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A RelA(p65) Thr505 phospho-site mutation reveals an important mechanism regulating NF- κ B-dependent liver regeneration and cancer

View Article: PubMed Central - PubMed

ABSTRACT

Post-translational modifications of nuclear factor (NF)-κB subunits provide a mechanism to differentially regulate their activity in response to the many stimuli that induce this pathway. However, the physiological significance of these modifications is largely unknown, and it remains unclear if these have a critical role in the normal and pathological functions of NF-κB in vivo. Among these, phosphorylation of the RelA(p65) Thr505 residue has been described as an important regulator of NF-κB activity in cell lines, but its physiological significance was not known. Therefore, to learn more about the role of this pathway in vivo, we generated a knockin mouse with a RelA T505A mutation. Unlike RelA knockout mice, the RelA T505A mice develop normally but exhibit aberrant hepatocyte proliferation following liver partial hepatectomy or damage resulting from carbon tetrachloride (CCl4) treatment. Consistent with these effects, RelA T505A mice exhibit earlier onset of cancer in the N-nitrosodiethylamine model of hepatocellular carcinoma. These data reveal a critical pathway controlling NF-κB function in the liver that acts to suppress the tumour-promoting activities of RelA.

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The phenotype of RelA T505A mice. (a) Schematic diagram of RelA showing the location of the Thr505 residue in transactivation domain 2. (b) Liver body weight ratio of wild type (WT), WT/T505A and T505A 12-week-old littermate mice. WT, n=4 males, 8 females; WT/T505A, n=2 males, 5 females; T505A, n=4 males, 5 females. Data represent mean±s.e.m. (c) Western blots showing relative levels of NF-κB subunits in WT and RelA T505A mice. (d) Western blot analysis of phospho-S536 RelA, RelA, IκB-α and β-actin from WT and RelA T505A hepatocytes. Hepatocytes were cultured in 0% FBS media and treated with 50 ng/ml of TNF-α for 10 or 30 min before preparation of cell lysates. Lanes represent independent experimental replicates from a single hepatocyte isolation. These results are representative of data from three separate hepatocyte preparations. In this and subsequent experiments, all mice were designated to an experimental group dependent on their strain. NS, not significant.
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fig1: The phenotype of RelA T505A mice. (a) Schematic diagram of RelA showing the location of the Thr505 residue in transactivation domain 2. (b) Liver body weight ratio of wild type (WT), WT/T505A and T505A 12-week-old littermate mice. WT, n=4 males, 8 females; WT/T505A, n=2 males, 5 females; T505A, n=4 males, 5 females. Data represent mean±s.e.m. (c) Western blots showing relative levels of NF-κB subunits in WT and RelA T505A mice. (d) Western blot analysis of phospho-S536 RelA, RelA, IκB-α and β-actin from WT and RelA T505A hepatocytes. Hepatocytes were cultured in 0% FBS media and treated with 50 ng/ml of TNF-α for 10 or 30 min before preparation of cell lysates. Lanes represent independent experimental replicates from a single hepatocyte isolation. These results are representative of data from three separate hepatocyte preparations. In this and subsequent experiments, all mice were designated to an experimental group dependent on their strain. NS, not significant.

Mentions: Among these pathways, phosphorylation of the RelA(p65) Thr505 residue (Figure 1a) provides a mechanism of crosstalk between NF-κB signalling and DNA replication stress. Our group had previously observed that phosphorylation of RelA at Thr505 by Chk1 correlated with a pro-apoptotic form of NF-κB in response to cisplatin treatment and following induction of the ARF tumour suppressor.11, 12, 15, 16 Additionally, we found that RelA Thr505 phosphorylation negatively regulates diverse cellular functions, such as proliferation, migration and autophagy.12 These data suggested that Thr505 mutation would remove the functions of RelA associated with tumour suppressor characteristics, resulting in a form of RelA with enhanced tumour-promoting activities, including the abilities to promote cell survival and proliferation. However, in common with many other NF-κB subunit PTMs, in vivo evidence for such an important functional role has been lacking. Therefore, to address this, we have generated a knockin mouse mutant where the RelA Thr505 residue has been changed to alanine. We hypothesised that selectively removing a specific mechanism of regulating NF-κB would provide important functional insights not seen with whole-gene deletion, where due to the pleiotropic functions of this pathway subtler effects are masked. In this report, we have investigated the effect of this mutation on the response to liver injury and chemically induced hepatocellular carcinoma, both processes requiring NF-κB activity.17, 18, 19, 20, 21 We find that mutation of RelA Thr505 leads to an aberrant proliferative response after liver injury and earlier onset of hepatocellular carcinoma, revealing an important mechanism normally acting to suppress the tumour-promoting functions of NF-κB.


