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Whole-transcriptome analysis of nuclear factor erythroid 2-related factor 2 (NRF2) modulation in bovine mammary cells.

H. Ford



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Whole-transcriptome analysis of nuclear factor erythroid 2-related factor 2 (NRF2) modulation in bovine mammary cells.
H. Ford*, M. Bionaz, S. Busato. Oregon State University Corvallis, OR.

The nuclear factor erythroid 2-related factor 2 (NRF2) has been well studied in non-ruminant models and has gained increased attention for its role as a potential target in promoting antioxidant defense and cell survival. The role of NRF2 in bovine and other ruminant models is not well studied. Given the extreme metabolic changes and associated oxidative stress encountered by dairy cattle as they transition from pregnancy to lactation, there is a clear need to better understand the role of NRF2 in ruminants. To accomplish this, MACT cells were treated with sulforaphane (SFN), a putative NRF2 activator, brusatol (BRU), a NRF2 inhibitor, and Morpholino (MOR), a synthetic oligonucleotide inhibitor of NRF2 translation. After 24h of treatment, both BRU and MOR decreased > 80% while SFN increased > 8-fold NRF2 activation as assessed via a gene reporter assay. RNA was extracted, prepared for sequencing using the QuantSeq kit (Lexogen), and sequenced using HighSeq 3500 (Illumina). The RNA-Seq data were normalized by RPM and statistically analyzed via ANOVA using JMP Genomics (SAS). Functional analysis of differentially expressed genes (DEG; FDR ≤ 0.05) compared with an untreated control was performed using DAVID and the Dynamic Impact Approach. The 943 DEG by BRU are associated with overall inhibition of metabolism, especially amino acid and energy metabolism. MOR had 421 DEG associated with activation of inflammatory and immune responses while inhibiting collagen and extracellular matrix. The 502 DEG by SFN are associated with induction of disulfide bonds, glycoproteins and selenoamino acid metabolism, but associated with inhibition of metabolism of other amino acids, ATPase activity, and intracellular trafficking. There were more DEG with opposite effects between BRU and SFN than MOR and SFN (151 vs. 48), indicating that BRU and SFN are modulating not only NRF2. Our study provided the first characterization of NRF2 role in bovine mammary cells highlighting a role of NRF2 that goes beyond the oxidative stress response.

Keywords: NRF2, cow, RNA-Seq.