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Title: Identification and characterization of a Nuclear Factor Kappa B p65 proteolytic fragment in nuclei of porcine hepatocytes in monolayer culture

item Caperna, Thomas
item Shannon, Amy
item Garrett, Wesley
item Ramsay, Timothy
item Blomberg, Le Ann
item Elsasser, Theodore

Submitted to: Domestic Animal Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/12/2013
Publication Date: 10/1/2013
Citation: Caperna, T.J., Shannon, A.E., Garrett, W.M., Ramsay, T.G., Blomberg, L., Elsasser, T.H. 2013. Identification and characterization of a Nuclear Factor Kappa B p65 proteolytic fragment in nuclei of porcine hepatocytes in monolayer culture. Domestic Animal Endocrinology. 45:154-162.

Interpretive Summary: The liver is a primary organ involved in integration of an animal’s response to environmental or metabolic stress. In this study, liver cells prepared from suckling pigs were isolated and maintained in culture and were used to investigate their response to inflammatory stress. By using this technique we showed that a specific set of proteins, known as transcription factors could be activated by tumor necrosis factor, a specific molecule produced by immune system cells. The transcription factor that responds to tumor necrosis factor resides in liver cells in an inactive state. Once tumor necrosis factor binds to the surface of the cell, the transcription factor is released from the inactive state and travels to the nucleus where it binds to the cell’s DNA. This action regulates the expression of numerous genes that allow the liver and thus the pig to respond to the inflammatory challenge. By using a combination of chemical and physical techniques we isolated and identified a specific fragment of the transcription factor that had not been previously identified. In addition, we showed that tumor necrosis factor was able to both enhance and inhibit the production of several proteins within the liver cells. These studies will allow us to better understand how the suckling pig handles inflammatory stress and will aid in our ability to treat and possibly prevent the decline in growth rate associated with early environmental stress in piglets.

Technical Abstract: Hepatocytes prepared from suckling pigs, and maintained in monolayer culture were used to investigate transcription factor activity at the cellular level. The hepatic response to proinflammatory signals is controlled by the activation of several transcription factors, including, Nuclear Factor-Kappa B (NF-'B). Nuclear F-'B is comprised of a family of several different heteromeric dimers which reside in the cytosol bound within an inhibitor complex, I'B. Following activation and phosphorylation of the I'B-NF-'B complex, the 65 kD protein (p65) migrates to the nucleus which induces transcriptional activity. In porcine hepatocytes, the immunoreactive p65 molecule appeared at the appropriate molecular weight in the cytosol but at a lower molecular weight in nuclei following short-term incubation with tumor necrosis factor–a (TNF). Incubation with TNF also resulted in phosphorylation of I'B, while incubation with other cytokines, interleukin-6 (IL-6), IL-17a or oncostatin M was not associated with either phosphorylation of I'B or nuclear translocation of p65. Western blot analysis of the nuclear protein revealed a protein of approximately 38kD (p38) which was not observed in the cytosol. The p38 protein was purified by immunoprecipitation, concentrated by electrophoresis and subsequently analyzed by mass spectrometry. Ten unique p65 peptides were identified following digestion with trypsin and chymotrypsin and all were mapped to the N-terminus and within the first 310 amino acid residues of the intact p65 protein. While a low molecular weight immunoreactive p65 has been previously observed, this is the first report to positively identify the p38 fragment within hepatic nuclei following cytokine induction.