SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.

Alcohol acts through numerous pathways leading to alcoholic liver disease (ALD). Cytochrome P450 (CYP2E1), an ethanol-inducible enzyme, metabolizes ethanol-producing toxic reactive oxygen species (ROS) and is regulated at the posttranslational level. Small ubiquitin-like modifier (SUMO)ylation is a posttranslational modification that involves the addition of SUMOs, which modulate protein stability, activity, and localization. We demonstrated that ubiquitin-conjugation enzyme 9, the SUMO-conjugating enzyme, is induced in the livers of an intragastric ethanol mouse model. Our aim is to examine whether SUMOylation could regulate ethanol-induced CYP2E1 expression in ALD and to elucidate the molecular mechanism(s). CYP2E1 and UBC9 expression in vitro and in vivo was detected by real-time PCR and immunoblotting/immunostaining. SUMOylation was assayed by mass spectrometry and coimmunoprecipitation. Ubc9 expression was induced in ethanol-fed mouse livers, and silencing inhibited ethanol-mediated CYP2E1 microsomal retention and enzymatic activity. CYP2E1 SUMOylation was found to be induced by ethanol in vitro and in vivo. Ubc9 silencing prevents ethanol-induced lipid accumulation and ROS production. UBC9 was highly expressed in human ALD livers. Finally, we found that lysine 410 is a key SUMOylated residue contributing to CYP2E1 protein stability and activity preventing CYP2E1 SUMOylation. Ethanol-mediated up-regulation of CYP2E1 via SUMOylation enhancing its protein stability and activity and may have important implications in ALD.-Tomasi, M. L., Ramani, K., Ryoo, M., Cossu, C., Floris, A., Murray, B. J., Iglesias-Ara, A., Spissu, Y., Mavila, N. SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.

S-adenosylmethionine and methylthioadenosine inhibit cancer metastasis by targeting microRNA 34a/b-methionine adenosyltransferase 2A/2B axis.

MicroRNA-34a (miR-34a) is down-regulated in colorectal cancers (CRC) and required for interleukin-6 (IL-6)-induced CRC metastasis. Mice lacking miR-34a developed more invasive cancer in a colitis-associated cancer model. In the same model, S-adenosylmethionine (SAMe) and methylthioadenosine (MTA) inhibited IL-6/STAT3 and lowered tumor burden. SAMe and MTA reduce the expression of methionine adenosyltransferase 2A (MAT2A) and there are consensus binding sites for miR-34a/b in the MAT2A 3'UTR. Here we examined whether SAMe/MTA influence miR-34a/b expression and cancer metastasis. We found SAMe and MTA raised miR-34a/b expression in CRC cell lines, inhibited migration and invasion in vitro and liver metastasis in vivo. Like CRC, MAT2A and MAT2B expression is induced in human pancreas and prostate cancers. Treatment with SAMe, MTA, miR-34a or miR-34b inhibited MAT2A expression mainly at the protein level. MAT2B protein level also fell because MAT2A and MAT2B enhance each other's protein stability. Overexpressing miR-34a or miR-34b inhibited while MAT2A or MAT2B enhanced CRC migration and invasion. Co-expressing either miR-34a/b had minimal to no effect on MAT2A/MAT2B's ability to increase migration, invasion and growth. Taken together, MAT2A and MAT2B are important targets of miR-34a/b and SAMe and MTA target this axis, suppressing MAT2A/MAT2B while raising miR-34a/b expression, inhibiting cancer metastasis.

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

Lipopolysaccharide (LPS), a bacterial endotoxin, induces inflammation in macrophages via activation of NF-κB 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-κB inhibitor α (Ikbα) via a SUMO2/3-ubiquitin pathway. However, UBC9 may also promote Ikbα stability by SUMO-1 conjugation that further regulates NF-κB 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 Ikbα in RAW264.7 macrophages. Hence, phospho-UBC9 may promote NF-κB signaling by regulating Ikbα and this may be a novel mechanism that deregulates liver inflammatory signaling.

Methionine adenosyltransferase α2 sumoylation positively regulate Bcl-2 expression in human colon and liver cancer cells.

Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and inhibits apoptosis via Bcl-2 by unknown mechanism. Methionine adenosyltransferase 2A (MAT2A) encodes for MATα2, the catalytic subunit of the MATII isoenzyme that synthesizes S-adenosylmethionine (SAMe). Ubc9, Bcl-2 and MAT2A expression are up-regulated in several malignancies. Exogenous SAMe decreases Ubc9 and MAT2A expression and is pro-apoptotic in liver and colon cancer cells. Here we investigated whether there is interplay between Ubc9, MAT2A and Bcl-2. We used human colon and liver cancer cell lines RKO and HepG2, respectively, and confirmed key finding in colon cancer specimens. We found MATα2 can regulate Bcl-2 expression at multiple levels. MATα2 binds to Bcl-2 promoter to activate its transcription. This effect is independent of SAMe as MATα2 catalytic mutant was also effective. MATα2 also directly interacts with Bcl-2 to enhance its protein stability. MATα2's effect on Bcl-2 requires Ubc9 as MATα2's stability is influenced by sumoylation at K340, K372 and K394. Overexpressing wild type (but not less stable MATα2 sumoylation mutants) protected from 5-fluorouracil-induced apoptosis in both colon and liver cancer cells. Colon cancer have higher levels of sumoylated MATα2, total MATα2, Ubc9 and Bcl-2 and higher MATα2 binding to the Bcl-2 P2 promoter. Taken together, Ubc9's protective effect on apoptosis may be mediated at least in part by sumoylating and stabilizing MATα2 protein, which in turn positively maintains Bcl-2 expression. These interactions feed forward to further enhance growth and survival of the cancer cell.

Molecular mechanisms of lipopolysaccharide-mediated inhibition of glutathione synthesis in mice.

Endotoxemia correlates with the degree of liver failure and may participate in worsening of liver diseases. Lipopolysaccharide (LPS; synonymous with endotoxin) treatment in mice lowered the hepatic glutathione (GSH) level, which in turn is a variable that determines susceptibility to LPS-induced injury. We previously showed that LPS treatment in mice lowered hepatic expression of the rate-limiting enzyme in GSH synthesis, glutamate-cysteine ligase (GCL). The aim of our current work was to determine the molecular mechanism(s) responsible for these changes. Studies were done using RAW cells (murine macrophages), in vivo LPS-treated mice, and mouse hepatocytes. We found that LPS treatment lowered GCL catalytic and modifier (Gclc and Gclm) subunit expression at the transcriptional level, which was unrelated to alterations in nitric oxide production or induction of NF-κB/p65 subunit. The key mechanism was a decrease in sumoylation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and MafG, which is required for their heterodimerization and subsequent binding and trans-activation of the antioxidant-response element (ARE) present in the promoter region of these genes that is essential for their expression. LPS treatment lowered markedly the expression of ubiquitin-conjugating enzyme 9 (Ubc9), which is required for sumoylation. Similar findings also occurred in liver after in vivo LPS treatment and in LPS-treated mouse hepatocytes. Overexpression of Ubc9 protected against LPS-mediated inhibition of Gclc and Gclm expression in RAW cells and hepatocytes. In conclusion, LPS-mediated lowering of GCL expression in hepatocytes and macrophages is due to lowering of sumoylation of Nrf2 and MafG, leading to reduced heterodimerization, binding, and trans-activation of ARE.

Polyamine and methionine adenosyltransferase 2A crosstalk in human colon and liver cancer.

Methionine adenosyltransferase (MAT) is an essential enzyme that is responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. MAT1A is expressed in normal liver and MAT2A is expressed in all extrahepatic tissues. MAT2A expression is increased in human colon cancer and in colon cancer cells treated with mitogens, whereas silencing MAT2A resulted in apoptosis. The aim of the current work was to examine the mechanism responsible for MAT2A-dependent growth and apoptosis. We found that in RKO (human adenocarcinoma cell line) cells, MAT2A siRNA treatment lowered cellular SAMe and putrescine levels by 70-75%, increased apoptosis and inhibited growth. Putrescine supplementation blunted significantly MAT2A siRNA-induced apoptosis and growth suppression. Putrescine treatment (100pmol/L) raised MAT2A mRNA level to 4.3-fold of control, increased the expression of c-Jun and c-Fos and binding to an AP-1 site in the human MAT2A promoter and the promoter activity. In human colon cancer specimens, the expression levels of MAT2A, ornithine decarboxylase (ODC), c-Jun and c-Fos are all elevated as compared to adjacent non-tumorous tissues. Overexpression of ODC in RKO cells also raised MAT2A mRNA level and MAT2A promoter activity. ODC and MAT2A are also overexpressed in liver cancer and consistently, similar MAT2A-ODC-putrescine interactions and effects on growth and apoptosis were observed in HepG2 cells. In conclusion, there is a crosstalk between polyamines and MAT2A. Increased MAT2A expression provides more SAMe for polyamines biosynthesis; increased polyamine (putrescine in this case) can activate MAT2A at the transcriptional level. This along with increased ODC expression in cancer all feed forward to further enhance the proliferative capacity of the cancer cell.