Role of Methionine Adenosyltransferase α2 and β Phosphorylation and Stabilization in Human Hepatic Stellate Cell Trans‐Differentiation

The American Journal of Pathology

Ryoo

2016

Self Cite

Lipopolysaccharide (LPS), a bacterial endotoxin, induces inflammation in macrophages via activation of NF-kB signaling. Sumoylation is a post-translational modification mediated by the small ubiquitin-like modifier, SUMO. Ubiquitin-conjugating enzyme 9 (UBC9) is the only known SUMO conjugating enzyme. LPS treatment lowers SUMO-1 and UBC9 mRNA levels in primary astrocytes. UBC9 can degrade NF-kB inhibitor a (Ikba) via a SUMO2/3-ubiquitin pathway. However, UBC9 may also promote Ikba stability by SUMO-1 conjugation that further regulates NF-kB signaling. The role of UBC9 in liver inflammation is unknown. We reported that CDK1-mediated phosphorylation of UBC9 enhanced its stability. Herein, we describe an anti-inflammatory role of UBC9 that is lost when it is phosphorylated during inflammation. LPS exposure caused induction in UBC9 phosphorylation and CDK1 activation specifically in Kupffer cells in vivo and in RAW264.7 macrophages in vitro. Silencing or overexpression experiments in vitro and in vivo showed that UBC9 was required to blunt the proinflammatory response elicited by LPS. LPS stimulation raised the binding of phospho-UBC9 but not the unphosphorylated counterpart, to Ikba in RAW264.7 macrophages. Hence, phospho-UBC9 may promote NF-kB signaling by regulating Ikba and this may be a novel mechanism that deregulates liver inflammatory signaling. (Am J Pathol 2016 http://dx

Section: Phos-tag Analysismentioning

confidence: 99%

“…Equal amounts of phosphorylated or unphosphorylated protein fractions were immunoprecipitated with two micrograms of UBC9 antibody, according to previously published protocols, 13,17 and then immunoblotted with Ikba antibody.…”

Section: Phospho-affinity Purification Chromatography and Coimmunoprementioning

confidence: 99%

Ubiquitin-Conjugating Enzyme 9 Phosphorylation as a Novel Mechanism for Potentiation of the Inflammatory Response

Tomasi

The American Journal of Pathology

Ryoo

2016

Self Cite

“…40 The phosphorylation of both MATα2 and MATβ is strongly induced during HSC activation. 41 Phospho-MATα2 and MATβ proteins are highly stable and the mutation of specific phosphorylation sites (Y371/Y374 in MATα2 and T257/Y259 in MATβ) inhibits HSC activation. 41 Chemical inhibitors, gene silencing and in vitro kinase assays have shown that mitogen-activated protein kinase/ERK kinase (MEK) might be involved in MATα2 phosphorylation whereas extracellular signal-regulated kinase (ERK) might phosphorylate the MATβ protein.…”

Section: Methionine Adenosyltransferasesmentioning

confidence: 99%

“…41 Chemical inhibitors, gene silencing and in vitro kinase assays have shown that mitogen-activated protein kinase/ERK kinase (MEK) might be involved in MATα2 phosphorylation whereas extracellular signal-regulated kinase (ERK) might phosphorylate the MATβ protein. 41 …”

Section: Methionine Adenosyltransferasesmentioning

confidence: 99%

Methionine adenosyltransferases in liver health and diseases

2017

Liver Research

Self Cite

Methionine adenosyltransferases (MATs) are essential for cell survival because they catalyze the biosynthesis of the biological methyl donor S-adenosylmethionine (SAMe) from methionine and adenosine triphosphate (ATP). Mammalian cells express two genes, MAT1A and MAT2A, which encode two MAT catalytic subunits, α1 and α2, respectively. The α1 subunit organizes into dimers (MATIII) or tetramers (MATI). The α2 subunit is found in the MATII isoform. A third gene MAT2B, encodes a regulatory subunit β, that regulates the activity of MATII by lowering the inhibition constant (Ki) for SAMe and the Michaelis constant (Km) for methionine. MAT1A expressed mainly in hepatocytes maintains the differentiated state of these cells whereas MAT2A and MAT2B are expressed in non-parenchymal cells of the liver (hepatic stellate cells [HSCs] and Kupffer cells) and extrahepatic tissues. A switch from the liver-specific MAT1A to MAT2A has been observed during conditions of active liver growth and de-differentiation. Liver injury, fibrosis, and cancer are associated with MAT1A silencing and MAT2A/MAT2B induction. Even though both MAT1A and MAT2A are involved in SAMe biosynthesis, they exhibit distinct molecular interactions in liver cells. This review provides an update on MAT genes and their roles in liver pathologies.

“…Antibodies used for Western blotting were: PHB1 and IGF2 (Abcam, Cambridge, MA), CTCF (Cell Signaling, Danvers, MA), and actin control (Sigma). For co-immunoprecipitation, 500 g of radioimmunoprecipitation assay extracts of cells or tissues were immunoprecipitated with 2 g of PHB1 antibody and immunoblotted with CTCF antibody according to previously established protocols (26). Western blots were developed using the chemiluminescence ECL system (Amersham Biosciences).…”

Section: ϫ⌬⌬Ctmentioning

confidence: 99%

Prohibitin 1 Regulates the H19-Igf2 Axis and Proliferation in Hepatocytes

Journal of Biological Chemistry

Mavila

et al. 2016

Self Cite

Edited by Xiao-Fan WangProhibitin 1 (PHB1) is a mitochondrial chaperone that regulates cell growth. Phb1 knock-out mice exhibit liver injury and hepatocellular carcinoma (HCC). Phb1 knock-out livers show induction of tumor growth-associated genes, H19 and insulinlike growth factor 2 (Igf2). These genes are controlled by the imprinting control region (ICR) containing CCCTC-binding transcription factor (CTCF)-binding sites. Because Phb1 knockout mice exhibited induction of H19 and Igf2, we hypothesized that PHB1-mediated regulation of the H19-Igf2 axis might control cell proliferation in normal hepatocytes. H19 and Igf2 were induced (8 -20-fold) in 3-week-old Phb1 knock-out livers, in Phb1 siRNA-treated AML12 hepatocytes (2-fold), and HCC cell lines when compared with control. Phb1 knockdown lowered CTCF protein in AML12 by ϳ30% when compared with control. CTCF overexpression lowered basal H19 and Igf2 expression by 30% and suppressed Phb1 knockdown-mediated induction of these genes. CTCF and PHB1 co-immunoprecipitated and colocalized on the ICR element, and Phb1 knockdown lowered CTCF ICR binding activity. The results suggest that PHB1 and CTCF cooperation may control the H19-Igf2 axis. Human HCC tissues with high levels of H19 and IGF2 exhibited a 40 -50% reduction in PHB1 and CTCF expression and their ICR binding activity. Silencing Phb1 or overexpressing H19 in the mouse HCC cell line, SAMe-D, induced cell growth. Blocking H19 induction prevented Phb1 knockdown-mediated growth, whereas H19 overexpression had the reverse effect. Interestingly H19 silencing induced PHB1 expression. Taken together, our results demonstrate that the H19-Igf2 axis is negatively regulated by CTCF-PHB1 cooperation and that H19 is involved in modulating the growthsuppressive effect of PHB1 in the liver.