14-3-3η Promotes Invadosome Formation via the FOXO3–Snail Axis in Rheumatoid Arthritis Fibroblast-like Synoviocytes

Erosive destruction of joint structures is a critical event in the progression of rheumatoid arthritis (RA), in which fibroblast-like synoviocytes (FLS) are the primary effectors. We previously reported that the ability of RA FLS to degrade extracellular matrix (ECM) components depends on the formation of actin-rich membrane protrusions, called invadosomes, through processes that remain elusive. 14-3-3η belongs to a family of scaffolding proteins involved in a wide range of cellular functions, and its expression is closely related to joint damage and disease activity in RA patients. In this study, we sought to assess the role of 14-3-3η in joint damage by examining its contribution to the invadosome formation phenotype of FLS. Using human primary FLS, we show that 14-3-3η expression is closely associated with their ability to form invadosomes. Furthermore, knockdown of 14-3-3η using shRNAs decreases the level of invadosome formation in RA FLS, whereas addition of the recombinant protein to FLS from healthy individuals promotes their formation. Mechanistic studies suggest that 14-3-3η regulates invadosome formation by increasing Snail expression, a mechanism that involves nuclear exclusion of the transcription repressor FOXO3. Our results implicate the 14-3-3η–FOXO3–Snail axis in promoting the aggressive ECM-degrading phenotype of RA FLS, and suggest a role for this scaffolding protein in cartilage degradation.

Accumulation of legacy fallout radionuclides in cryoconite on Isfallsglaciären (Arctic Sweden) and their downstream spatial distribution

The release of legacy contaminants such as fallout radionuclides (FRNs) in response to glacier retreat is a process that has received relatively little attention to date, yet may have consequences as a source of secondary contamination as glaciers melt and down-waste in response to a warming climate. The presence of FRNs in glacier-fed catchments is poorly understood in comparison to other contaminants, yet there is now emerging evidence from multiple regions of the global cryosphere for substantially augmented FRN activities in cryoconite. Here we report concentrated FRNs in both cryoconite and proglacial sediments from the Isfallsglaciären catchment in Arctic Sweden. Activities of some FRNs in cryoconite are 2 orders of magnitude above those found elsewhere in the catchment, and above the activities found in other environmental matrices outside of nuclear exclusion zones. We also describe the presence of the short-lived cosmogenic radionuclide 7Be in cryoconite samples, highlighting the importance of meltwater–sediment interactions in radionuclide accumulation in the ice surface environment. It is currently unknown whether high accumulations of fallout radionuclides in glaciers have the potential to impact local environmental quality through down-wasting and downstream transport of contaminants to the proglacial environment through interaction with sediments and meltwater. We thus recommend that future research in this field focusses on processes of accumulation of FRNs and other environmental contaminants in cryoconite and whether these contaminants are present in quantities harmful for downstream ecosystems.

Adipose MDM2 regulates systemic insulin sensitivity

The intimate association between obesity and type II diabetes urges for a deeper understanding of adipocyte function. We and others have previously delineated a role for the tumor suppressor p53 in adipocyte biology. Here, we show that mice haploinsufficient for MDM2, a key regulator of p53, in their adipose stores suffer from overt obesity, glucose intolerance, and hepatic steatosis. These mice had decreased levels of circulating palmitoleic acid [non-esterified fatty acid (NEFA) 16:1] concomitant with impaired visceral adipose tissue expression of Scd1 and Ffar4. A similar decrease in Scd and Ffar4 expression was found in in vitro differentiated adipocytes with perturbed MDM2 expression. Lowered MDM2 levels led to nuclear exclusion of the transcriptional cofactors, MORC2 and LIPIN1, and thereby possibly hampered adipocyte function by antagonizing LIPIN1-mediated PPARγ coactivation. Collectively, these data argue for a hitherto unknown interplay between MDM2 and MORC2/LIPIN1 involved in balancing adipocyte function.

Calpain7 Negatively Regulates Human Endometrial Stromal Cell Decidualization in Endometriosis by Promoting FoxO1’s Phosphorylation and Nuclear Exclusion via Hydrolyzing AKT1

