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Validation of putative target genes of nuclear factor erythroid 2-related factor 2 (NRF2) in bovine.

H. Ford



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Validation of putative target genes of nuclear factor erythroid 2-related factor 2 (NRF2) in bovine.
H. Ford*, M. Bionaz. Oregon State University Corvallis, OR.

Finding ways to ameliorate the oxidative stress experienced by high-producing dairy cattle in the periparturient period is one of the many challenges faced by researchers and producers focusing on dairy production. The nuclear receptor transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) has been studied in non-ruminant models and may be utilized in ruminants to reduce oxidative stress through the upregulation of genes associated with antioxidant defense and cell survival. This is extremely important in the mammary gland where heightened oxidative stress can cause cell death and reduce milk production. Despite this, research on NRF2 in ruminants is limited and there is a need to identify the genes affected by NRF2 modulation and the associated biological outcomes. In this study, the aim was to validate in bovine a set of 7 putative NRF2 target genes as established in monogastric animals. This was achieved by using a combination of 10 μM of the NRF2 agonist sulforaphane (SFN), 100 nM of the NRF2 antagonist brusatol (BRU), NRF2-translation inhibition via Morpholino, and an untreated control in immortalized bovine mammary epithelial cells (MAC-T). RNA was extracted for RT-qPCR. Genes upregulated by SFN were GSR, KEAP1, NFE2L2, GCLC and NQO1 while treatment with BRU resulted in significant downregulation of GSTM1 and NQO1, but upregulation of GCLC and NFE2L2. Furthermore, treatment of cells with Morpholino resulted in downregulation of GSTM1, GPX1, and NQO1 but upregulation of GCR, GCLC, and KEAP1. Data indicated that transcription of GSTM1 and GPX1 requires a basic activation of NRF2 but is not induced by it. Transcription of GCLC and NFE2L2 is partly controlled by NRF2 but the regulation is complex, likely involving other transcription factors. The data revealed that NQO1 is the only transcript among the ones tested that is tightly regulated by NRF2 (i.e., true NRF2 target). All the other putative NRF2 targets are likely controlled by additional transcription factors in bovine mammary cells. Our data highlighted the problem of using in ruminant target genes previously identified in monogastric animals and, thus, the need to identify reliable NRF2 targets in bovine.

Keywords: NRF2, cow, RT-qPCR.