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The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK α1/α2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury.

Dysfunctions in the intestinal barrier, associated with an altered paracellular pathway, are commonly observed in inflammatory bowel disease (IBD). The AMP-activated protein kinase (AMPK), principally known as a cellular energy sensor, has also been shown to play a key role in the stabilization and assembly of tight junctions. Here, we aimed to investigate the contribution of intestinal epithelial AMPK to the initiation, progression and resolution of acute colitis. We also tested the hypothesis that protection mediated by metformin administration on intestinal epithelium damage required AMPK activation. A dextran sodium sulfate (DSS)-induced colitis model was used to assess disease progression in WT and intestinal epithelial cell (IEC)-specific AMPK KO mice. Barrier integrity was analyzed by measuring paracellular permeability following dextran-4kDa gavage and pro-inflammatory cytokines and tight junction protein expression. The deletion of intestinal epithelial AMPK delayed intestinal injury repair after DSS exposure and was associated with a slower re-epithelization of the intestinal mucosa coupled with severe ulceration and inflammation, and altered barrier function. Following intestinal injury, IEC AMPK KO mice displayed a lower goblet cell counts with concomitant decreased Muc2 gene expression, unveiling an impaired restitution of goblet cells and contribution to wound healing process. Metformin administration during the recovery phase attenuated the severity of DSS-induced colitis through improvement in intestinal repair capacity in both WT and IEC AMPK KO mice. Taken together, these findings demonstrate a critical role for IEC-expressed AMPK in regulating mucosal repair and epithelial regenerative capacity following acute colonic injury. Our studies further underscore the therapeutic potential of metformin to support repair of the injured intestinal epithelium, but this effect is conferred independently of intestinal epithelial AMPK.

Coordination of gene expression with nutrient availability supports proliferation and homeostasis and is shaped by protein acetylation. Yet how physiological/pathological signals link acetylation to specific gene expression programs and whether such responses are cell-type-specific is unclear. AMP-activated protein kinase (AMPK) is a key energy sensor, activated by glucose limitation to resolve nutrient supply-demand imbalances, critical for diabetes and cancer. Unexpectedly, we show here that, in gastrointestinal cancer cells, glucose activates AMPK to selectively induce EP300, but not CREB-binding protein (CBP). Consequently, EP300 is redirected away from nuclear receptors that promote differentiation towards [beta]-catenin, a driver of proliferation and colorectal tumorigenesis. Importantly, blocking glycogen synthesis permits reactive oxygen species (ROS) accumulation and AMPK activation in response to glucose in previously nonresponsive cells. Notably, glycogen content and activity of the ROS/AMPK/EP300/[beta]-catenin axis are opposite in healthy versus tumor sections. Glycogen content reduction from healthy to tumor tissue may explain AMPK switching from tumor suppressor to activator during tumor evolution.

AMP activated protein kinase (AMPK) is a critical energy sensor, regulating signaling networks involved in pathology including metabolic diseases and cancer. This increasingly recognized role of AMPK has prompted tremendous research efforts to develop new pharmacological AMPK activators. To precisely study the role of AMPK, and the specificity and activity of AMPK activators in cellular models, genetic AMPK inactivating tools are required. We report here methods for genetic inactivation of AMPK \\(1/ 2\\) catalytic subunits in human cell lines by the CRISPR/Cas9 technology, a recent breakthrough technique for genome editing.

Elevated levels of type I interferon (IFN) during pregnancy are associated with intrauterine growth retardation, preterm birth, and fetal demise through mechanisms that are not well understood. A critical step of placental development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast (ST) layer. Fusion is mediated by syncytins, proteins deriving from ancestral endogenous retroviral envelopes. Using cultures of human trophoblasts or mouse cells, we show that IFN-induced transmembrane proteins (IFITMs), a family of restriction factors blocking the entry step of many viruses, impair ST formation and inhibit syncytin-mediated fusion. Moreover, the IFN inducer polyinosinic:polycytidylic acid promotes fetal resorption and placental abnormalities in wild-type but not in Ifitm-deleted mice. Thus, excessive levels of IFITMs may mediate the pregnancy complications observed during congenital infections and other IFN-induced pathologies.

Phthalates are environmental contaminants commonly used as plasticizers in polyvinyl chloride (PVC) products. Recently, exposure to phthalates has been associated with preterm birth, low birth weight, and pregnancy loss. There is limited information about the possible mechanisms linking maternal phthalate exposure and placental development, but one such mechanism may be mediated by peroxisome proliferator–activated receptor γ (PPARγ). PPARγ belongs to the nuclear receptor superfamily that regulates, in a ligand-dependent manner, the transcription of target genes. Studies of PPARγ-deficient mice have demonstrated its essential role in lipid metabolism and placental development. In the human placenta, PPARγ is expressed in the villous cytotrophoblast (VCT) and is activated during its differentiation into syncytiotrophoblast. The goal of this study was to investigate the action of mono(2-ethylhexyl) phthalate (MEHP) on PPARγ activity during differentiation of VCTs. We combined immunofluorescence, PPARγ activity/hCG assays, western blotting, and lipidomics analyses to characterize the impacts of physiologically relevant concentrations of MEHP (0.1, 1, and 10 μM) on cultured VCTs isolated from human term placentas. Doses of 0.1 and 1 μM MEHP showed significantly lower PPARγ activity and less VCT differentiation in comparison with controls, whereas, surprisingly, a 10 μM dose had the opposite effect. MEHP exposure inhibited hCG production and significantly altered lipid composition. In addition, MEHP had significant effects on the mitogen-activated protein kinase (MAPK) pathway. This study suggests that MEHP has a U-shaped dose–response effect on trophoblast differentiation that is mediated by the PPARγ pathway and acts as an endocrine disruptor in the human placenta. https://doi.org/10.1289/EHP3730.

