Rachel J. Roth Flach

The Conserved Misshapen-Warts-Yorkie Pathway Acts in Enteroblasts to Regulate Intestinal Stem Cells in Drosophila

Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. Here, we show that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP.

IL-1 signaling in obesity-induced hepatic lipogenesis and steatosis.

Non-alcoholic fatty liver disease is prevalent in human obesity and type 2 diabetes, and is characterized by increases in both hepatic triglyceride accumulation (denoted as steatosis) and expression of pro-inflammatory cytokines such as IL-1β. We report here that the development of hepatic steatosis requires IL-1 signaling, which upregulates Fatty acid synthase to promote hepatic lipogenesis. Using clodronate liposomes to selectively deplete liver Kupffer cells in ob/ob mice, we observed remarkable amelioration of obesity-induced hepatic steatosis and reductions in liver weight, triglyceride content and lipogenic enzyme expressions. Similar results were obtained with diet-induced obese mice, although visceral adipose tissue macrophage depletion also occurred in response to clodronate liposomes in this model. There were no differences in the food intake, whole body metabolic parameters, serum β-hydroxybutyrate levels or lipid profiles due to clodronate-treatment, but hepatic cytokine gene expressions including IL-1β were decreased. Conversely, treatment of primary mouse hepatocytes with IL-1β significantly increased triglyceride accumulation and Fatty acid synthase expression. Furthermore, the administration of IL-1 receptor antagonist to obese mice markedly reduced obesity-induced steatosis and hepatic lipogenic gene expression. Collectively, our findings suggest that IL-1β signaling upregulates hepatic lipogenesis in obesity, and is essential for the induction of pathogenic hepatic steatosis in obese mice.

MAP kinase phosphatase-1 deficiency impairs skeletal muscle regeneration and exacerbates muscular dystrophy

In skeletal muscle, the mitogen‐activated protein kinase (MAPK) phosphatase‐1 (MKP‐1) is a critical negative regulator of the MAPKs. Since the MAPKs have been reported to be both positive and negative for myogenesis, the physiological role of MKP‐1 in skeletal muscle repair and regeneration has remained unclear. Here, we show that MKP‐1 plays an essential role in adult regenerative myogenesis. In a cardiotoxin‐induced muscle injury model, lack of MKP‐1 impaired muscle regeneration. In mdx mice, MKP‐1 deficiency reduced body weight, muscle mass, and muscle fiber cross‐sectional area. In addition, MKP‐1‐deficient muscles exhibit exacerbated myopathy accompanied by increased inflammation. Lack of MKP‐1 compromised myoblast proliferation and induced precocious differentiation, phenotypes that were rescued by pharmacological inhibition of p38α/β MAPK. MKP‐1 coordinates both myoblast proliferation and differentiation. Mechanistically, MyoD bound to the MKP‐1 promoter and activated MKP‐1 expression in proliferating myoblasts. Later, during myogenesis, MyoD uncoupled from the MKP‐1 promoter leading to the down‐regulation of MKP‐1 and facilitation of promyogenic p38α/β MAPK signaling. Hence, MKP‐1 plays a critical role in muscle stem cells and in the immune response to coordinate muscle repair and regeneration.—Shi, H., Boadu, E., Mercan, F., Le, A. M., Roth Flach, R. J., Zhang, L., Tyner, K. J., Olwin, B. B., Bennett, A. M. MAP kinase phosphatase‐1 deficiency impairs skeletal muscle regeneration and exacerbates muscular dystrophy. FASEB J. 24, 2985–2997 (2010). www.fasebj.org

β3-Adrenergic Receptor Stimulation Induces E-Selectin-mediated Adipose Tissue Inflammation

