Abstract
Recent studies indicated that intramammary administration of active vitamin D3 hormone (1,25D3) inhibits the inflammatory process associated with mastitis. We hypothesized that attenuation of endoplasmic reticulum (ER) stress by 1,25D3 in mammary epithelial cells (MECs) is an important cellular mechanism contributing to this beneficial effect of intramammary treatment with 1,25D3. To test this hypothesis, the effect of 1,25D3 was studied on induction of ER stress in a transformed human MEC line, MCF-7 cells. Treatment with two different ER stress inducers, thapsigargin (TG) and tunicamycin (TM), caused a dose-dependent induction of ER stress as evident from up-regulation of protein kinase RNA-like ER kinase (PERK), heat shock protein family A (Hsp70) member 5 (HSPA5), activating transcription factor (ATF4), ATF6, DNA damage inducible transcript 3 (DDIT3) and spliced X-box binding protein 1 (sXBP1) and impaired cell viability and decreased expression of vitamin D receptor (VDR) in MCF-7 cells (P < 0.05). Treatment with 1,25D3 (100 nM) inhibited TG (10 nM)- and TM (1 μg/mL)-induced mRNA and/or protein levels of ATF4, ATF6, DDIT3 and HSPA5 in MCF-7 cells (P < 0.05). In addition, 1,25D3 (100 nM) antagonized the effect of TG (10 nM) and TM (1 μg/mL) on mRNA and protein levels of VDR and mRNA levels of genes involved in production and degradation of 1,25D3 in MCF-7 cells (P < 0.05). Moreover, 1,25D3 (100 nM) inhibited nuclear factor-κB (NF-κB) activation in response to TM (10 nM) and TG (1 μg/mL) in MCF-7 cells. In conclusion, the present findings show that 1,25D3 is effective in attenuating ER stress and the NF-κB-driven inflammatory response in MCF-7 cells. This indicates that attenuation of ER stress by 1,25D3 in MECs may contribute to the recently observed inhibitory effect of intramammary treatment of dairy cows with 1,25D3 on the inflammatory process associated with mastitis.
Highlights
Mastitis refers to an inflammation of mammary tissue mostly caused by infections with different pathogenic bacteria
A general limitation of this study is the use of a transformed human breast cancer cell line, which displays differences from normal mammary epithelial cells (MECs) with regard to the abundance of certain receptors, such as estrogen receptors [55], and the response to non-physiological stimuli [56], both MCF-7 cells and normal MECs cells exhibit a similar regulation by important lactogenic hormones including oxytocin and prolactin [57,58,59]
This indicates that attenuation of endoplasmic reticulum (ER) stress by 1,25D3 in MECs may contribute to the recently observed inhibitory effect of intramammary treatment of dairy cows with 1,25D3 on the inflammatory process associated with mastitis [6,7,8]
Summary
Mastitis refers to an inflammation of mammary tissue mostly caused by infections with different pathogenic bacteria. Previous in vitro-studies revealed that the active vitamin D3 hormone, 1,25-hydroxyvitamin D3 (1,25D3), improves bactericidal capacity of human and bovine monocytes against common bacterial pathogens involved in mastitis development [3,4,5]. This effect likely contributes to a decreased bacterial growth in mammary glands experimentally infected with Streptococcus uberis [6] and an increased expression of host-defense genes in mammary immune cells [7, 8] of dairy cows subjected to intramammary treatment with 1,25D3 or its metabolite 25D3. Despite the NF-κB-regulated acute inflammatory response is important to effectively combat the infectious bacteria causing mastitis, it is important that the inflammatory process is rapidly attenuated because prolonged production of ROS, cytokines and other inflammatory molecules causes structural damage of the mammary gland through injurious action on cellular components (lipids, proteins, DNA), thereby, decreasing cell viability, and inducing cell death [14]
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