Orai calcium release-activated calcium modulator 1 (ORAI1) plays a role in endoplasmic reticulum stress in bovine mammary epithelial cells challenged with physiological levels of ketone bodies

摘要

The Orai calcium release-activated calcium modulator1 (ORAI1) is a key component of the store-operatedCa~(2+) entry mechanism regulating cellular Ca~(2+) balancein nonruminants. Alterations in ORAI1 abundancehave been associated with endoplasmic reticulum (ER)stress and changes in lipid metabolism in hepatocytes,an important lipogenic organ in nonruminants. Objectiveswere to (1) determine abundance of ORAI1 andcomponents of the ER stress response in mammarytissue of ketotic cows, and (2) the potential role ofORAI1 on mammary cell responses to high levels ofβ-hydroxybutyrate (BHB). Healthy (n = 6, plasmaBHB < 0.60 mmol/L) and clinically ketotic (n = 6,plasma BHB > 2.0 mmol/L) Holstein cows (days in milk= 10.13 ± 1.90) were used for mammary gland tissueand blood sample collection. Although milk production(22.5 ± 1.26, 33 ± 1.59, kg of milk/cow per day) anddry matter intake (19.5 ± 1.05, 21.9 ± 0.95, kg/d) werelower in ketotic cows, abundance of ORAI1 proteinwas greater and was associated with greater mRNAabundance of ER stress proteins (PERK, IRE1, ATF6,and GRP78) and lipogenic genes (FASN, SREBP1,and ACACA). Cellular mechanisms to establish linksbetween BHB and mammary cell responses were evaluatedusing the immortalized cell line bovine mammaryepithelial cells (MAC-T). First, a dose response studywas performed with 0, 0.6, 1.2, 1.8, 2.4, or 4.8 mM BHBfor 24 h. The mRNA abundance of FASN, SREBP1,and ACACA and lipid droplet formation peaked at1.2 mM BHB. A subsequent study involved transfectingMAC-T with small interfering Orai 1 (siORAI1)or the ORAI1 inhibitor BTP2 for 24 h followed by achallenge with 1.2 mM BHB for 24 h. Transcriptionand protein abundance of FASN, SREBP1, ACACA,and ER stress proteins returned to basal levels whenORAI1 was silenced or inhibited. Furthermore, theCa~(2+) ionophore ionomycin (raises the intracellular levelof Ca~(2+)) also increased abundance of ORAI1, FASN,SREBP1, ACACA, and ER stress proteins. Data suggestthat the mammary gland experiences ER stressduring ketosis, partly due to the greater supply of BHBoriginating from ketogenesis in the liver. IntracellularCa~(2+) signaling and ORAI1 seem to mediate in part theBHB-induced ER stress in mammary cells.