Background: Studies suggest that lipolysis induces adipose tissue inflammation, commonly associated with type 2 diabetes. Results: Activation of adipose-resident endothelium is required for β3-adrenergic receptor-mediated but not fasting-induced inflammation. Conclusion: Both β3-adrenergic receptor stimulation and fasting induce adipose tissue inflammation, but by different mechanisms. Significance: The study shows heterogeneity of immune cell dynamics in adipose tissue. Inflammation induced by wound healing or infection activates local vascular endothelial cells to mediate leukocyte rolling, adhesion, and extravasation by up-regulation of leukocyte adhesion molecules such as E-selectin and P-selectin. Obesity-associated adipose tissue inflammation has been suggested to cause insulin resistance, but weight loss and lipolysis also promote adipose tissue immune responses. While leukocyte-endothelial interactions are required for obesity-induced inflammation of adipose tissue, it is not known whether lipolysis-induced inflammation requires activation of endothelial cells. Here, we show that β3-adrenergic receptor stimulation by CL 316,243 promotes adipose tissue neutrophil infiltration in wild type and P-selectin-null mice but not in E-selectin-null mice. Increased expression of adipose tissue cytokines IL-1β, CCL2, and TNF-α in response to CL 316,243 administration is also dependent upon E-selectin but not P-selectin. In contrast, fasting increases adipose-resident macrophages but not neutrophils, and does not activate adipose-resident endothelium. Thus, two models of lipolysis-induced inflammation induce distinct immune cell populations within adipose tissue and exhibit distinct dependences on endothelial activation. Importantly, our results indicate that β3-adrenergic stimulation acts through up-regulation of E-selectin in adipose tissue endothelial cells to induce neutrophil infiltration.

Loss of Mitogen-activated Protein Kinase Phosphatase-1 Protects from Hepatic Steatosis by Repression of Cell Death-inducing DNA Fragmentation Factor A (DFFA)-like Effector C (CIDEC)/Fat-specific Protein 27

The integration of metabolic signals required for the regulation of hepatic lipid homeostasis is complex. Previously, we showed that mice lacking expression of the mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) have increased fatty acid oxidation and are protected from the development of hepatic steatosis. Here, we show that leptin receptor-deficient (db/db) mice lacking MKP-1 are also resistant to the development of hepatic steatosis. Microarray analyses of livers from db/db mice lacking MKP-1 showed suppression of peroxisome proliferator-activated receptor γ (PPARγ) target genes. We identified the fat-specific protein 27 (Fsp27), which promotes PPARγ-mediated hepatic steatosis, as repressed in livers of both db/db and high fat diet-fed mice lacking MKP-1. Hepatocytes from MKP-1-deficient mice exhibited reduced PPARγ-induced lipid droplet formation. Mechanistically, loss of MKP-1 inhibited PPARγ function by increasing MAPK-dependent phosphorylation on PPARγ at its inhibitory residue of serine 112. These results demonstrate that in addition to inhibiting hepatic fatty acid oxidation, MKP-1 promotes hepatic lipogenic gene expression through PPARγ. Hence, MKP-1 plays an important role in MAPK-mediated control of hepatic lipid homeostasis.

Identification of Map4k4 as a novel suppressor of skeletal muscle differentiation.

ABSTRACT Myoblast differentiation into mature myotubes is a critical step in the development and repair of human skeletal muscle. Here we show that small interfering RNA (siRNA)-based silencing of the Ste20-like mitogen-activated protein 4 kinase 4 (Map4k4) in C2C12 myoblasts markedly enhances expression of myogenic differentiation genes, myoblast fusion, and myotube diameter. In contrast, adenovirus-mediated expression of native Map4k4 in C2C12 cells attenuates each of these processes, indicating that Map4k4 is a negative regulator of myogenic differentiation and hypertrophy. Expression of a Map4k4 kinase-inactive mutant enhances myotube formation, suggesting that the kinase activity of Map4k4 is essential for its inhibition of muscle differentiation. Map4k4 regulation of myogenesis is unlikely to be mediated by classic mitogen-activated protein kinase (MAPK) signaling pathways, because no significant difference in phosphorylation of extracellular signal-regulated kinase (ERK), p38, or c-Jun N-terminal kinase (JNK) is observed in Map4k4-silenced cells. Furthermore, silencing of these other MAPKs does not result in a hypertrophic myotube phenotype like that seen with Map4k4 depletion. Uniquely, Map4k4 silencing upregulates the expression of the myogenic regulatory factor Myf5, whose depletion inhibits myogenesis. Furthermore, Myf5 is required for enhancement of myotube formation in Map4k4-silenced cells, while Myf5 overexpression rescues Map4k4-mediated inhibition of myogenic differentiation. These results demonstrate that Map4k4 is a novel suppressor of skeletal muscle differentiation, acting through a Myf5-dependent mechanism.

