(3-Br PA, an inhibitor of glucose metabolism) treatment to determine its dependence on glucose availability. Cell proliferation ratios, glucose consumption and enzyme activities were measured with commercial kits, and real-time quantitative polymerase chain reaction(RT-q PCR), and western blots were used to determine the expression of genes and proteins associated with mammary calcium transport in dairy goats, respectively.Results: 1,25-(OH)2D3promoted cell proliferation and the expression of genes involved in calcium transport in a dose-dependent manner when the concentration did not exceed 10.0 nmol/L. In addition, 100.0 nmol/L 1,25-(OH)2D3inhibited cell proliferation and the expression of associated genes compared with the 10.0 nmol/L treatment.The inhibition of hexokinase 2(HK2), a rate-limiting enzyme in glucose metabolism, decreased the expression of PMCA1 b and PMCA2 b at the m RNA and protein levels as well as the transcription of Orai1, indicating that glucose availability was required for goat mammary calcium transport. The optimal concentration of 1,25-(OH)2D3that facilitated calcium transport in this study was 10.0 nmol/L.Conclusions: Supplementation with 1,25-(OH)2D3influenced cell proliferation and regulated the expression of calcium transport modulators in a dose- and energy-dependent manner, thereby highlighting the role of 1,25-(OH)2D3as an efficacious regulatory agent that produces calcium-enriched milk in ruminants when a suitable energy status was guaranteed.