Nuria Martinez-Lopez

Autophagy proteins regulate ERK phosphorylation

Autophagy is a conserved pathway that maintains cellular quality control. Extracellular signal-regulated kinase (ERK) controls various aspects of cell physiology including proliferation. Multiple signalling cascades, including ERK, have been shown to regulate autophagy, however whether autophagy proteins (ATG) regulate cell signalling is unknown. Here we show that growth factor exposure increases the interaction of ERK cascade components with ATG proteins in the cytosol and nucleus. ERK and its upstream kinase MEK localize to the extra-luminal face of autophagosomes. ERK2 interacts with ATG proteins via its substrate-binding domains. Deleting Atg7 or Atg5 or blocking LC3 lipidation or ATG5–ATG12 conjugation decreases ERK phosphorylation. Conversely, increasing LC3-II availability by silencing the cysteine protease ATG4B or acute trehalose exposure increases ERK phosphorylation. Decreased ERK phosphorylation in Atg5−/− cells does not occur from overactive phosphatases. Our findings thus reveal an unconventional function of ATG proteins as cellular scaffolds in the regulation of ERK phosphorylation.

Autophagy, lipophagy and lysosomal lipid storage disorders.

Autophagy is a catabolic process with an essential function in the maintenance of cellular and tissue homeostasis. It is primarily recognised for its role in the degradation of dysfunctional proteins and unwanted organelles, however in recent years the range of autophagy substrates has also been extended to lipids. Degradation of lipids via autophagy is termed lipophagy. The ability of autophagy to contribute to the maintenance of lipo-homeostasis becomes particularly relevant in the context of genetic lysosomal storage disorders where perturbations of autophagic flux have been suggested to contribute to the disease aetiology. Here we review recent discoveries of the molecular mechanisms mediating lipid turnover by the autophagy pathways. We further focus on the relevance of autophagy, and specifically lipophagy, to the disease mechanisms. Moreover, autophagy is also discussed as a potential therapeutic target in several key lysosomal storage disorders.

Murine double minute 2 regulates Hu antigen R stability in human liver and colon cancer through NEDDylation

Hu antigen R (HuR) is a central RNA‐binding protein regulating cell dedifferentiation, proliferation, and survival, which are well‐established hallmarks of cancer. HuR is frequently overexpressed in tumors correlating with tumor malignancy, which is in line with a role for HuR in tumorigenesis. However, the precise mechanism leading to changes in HuR expression remains unclear. In the liver, HuR plays a crucial role in hepatocyte proliferation, differentiation, and transformation. Here, we unraveled a novel mean of regulation of HuR expression in hepatocellular carcinoma (HCC) and colon cancer. HuR levels correlate with the abundance of the oncogene, murine double minute 2 (Mdm2), in human HCC and colon cancer metastases. HuR is stabilized by Mdm2‐mediated NEDDylation in at least three lysine residues, ensuring its nuclear localization and protection from degradation. Conclusion: This novel Mdm2/NEDD8/HuR regulatory framework is essential for the malignant transformation of tumor cells, which, in turn, unveils a novel signaling paradigm that is pharmacologically amenable for cancer therapy. (Hepatology 2012)

Evidence for LKB1/AMP-activated protein kinase/ endothelial nitric oxide synthase cascade regulated by hepatocyte growth factor, S-adenosylmethionine, and nitric oxide in hepatocyte proliferation.