Background Endometrial receptivity damage caused by impaired decidualization may be one of the mechanisms of infertility in endometriosis (EMs). Our previous study demonstrated that Calpain-7 (CAPN7) is abnormally overexpressed in EMs. Whether CAPN7 affects the regulation of decidualization and by what mechanism CAPN7 regulates decidualization remains to be determined. Methods Immunohistochemistry (IHC) was used to assess the CAPN7 expression in human endometria. Quantitative real-time PCR (qRT-PCR), western blotting, ELIFA and ELISA were applied to explore PRL and IGFBP-1 expressions in decidualized human endometrial stromal cells (HESC). Immunofluorescence analysis and the nuclear and cytoplasmic protein extract assay were employed to test CAPN7’s affection on FoxO1’s location in HESC. Western blotting was used to explore the regulatory mechanism of CAPN7 to AKT1/FoxO1 signalling pathway. Results In this study, we found CAPN7 expression decreased during human endometrial stromal cell (HESC) decidualization in vitro. CAPN7 negatively regulated decidualization in vitro and in vivo. We also identified one conserved potential PEST sequence in the AKT1 protein and found that CAPN7 was able to hydrolyse AKT1 and enhance AKT1’s phosphorylation. Correspondingly, CAPN7 notably promoted the phosphorylation of Forkhead Box O1 (FoxO1), the downstream of AKT1 protein, at Ser319, leading to increased FoxO1 exclusion from nuclei and attenuated FoxO1 transcriptional activity in decidualized HESC. In addition, we detected endometrium CAPN7, p-AKT1 and p-FoxO1 expressions were increased in EMs. Conclusions These data demonstrate that CAPN7 negatively regulates HESC decidualization in EMs probably by promoting FoxO1’s phosphorylation and FoxO1 nuclear exclusion via hydrolyzing AKT1. The dysregulation of CAPN7 may be a novel cause of EMs.

Hyper-accumulation of legacy fallout radionuclides in cryoconite on Isfallsglaciären (Arctic Sweden) and their downstream distribution

The release of legacy fallout radionuclides (FRNs) in response to glacier retreat is a process that has received relatively little attention to date, yet may have important consequences as a source of secondary contamination as glaciers melt and down-waste in response to a warming climate. The prevalence of FRNs in glacier-fed catchments is poorly understood in comparison to other contaminants, yet there is now emerging evidence from multiple regions of the global cryosphere for substantially augmented FRN activities in cryoconite. Here we report concentrated FRNs in both cryoconite and proglacial sediments from the Isfallsglaciären catchment in Arctic Sweden. Activities of some FRNs in cryoconite are two orders of magnitude above those found elsewhere in the catchment, and above the activities found in other environmental matrices outside of nuclear exclusion zones. We also describe the presence of the short-lived cosmogenic radionuclide 7Be in cryoconite samples, highlighting the importance of meltwater-sediment interactions in radionuclide accumulation in the ice surface environment. The presence of fallout radionuclides in glaciers may have the potential to impact local environmental quality through both isolated hotspots of radioactivity caused by glacier down-wasting, and downstream transport of contaminants to the proglacial environment through interaction with sediments and meltwater. We thus recommend that future research in this field focusses on processes of accumulation of FRNs and other environmental contaminants in cryoconite, and whether these contaminants are present in quantities harmful for both local and downstream ecosystems.

Abstract 17034: The AKT/AMPK/FOXO3 Axis in Pulmonary Arterial Hypertension

Like cancer, pulmonary arterial hypertension (PAH) is characterized by exaggerated proliferation and resistance to apoptosis related to metabolic alterations (Warburg effect) of pulmonary smooth muscle cells (PASMCs). These anomalies result in a progressive narrowing of the pulmonary arteries, increasing pulmonary resistance and leading to right heart failure and premature death. In cancer cells, unphosphorylated and nuclear FOXO3 has been extensively studied as a crucial protein that functions as a tumor suppressor by regulating expression of genes involved in apoptosis and cell cycle arrest. These functions combined with other FOXO3 attributes, including its key role in communicating mitochondrial-nuclear signals, make the FOXO3 a suitable candidate for controlling the cancer-like phenotype of PAH-PASMCs. Interestingly, AKT and AMPK known to be implicated in PAH exert antagonistic effects on FOXO3; AKT promoting its nuclear exclusion while AMPK favors its nuclear and mitochondrial accumulation. The thus made the hypothesis that FOXO3’s nuclear exclusion (secondary to AKT/AMPK imbalance) promotes metabolic reprogramming towards glycolysis leading to enhanced proliferation/resistance to apoptosis of PAH-PASMCs and vascular remodeling. Using Western blot and immunofluorescence in isolated PASMCs from both PAH and control patients (n=10), we found that nuclear and mitochondrial exclusion of FOXO3 due to its phosphorylation is a feature of PAH-PASMCs. In vitro, we demonstrated that nuclear localization of FOXO3 using an adenovirus expressing a constitutively active, non-phosphorylable form of FOXO3 or trifluoperazine (TFP) resulted in reduced PAH-PASMC proliferation (Ki67 labeling, p<0,0005) and resistance to apoptosis (Annexin V assay, p<0,05). These effects were accompanied by increased expression of P27 and SOD2 and diminished expression of Survivin (p<0,05). In vivo, we showed that FOXO3 activation using TPF improved established PAH in the monocrotaline rats (reduced RVSP and increased Sv and CO, by right catheterization, p<0,01, n=29) without any sign of toxicity. We showed that FOXO3 is implicated in pulmonary vascular remodeling. Pharmacological activation of FOXO3 may represent a novel avenue to improve PAH.

Nuclear exclusion of YAP exacerbates podocyte apoptosis and disease progression in Adriamycin-induced focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a chronic glomerular disease with poor clinical outcomes. Podocyte loss via apoptosis is one important mechanism underlying the pathogenesis of FSGS. Recently, Yes-associated-protein (YAP), a key downstream protein in the Hippo pathway, was identified as an activator for multiple gene transcriptional factors in the nucleus to control cell proliferation and apoptosis. To investigate the potential role of YAP in the progression of FSGS, we examined kidney samples from patients with minimal change disease or FSGS and found that increases in podocyte apoptosis is positively correlated with the cytoplasmic distribution of YAP in human FSGS. Utilizing an established mT/mG transgenic mouse model and primary cultured podocytes, we found that YAP was distributed uniformly in nucleus and cytoplasm in the podocytes of control animals. Adriamycin treatment induced gradual nuclear exclusion of YAP with enhanced phospho-YAP/YAP ratio, accompanied by the induction of podocyte apoptosis both in vivo and in vitro. Moreover, we used verteporfin to treat an Adriamycin-induced FSGS mouse model, and found YAP inhibition by verteporfin induced nuclear exclusion of YAP, thus increasing podocyte apoptosis and accelerating disease progression. Therefore, our findings suggest that YAP nuclear distribution and activation in podocytes is an important endogenous anti-apoptotic mechanism during the progression of FSGS.

MON-635 FDXR Regulates Iron Metabolism and Glucose Metabolism in Liver

Iron is an essential cofactor for many proteins that function in electron transport or oxygen transport as heme or iron-sulfur cluster. On the contrary, iron also has the potential to cause oxidative damage if not carefully regulated and when in labial iron excess. Clinical studies show that elevated serum ferritin levels are observed in most patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). In this context, p53 is shown to induces some mitochondrial iron regulatory genes. The role of crosstalk between p53 and iron metabolism has not been sufficiently examined in the pathogenesis of diabetes and NAFLD. Here, we examined the role of ferredoxin reductase (FDXR), a key mitochondrial regulator for iron metabolism, as p53-inducible gene with focusing on the hepatocyte and liver. We confirmed that p53 induced FDXR expression in HepG2 cells and SKEHP1 cells. Biochemical analysis demonstrated that FDXR regulated ROS levels via iron metabolism. In vivo analysis, high-fat diet activated the p53-FDXR pathway in mice liver. We generated transgene expression in mice liver using adenovirus infection carrying shRNA or CRISPR Cas9 system. Treatment with the FDXR knockdown increased hepatic iron content and aggravated glucose intolerance. Besides, forkhead box protein O1 (FOXO1), a key transcriptional factor that induces phosphoenolpyruvate carboxylase and glucose-6-phosphatase increased ratio of nuclear localization, indicating hepatic gluconeogenesis activation. Consistently, biochemical analysis in HepG2 cells demonstrated that FDXR regulated insulin-dependent FOXO1 nuclear exclusion through oxidative stress. In conclusion, p53-inducible FDXR regulates iron metabolism and oxidative stress. FDXR inhibits iron accumulation and oxidative stress in liver and links to suppression of hepatic gluconeogenesis via insulin-dependent FOXO1 nuclear exclusion. The results of this study provide important new insights into relationship between iron metabolism and glucose metabolism as well as potentially identify novel therapeutic targets for the treatment of diabetes and NAFLD.

Spatiotemporal gating of SIRT1 functions by O-GlcNAcylation is essential for liver metabolic switching and prevents hyperglycemia

Inefficient physiological transitions are known to cause metabolic disorders. Therefore, investigating mechanisms that constitute molecular switches in a central metabolic organ like the liver becomes crucial. Specifically, upstream mechanisms that control temporal engagement of transcription factors, which are essential to mediate physiological fed–fast–refed transitions are less understood. SIRT1, a NAD+-dependent deacetylase, is pivotal in regulating hepatic gene expression and has emerged as a key therapeutic target. Despite this, if/how nutrient inputs regulate SIRT1 interactions, stability, and therefore downstream functions are still unknown. Here, we establish nutrient-dependent O-GlcNAcylation of SIRT1, within its N-terminal domain, as a crucial determinant of hepatic functions. Our findings demonstrate that during a fasted-to-refed transition, glycosylation of SIRT1 modulates its interactions with various transcription factors and a nodal cytosolic kinase involved in insulin signaling. Moreover, sustained glycosylation in the fed state causes nuclear exclusion and cytosolic ubiquitin-mediated degradation of SIRT1. This mechanism exerts spatiotemporal control over SIRT1 functions by constituting a previously unknown molecular relay. Of note, loss of SIRT1 glycosylation discomposed these interactions resulting in aberrant gene expression, mitochondrial dysfunctions, and enhanced hepatic gluconeogenesis. Expression of nonglycosylatable SIRT1 in the liver abrogated metabolic flexibility, resulting in systemic insulin resistance, hyperglycemia, and hepatic inflammation, highlighting the physiological costs associated with its overactivation. Conversely, our study also reveals that hyperglycosylation of SIRT1 is associated with aging and high-fat–induced obesity. Thus, we establish that nutrient-dependent glycosylation of SIRT1 is essential to gate its functions and maintain physiological fitness.