Sodium salicylate reduced mRNA abundance of hypoxia-associated genes in MAC-T cells

Abstract

Hypoxia is an oxygen deficiency commonly found in growing tissues and is speculated to occur in the rapidly developing mammary gland in peripartum dairy cattle. Low oxygen concentrations can activate hypoxia-inducible factor-1 (HIF-1), which increases transcription of genes involved in angiogenesis (VEGFA) and glucose transport (GLUT1), among other processes. The mRNA stability of these genes is positively regulated by heterogeneous nuclear ribonucleoprotein D (HNRNPD; also known as AUF1). In our previous research, postpartum administration of sodium salicylate (SS) increased whole-lactation milk yield in multiparous cows but tended to reduce milk yield in primiparous cows. Because rapid mammary tissue development likely occurs in cows approaching first lactation, we hypothesized that SS inhibited the activation of HIF-1α and decreased transcription of downstream targets. MAC-T cells were treated with SS (100 μM) or control medium before incubation under either hypoxic (1% O2) or normoxic conditions for 12 h. Additionally, cells were transfected with either HIF1A small interfering RNA (siRNA) or a scrambled siRNA negative control 48 h before hypoxia treatments. HIF1A, GLUT1, VEGFA, and HNRNPD were quantified relative to the internal control gene NENF. Transcript abundance was assessed using a linear mixed model with the fixed effects of SS, hypoxia, siRNA, and all 2- and 3-way interaction terms and the random effect of plate nested within hypoxia. Treatment with SS interacted with hypoxia for GLUT1, as SS reduced GLUT1 when MAC-T cells were cultured in normoxic conditions; however, no effect of SS was found in hypoxia-treated cells. Regardless of oxygen status, SS reduced HNRNPD and tended to decrease VEGFA mRNA relative to untreated cells. Hypoxia increased GLUT1, yet no effect was observed on VEGFA or HNRNPD. Small interfering RNA knocked down HIF1A, but no effect was found on GLUT1, VEGFA, or HNRNPD. In conclusion, SS reduced transcript abundance of genes involved with mammary gland development but generally did not interact with oxygen status.