S‐adenosylmethionine (SAMe) is involved in numerous complex hepatic processes such as hepatocyte proliferation, death, inflammatory responses, and antioxidant defense. One of the most relevant actions of SAMe is the inhibition of hepatocyte proliferation during liver regeneration. In hepatocytes, SAMe regulates the levels of cytoplasmic HuR, an RNA‐binding protein that increases the half‐life of target messenger RNAs such as cyclin D1 and A2 via inhibition of hepatocyte growth factor (HGF)‐mediated adenosine monophosphate–activated protein kinase (AMPK) phosphorylation. Because AMPK is activated by the tumor suppressor kinase LKB1, and AMPK activates endothelial nitric oxide (NO) synthase (eNOS), and NO synthesis is of great importance for hepatocyte proliferation, we hypothesized that in hepatocytes HGF may induce the phosphorylation of LKB1, AMPK, and eNOS through a process regulated by SAMe, and that this cascade might be crucial for hepatocyte growth. We demonstrate that the proliferative response of hepatocytes involves eNOS phosphorylation via HGF‐mediated LKB1 and AMPK phosphorylation, and that this process is regulated by SAMe and NO. We also show that knockdown of LKB1, AMPK, or eNOS with specific interference RNA (iRNA) inhibits HGF‐mediated hepatocyte proliferation. Finally, we found that the LKB1/AMPK/eNOS cascade is activated during liver regeneration after partial hepatectomy and that this process is impaired in mice treated with SAMe before hepatectomy, in knockout mice deficient in hepatic SAMe, and in eNOS knockout mice. Conclusion: We have identified an LKB1/AMPK/eNOS cascade regulated by HGF, SAMe, and NO that functions as a critical determinant of hepatocyte proliferation during liver regeneration after partial hepatectomy. (HEPATOLOGY 2009;49:608–617.)

Autophagy and Lipid Droplets in the Liver.

Autophagy is a conserved quality-control pathway that degrades cytoplasmic contents in lysosomes. Autophagy degrades lipid droplets through a process termed lipophagy. Starvation and an acute lipid stimulus increase autophagic sequestration of lipid droplets and their degradation in lysosomes. Accordingly, liver-specific deletion of the autophagy gene Atg7 increases hepatic fat content, mimicking the human condition termed nonalcoholic fatty liver disease. In this review, we provide insights into the molecular regulation of lipophagy, discuss fundamental questions related to the mechanisms by which autophagosomes recognize lipid droplets and how ATG proteins regulate membrane curvature for lipid droplet sequestration, and comment on the possibility of cross talk between lipophagy and cytosolic lipases in lipid mobilization. Finally, we discuss the contribution of lipophagy to the pathophysiology of human fatty liver disease. Understanding how lipophagy clears hepatocellular lipid droplets could provide new ways to prevent fatty liver disease, a major epidemic in developed nations.

Autophagy and Aging

Autophagy is a critical quality control pathway that is conserved across diverse systems ranging from simple unicellular organisms like yeast to more complex systems, for instance mammals. Although, the fundamental role of autophagy is to maintain cellular quality control through lysosomal degradation of unwanted proteins and organelles, recent studies have mapped several new functions of this pathway that range from fuel utilization, cellular differentiation to protection against cell death. Given the importance of this pathway in maintaining cellular homeostasis, it has been considered that compromised autophagy could contribute to several of the commonly observed age-associated pathologies including neurodegeneration, reduction of muscle mass, cardiac malfunction, excessive lipid accumulation in tissues and glucose intolerance. The present chapter describes the two best-characterized autophagy pathways—macroautophagy and chaperone-mediated autophagy, and discusses how changes in these pathways associate with age-associated disorders. Understanding how to maintain “clean cells” by activation of autophagy could be an attractive strategy to maintain healthspan in aged individuals.

Fatty liver and fibrosis in glycine N-methyltransferase knockout mice is prevented by nicotinamide

Deletion of glycine N‐methyltransferase (GNMT), the main gene involved in liver S‐adenosylmethionine (SAM) catabolism, leads to the hepatic accumulation of this molecule and the development of fatty liver and fibrosis in mice. To demonstrate that the excess of hepatic SAM is the main agent contributing to liver disease in GNMT knockout (KO) mice, we treated 1.5‐month‐old GNMT‐KO mice for 6 weeks with nicotinamide (NAM), a substrate of the enzyme NAM N‐methyltransferase. NAM administration markedly reduced hepatic SAM content, prevented DNA hypermethylation, and normalized the expression of critical genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis. More importantly, NAM treatment prevented the development of fatty liver and fibrosis in GNMT‐KO mice. Because GNMT expression is down‐regulated in patients with cirrhosis, and because some subjects with GNMT mutations have spontaneous liver disease, the clinical implications of the present findings are obvious, at least with respect to these latter individuals. Because NAM has been used for many years to treat a broad spectrum of diseases (including pellagra and diabetes) without significant side effects, it should be considered in subjects with GNMT mutations. Conclusion: The findings of this study indicate that the anomalous accumulation of SAM in GNMT‐KO mice can be corrected by NAM treatment leading to the normalization of the expression of many genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis, as well as reversion of the appearance of the pathologic phenotype. (HEPATOLOGY 2010)

S-adenosylmethionine and proliferation: new pathways, new targets.

SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP, and SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle. SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death. Recent results show a new signalling pathway implicated in the proliferation of the hepatocyte, where AMPK (AMP-activated protein kinase) and HuR, modulated by SAMe, take place in HGF (hepatocyte growth factor)-mediated cell growth. Abnormalities in methionine metabolism occur in several animal models of alcoholic liver injury, and it is also altered in patients with liver disease. Both high and low levels of SAMe predispose to liver injury. In this regard, knockout mouse models have been developed for the enzymes responsible for SAMe synthesis and catabolism, MAT1A and GNMT (glycine N-methyltransferase) respectively. These knockout mice develop steatosis and HCC (hepatocellular carcinoma), and both models closely replicate the pathologies of human disease, which makes them extremely useful to elucidate the mechanism underlying liver disease. These new findings open a wide range of possibilities to discover novel targets for clinical applications.

Activation of LKB1‐Akt pathway independent of phosphoinositide 3‐kinase plays a critical role in the proliferation of hepatocellular carcinoma from nonalcoholic steatohepatitis

LKB1, originally considered a tumor suppressor, plays an important role in hepatocyte proliferation and liver regeneration. Mice lacking the methionine adenosyltransferase (MAT) gene MAT1A exhibit a chronic reduction in hepatic S‐adenosylmethionine (SAMe) levels, basal activation of LKB1, and spontaneous development of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). These results are relevant for human health because patients with liver cirrhosis, who are at risk to develop HCC, have a marked reduction in hepatic MAT1A expression and SAMe synthesis. In this study, we isolated a cell line (SAMe‐deficient [SAMe‐D]) from MAT1A knockout (MAT1A‐KO) mouse HCC to examine the role of LKB1 in the development of liver tumors derived from metabolic disorders. We found that LKB1 is required for cell survival in SAMe‐D cells. LKB1 regulates Akt‐mediated survival independent of phosphoinositide 3‐kinase, adenosine monophosphate protein–activated kinase (AMPK), and mammalian target of rapamycin complex (mTORC2). In addition, LKB1 controls the apoptotic response through phosphorylation and retention of p53 in the cytoplasm and the regulation of herpesvirus‐associated ubiquitin‐specific protease (HAUSP) and Hu antigen R (HuR) nucleocytoplasmic shuttling. We identified HAUSP as a target of HuR. Finally, we observed cytoplasmic staining of p53 and p‐LKB1(Ser428) in a NASH‐HCC animal model (from MAT1A‐KO mice) and in liver biopsies obtained from human HCC derived from both alcoholic steatohepatitis and NASH. Conclusion: The SAMe‐D cell line is a relevant model of HCC derived from NASH disease in which LKB1 is the principal conductor of a new regulatory mechanism and could be a practical tool for uncovering new therapeutic strategies. (HEPATOLOGY 2010)

Autophagy in the CNS and Periphery Coordinate Lipophagy and Lipolysis in the Brown Adipose Tissue and Liver.

The integrative physiology of inter-organ communication in lipophagy regulation is not well understood. Lipophagy and the cytosolic lipases ATGL and HSL contribute to lipid droplet (LD) mobilization; however, whether autophagy proteins engage with lipases to promote lipid utilization remains unknown. Here, we show that cold induces autophagy in proopiomelanocortin (POMC) neurons and activates lipophagy in brown adipose tissue (BAT) and liver in mice. Targeted activation of autophagy in POMC neurons via intra-hypothalamic rapamycin is sufficient to trigger lipid utilization in room temperature-housed mice. Conversely, inhibiting autophagy in POMC neurons or in peripheral tissues or denervating BAT blocks lipid utilization. Unexpectedly, the autophagosome marker LC3 is mechanistically coupled to ATGL-mediated lipolysis. ATGL exhibits LC3-interacting region (LIR) motifs, and mutating a single LIR motif on ATGL displaces ATGL from LD and disrupts lipolysis. Thus, cold-induced activation of central autophagy activates lipophagy and cytosolic lipases in a complementary manner to mediate lipolysis in peripheral tissues.