ß-Hydroxybutyrate affects cell physiological parameters, inflammatory markers and hormone receptor expression in bovine endometrial gland cells in vitro.

INTRODUCTION: After calving, dairy cows are commonly affected by negative energy balance (NEB), indicated by high ß-Hydroxybutyrate (BHBA) blood levels. These are associated with subfertility frequently related to uterine inflammation. Since this could compromise functionality of endometrial glands that are essential for proper embryo implantation in sheep, we investigated effects of BHBA on bovine endometrial gland cells (BEGC) in vitro. MATERIAL AND METHODS: BEGC were stimulated with different concentrations of BHBA over different periods. Cell metabolism and motility were examined by MTT-assay and Live-cell-imaging. The mRNA expression of the receptors for estrogen (ESR1, ESR2), progesterone (PR) and IFNτ (IFNAR1, IFNAR2), and the inflammatory cytokines TNFα and IL-6 was determined by RT-qPCR. Protein expression for PR and ESR1 was analyzed by semiquantitative Western Blot. RESULTS: BEGC metabolism was significantly decreased after stimulation with 1.2, 1.8 and 2.4 mM BHBA over 24 and 36 h. Cell motility was significantly reduced by 1.8 and 2.4 mM BHBA already after 11 h. After 24 h stimulation, the ESR1 mRNA expression was significantly increased in BEGC stimulated with 0.6 mM BHBA. PR and TNFα mRNA expressions were increased in cells stimulated with 2.4 mM BHBA. Protein expression of ESR1 and PR was not altered. DISCUSSION: Treatment with BHBA leads to restriction of BEGC metabolism and motility, and increased expression of TNFα, ESR1 and PR in vitro. This could explain how increased BHBA blood levels might compromise functionality of uterine glands in vivo and thus could contribute to compromised reproductive success of cows suffering from NEB.

The importin α/ß-specific inhibitor Ivermectin affects HIF-dependent hypoxia response pathways.

Hypoxia-inducible transcription factors (HIFs) regulate hundreds of genes involved in cellular adaptation to reduced oxygen availability. HIFs consist of an O2-labile α-subunit (primarily HIF-1α and HIF-2α) and a constitutive HIF-1ß subunit. In normoxia the HIF-α subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1-3, resulting in recognition by von Hippel-Lindau protein, ubiquitination and proteasomal degradation. In contrast, reduced oxygen availability inhibits PHD activity resulting in HIF-1α stabilisation and nuclear accumulation. Nuclear import of HIF-1α mainly depends on classical nuclear localisation signals (NLS) and involves importin α/ß heterodimers. Recently, a specific inhibitor of nuclear import has been identified that inhibits importin α/ß-dependent import with no effects on a range of other nuclear transport pathways involving members of the importin protein family. In this study we evaluated the physiological activity of this importin α/ß-inhibitor (Ivermectin) in the hypoxia response pathway. Treatment with Ivermectin decreases binding activity of HIF-1α to the importin α/ß-heterodimer. Moreover, HIF-1α nuclear localisation, nuclear HIF-1α protein levels, HIF-target gene expression, as well as HIF-transcriptional activity are reduced upon Ivermectin treatment. For the first time, we demonstrate the effect of specific importin α/ß-inhibition on the hypoxic response on the molecular level.