A RelA(p65) Thr505 phospho-site mutation reveals an important mechanism regulating NF- κ B-dependent liver regeneration and cancer
The phenotype of RelA T505A mice. (a) Schematic diagram of RelA showing the location of the Thr505 residue in transactivation domain 2. (b) Liver body weight ratio of wild type (WT), WT/T505A and T505A 12-week-old littermate mice. WT, n=4 males, 8 females; WT/T505A, n=2 males, 5 females; T505A, n=4 males, 5 females. Data represent mean±s.e.m. (c) Western blots showing relative levels of NF-κB subunits in WT and RelA T505A mice. (d) Western blot analysis of phospho-S536 RelA, RelA, IκB-α and β-actin from WT and RelA T505A hepatocytes. Hepatocytes were cultured in 0% FBS media and treated with 50 ng/ml of TNF-α for 10 or 30 min before preparation of cell lysates. Lanes represent independent experimental replicates from a single hepatocyte isolation. These results are representative of data from three separate hepatocyte preparations. In this and subsequent experiments, all mice were designated to an experimental group dependent on their strain. NS, not significant.
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fig1: The phenotype of RelA T505A mice. (a) Schematic diagram of RelA showing the location of the Thr505 residue in transactivation domain 2. (b) Liver body weight ratio of wild type (WT), WT/T505A and T505A 12-week-old littermate mice. WT, n=4 males, 8 females; WT/T505A, n=2 males, 5 females; T505A, n=4 males, 5 females. Data represent mean±s.e.m. (c) Western blots showing relative levels of NF-κB subunits in WT and RelA T505A mice. (d) Western blot analysis of phospho-S536 RelA, RelA, IκB-α and β-actin from WT and RelA T505A hepatocytes. Hepatocytes were cultured in 0% FBS media and treated with 50 ng/ml of TNF-α for 10 or 30 min before preparation of cell lysates. Lanes represent independent experimental replicates from a single hepatocyte isolation. These results are representative of data from three separate hepatocyte preparations. In this and subsequent experiments, all mice were designated to an experimental group dependent on their strain. NS, not significant.
Mentions: Among these pathways, phosphorylation of the RelA(p65) Thr505 residue (Figure 1a) provides a mechanism of crosstalk between NF-κB signalling and DNA replication stress. Our group had previously observed that phosphorylation of RelA at Thr505 by Chk1 correlated with a pro-apoptotic form of NF-κB in response to cisplatin treatment and following induction of the ARF tumour suppressor.11, 12, 15, 16 Additionally, we found that RelA Thr505 phosphorylation negatively regulates diverse cellular functions, such as proliferation, migration and autophagy.12 These data suggested that Thr505 mutation would remove the functions of RelA associated with tumour suppressor characteristics, resulting in a form of RelA with enhanced tumour-promoting activities, including the abilities to promote cell survival and proliferation. However, in common with many other NF-κB subunit PTMs, in vivo evidence for such an important functional role has been lacking. Therefore, to address this, we have generated a knockin mouse mutant where the RelA Thr505 residue has been changed to alanine. We hypothesised that selectively removing a specific mechanism of regulating NF-κB would provide important functional insights not seen with whole-gene deletion, where due to the pleiotropic functions of this pathway subtler effects are masked. In this report, we have investigated the effect of this mutation on the response to liver injury and chemically induced hepatocellular carcinoma, both processes requiring NF-κB activity.17, 18, 19, 20, 21 We find that mutation of RelA Thr505 leads to an aberrant proliferative response after liver injury and earlier onset of hepatocellular carcinoma, revealing an important mechanism normally acting to suppress the tumour-promoting functions of NF-κB.

View Article: PubMed Central - PubMed

ABSTRACT

Post-translational modifications of nuclear factor (NF)-κB subunits provide a mechanism to differentially regulate their activity in response to the many stimuli that induce this pathway. However, the physiological significance of these modifications is largely unknown, and it remains unclear if these have a critical role in the normal and pathological functions of NF-κB in vivo. Among these, phosphorylation of the RelA(p65) Thr505 residue has been described as an important regulator of NF-κB activity in cell lines, but its physiological significance was not known. Therefore, to learn more about the role of this pathway in vivo, we generated a knockin mouse with a RelA T505A mutation. Unlike RelA knockout mice, the RelA T505A mice develop normally but exhibit aberrant hepatocyte proliferation following liver partial hepatectomy or damage resulting from carbon tetrachloride (CCl4) treatment. Consistent with these effects, RelA T505A mice exhibit earlier onset of cancer in the N-nitrosodiethylamine model of hepatocellular carcinoma. These data reveal a critical pathway controlling NF-κB function in the liver that acts to suppress the tumour-promoting activities of RelA.

No MeSH data available.


Related in: MedlinePlus