NLRP3 controls the secretion of inflammatory cytokines IL-1β/18 and pyroptosis by assembling the inflammasome. Upon coordinated priming and activation stimuli, NLRP3 recruits NEK7 within hetero-oligomers that nucleate ASC and caspase-1 filaments, but the apical molecular mechanisms underlying inflammasome assembly remain elusive. Here we show that NEK7 recruitment to NLRP3 is controlled by the phosphorylation status of NLRP3 S803 located within the interaction surface, in which NLRP3 S803 is phosphorylated upon priming and later dephosphorylated upon activation. Phosphomimetic substitutions of S803 abolish NEK7 recruitment and inflammasome activity in macrophages in vitro and in vivo. In addition, NLRP3-NEK7 binding is also essential for NLRP3 deubiquitination by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3. Finally, we identify CSNK1A1 as the kinase targeting NLRP3 S803. Our findings thus reveal NLRP3 S803 phosphorylation status as a druggable apical molecular mechanism controlling inflammasome assembly. Nlrp3 inflammasome activation requires Nek7 recruitment to drive ASC speck formation. Here the authors show how Nlrp3 phosphorylation events control this Nek7 recruitment.

Due to rising costs and the difficulty to identify new targets, drug repurposing appears as a viable strategy for the development of new anti-cancer treatments. Although the interest of disulfiram (DSF), an anti-alcohol drug, to treat cancer was reported for many years, it is only very recently that one anticancer mechanism-of-action was highlighted. This would involve the inhibition of the p97 segregase adaptor NPL4, which is essential for the turnover of proteins involved in multiple regulatory and stress-response intracellular pathways. However, recently DSF was also reported as one of the first phosphoglycerate dehydrogenase (PHGDH) inhibitors, a tetrameric enzyme catalyzing the initial step of the serine synthetic pathway that is highly expressed in numerous cancer types. Here, we investigated the structure-activity relationships (SAR) of PHGDH inhibition by disulfiram analogues as well as the mechanism of action of DSF on PHGDH via enzymatic and cell-based evaluation, mass spectrometric and mutagenesis experiments.

Hypertension prevalence increases when socioeconomic status decreases but gender differences in the relationship between socioeconomic status and hypertension have been less studied. This work aimed to explore the pattern of associations between three indicators of socioeconomic status at individual, household, and municipal levels with hypertension across genders in a large sample of French adults from the CONSTANCES cohort. Using data at inclusion from 59 805 participants (52% women) aged 25-69 years and recruited between 2012 and 2015, multilevel log-Poisson regressions with robust variance estimates were used to assess the associations of Relative Index of Inequality in education, monthly income per consumption unit and residential deprivation with hypertension. Modifying effects of gender and age in those associations were tested. Hypertension prevalence was higher in men than in women. Steep socioeconomic gradients of hypertension were observed for the three socioeconomic indicators in both genders and from the youngest to the oldest age class. Socioeconomic inequalities, especially educational inequalities, were larger among women than men: Relative Index of Inequality for highest versus lowest education among the 25-34 years were 0.43 [95%-confidence interval = 0.28-0.67] in women and 0.70 [95%-confidence interval = 0.53-0.92] in men. With increasing age, socioeconomic gradients of hypertension eased in men and even more in women so that gender differences decreased. In this cross-sectional analysis of a large sample of adults, prevalence of hypertension was higher in men than in women. Moreover, socioeconomic status and especially education displayed a stronger association with hypertension prevalence in women compared to men. Reducing inequalities in hypertension may require gender-specific approaches.

In shade‐intolerant plants such as Arabidopsis , a reduction in the red/far‐red (R/FR) ratio, indicative of competition from other plants, triggers a suite of responses known as the shade avoidance syndrome (SAS). The phytochrome photoreceptors measure the R/FR ratio and control the SAS. The phytochrome‐interacting factors 4 and 5 (PIF4 and PIF5) are stabilized in the shade and are required for a full SAS, whereas the related bHLH factor HFR1 (long hypocotyl in FR light) is transcriptionally induced by shade and inhibits this response. Here we show that HFR1 interacts with PIF4 and PIF5 and limits their capacity to induce the expression of shade marker genes and to promote elongation growth. HFR1 directly inhibits these PIFs by forming non‐DNA‐binding heterodimers with PIF4 and PIF5. Our data indicate that PIF4 and PIF5 promote SAS by directly binding to G‐boxes present in the promoter of shade marker genes, but their action is limited later in the shade when HFR1 accumulates and forms non‐DNA‐binding heterodimers. This negative feedback loop is important to limit the response of plants to shade.