Mitogen-activated protein kinase phosphatase-1 - a potential therapeutic target in metabolic disease.

Importance of the field: Metabolic disease, which is associated with obesity and cardiovascular disease, is a worldwide epidemic. There continues to be a tremendous effort towards the development of therapies to curtail obesity and its associated pathophysiological sequelae. MAPKs have been implicated in metabolic disease suggesting that these enzymes, and those that regulate them, can potentially serve as therapeutic targets to combat this disease. The MAPK phosphatase-1 (MKP-1) mediates the dephosphorylation and inactivation of MAPKs in insulin-responsive tissues. Therefore, the actions of MKP-1 may play an important role in the maintenance of metabolic homeostasis. Areas covered in this review: The functional effects of MKP-1 in MAPK regulation with emphasis on its role in physiological and pathophysiological signaling functions that have been elucidated through the use of mouse genetics. What the reader will gain: The reader will learn that MAPK inactivation through the effects of MKP-1 is essential for the maintenance of metabolic homeostasis. We will convey the idea that MKP-1 acts as a critical signaling node in MAPK-mediated regulation of cell signaling and metabolism. Take home message: Pharmacological inactivation of MKP-1 may be of therapeutic value in the treatment of obesity and possibly other metabolic disorders.

Tenomodulin promotes human adipocyte differentiation and beneficial visceral adipose tissue expansion

Proper regulation of energy storage in adipose tissue is crucial for maintaining insulin sensitivity and molecules contributing to this process have not been fully revealed. Here we show that type II transmembrane protein tenomodulin (TNMD) is upregulated in adipose tissue of insulin-resistant versus insulin-sensitive individuals, who were matched for body mass index (BMI). TNMD expression increases in human preadipocytes during differentiation, whereas silencing TNMD blocks adipogenesis. Upon high-fat diet feeding, transgenic mice overexpressing Tnmd develop increased epididymal white adipose tissue (eWAT) mass, and preadipocytes derived from Tnmd transgenic mice display greater proliferation, consistent with elevated adipogenesis. In Tnmd transgenic mice, lipogenic genes are upregulated in eWAT, as is Ucp1 in brown fat, while liver triglyceride accumulation is attenuated. Despite expanded eWAT, transgenic animals display improved systemic insulin sensitivity, decreased collagen deposition and inflammation in eWAT, and increased insulin stimulation of Akt phosphorylation. Our data suggest that TNMD acts as a protective factor in visceral adipose tissue to alleviate insulin resistance in obesity.

Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe−/− mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe−/− and Ldlr−/− mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis.

The lipid droplet protein Hypoxia-inducible gene 2 promotes hepatic triglyceride deposition by inhibiting lipolysis

Background: Excess hepatic triglyceride accumulation is associated with metabolic disease. Results: Hig2 localizes to hepatic lipid droplets, enhancing their lipid content, and its deficiency increases triglyceride lipolysis. Conclusion: Hig2 is a lipid droplet protein in hepatocytes that promotes liver triglyceride deposition by reducing its rate of degradation. Significance: Hig2 is revealed as a critical lipid droplet protein controlling liver fat. The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation.