Abstract
Using real-time RT-PCR and Western blot analysis in bovine kidney epithelial cells, we systematically investigated the effects of butyrate on patterns of gene expression relevant to DNA replication apparatus. The real-time PCR and Western blot data generally confirmed previously reported microarray data. Of the five genes tested by quantitative RT-PCR, CDKN1A (p21(waf1)) was up regulated, CDC2/cdk1, MCM6, ORC1L were down regulated, while ORC3L expression remained unchanged following butyrate treatment. Also consistent with RT-PCR results, Western blot analysis confirmed that butyrate up-regulated cyclin-kinase inhibitor p21(waf1) in a does-dependent manner. In contrast, butyrate treatment had no effect on the expression of ERK 1/2 proteins. Also consistent with mRNA results, ORC1 and MCM3 proteins were down-regulated by butyrate treatment, while ORC2 protein remained unchanged. The present results suggest that ORC1, not ORC2 or ORC3, along with MCM proteins play a critical role in regulating the initiation of DNA replication and cell cycle progression in MDBK cells and are targets of butyrate regulation.
Highlights
In cattle, few definitive studies, if any at all, have sought to address the capabilities of nutrients to modulate gene expression and proteomic outcomes as a means of arresting metabolic stress
The present results suggest that ORC1, not ORC2 or ORC3, along with MCM proteins play a critical role in regulating the initiation of DNA replication and cell cycle progression in MDBK cells
Initiation of eukaryotic DNA replication depends on the function of pre-replication complexes, one of its key components being the six subunits of the origin recognition complex (ORC)
Summary
Ruminant species metabolize SCFAs to fulfill up to 70% of their nutrient energy requirements Beyond their nutritional impact; SCFAs, especially butyrate, modulate cell differentiation, proliferation, motility, and in particular, induce cell cycle arrest and apoptosis. Because short-chain fatty acids are common nutrients, understanding their important biological functions additional to a simple energy supply will certainly help us to understand critical control points in the cell cycle that could lead to improvements in the efficient production of food animals. During periods of naturally encountered stresses, such as the weaning period when production and uptake of short-chain fatty acids are significantly increased, short-chain fatty acids-induced apoptosis and cell cycle arrest may affect growth slumps of young cattle. MDBK as an established bovine cell line with inducible apoptosis and cell cycle regulatory events certainly is ideal and an invaluable tool for functional genomic studies on homeostasis in animals
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