Restoration of the ER stress response protein TDAG51 in hepatocytes mitigates NAFLD in mice.

Endoplasmic reticulum stress is associated with insulin resistance and the development of nonalcoholic fatty liver disease. Deficiency of the endoplasmic reticulum stress response T-cell death-associated gene 51 (TDAG51) (TDAG51-/-) in mice promotes the development of high-fat diet (HFD)-induced obesity, fatty liver, and hepatic insulin resistance. However, whether this effect is due specifically to hepatic TDAG51 deficiency is unknown. Here, we report that hepatic TDAG51 protein levels are consistently reduced in multiple mouse models of liver steatosis and injury as well as in liver biopsies from patients with liver disease compared to normal controls. Delivery of a liver-specific adeno-associated virus (AAV) increased hepatic expression of a TDAG51-GFP fusion protein in WT, TDAG51-/-, and leptin-deficient (ob/ob) mice. Restoration of hepatic TDAG51 protein was sufficient to increase insulin sensitivity while reducing body weight and fatty liver in HFD fed TDAG51-/- mice and in ob/ob mice. TDAG51-/- mice expressing ectopic TDAG51 display improved Akt (Ser473) phosphorylation, post-insulin stimulation. HFD-fed TDAG51-/- mice treated with AAV-TDAG51-GFP displayed reduced lipogenic gene expression, increased beta-oxidation and lowered hepatic and serum triglycerides, findings consistent with reduced liver weight. Further, AAV-TDAG51-GFP-treated TDAG51-/- mice exhibited reduced hepatic precursor and cleaved sterol regulatory-element binding proteins (SREBP-1 and SREBP-2). In vitro studies confirmed the lipid-lowering effect of TDAG51 overexpression in oleic acid-treated Huh7 cells. These studies suggest that maintaining hepatic TDAG51 protein levels represents a viable therapeutic approach for the treatment of obesity and insulin resistance associated with nonalcoholic fatty liver disease.

Repurposing Two Old Friends to Fight Cancer: Caffeine and Statins.

Statins are a class of cholesterol-lowering drugs that inhibit 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme of the mevalonate pathway. Evidence suggests that certain cancers depend on the mevalonate pathway for growth and survival, and thus blocking the mevalonate pathway with statins may offer a viable therapeutic approach for treating cancer, or at least enhance the efficacy of existing cancer drugs. In this issue of Cancer Research, Tran and colleagues showed that caffeine works jointly with FOXM1 inhibition to enhance the antitumor activity of statins in neuroblastoma cells. They found that caffeine synergizes with statins by suppressing statin-induced feedback activation of the mevalonate pathway. Here, we reflect on the potential of combining caffeine and statin drugs as a strategy for potentiating anticancer activity. See related article by Tran et al., p. 2248.

Scratching the Surface-An Overview of the Roles of Cell Surface GRP78 in Cancer.

The 78 kDa glucose-regulated protein (GRP78) is considered an endoplasmic reticulum (ER)-resident molecular chaperone that plays a crucial role in protein folding homeostasis by regulating the unfolded protein response (UPR) and inducing numerous proapoptotic and autophagic pathways within the eukaryotic cell. However, in cancer cells, GRP78 has also been shown to migrate from the ER lumen to the cell surface, playing a role in several cellular pathways that promote tumor growth and cancer cell progression. There is another insidious consequence elicited by cell surface GRP78 (csGRP78) on cancer cells: the accumulation of csGRP78 represents a novel neoantigen leading to the production of anti-GRP78 autoantibodies that can bind csGRP78 and further amplify these cellular pathways to enhance cell growth and mitigate apoptotic cell death. This review examines the current body of literature that delineates the mechanisms by which ER-resident GRP78 localizes to the cell surface and its consequences, as well as potential therapeutics that target csGRP78 and block its interaction with anti-GRP78 autoantibodies, thereby inhibiting further amplification of cancer cell progression.

Caffeine blocks SREBP2-induced hepatic PCSK9 expression to enhance LDLR-mediated cholesterol clearance.

Evidence suggests that caffeine (CF) reduces cardiovascular disease (CVD) risk. However, the mechanism by which this occurs has not yet been uncovered. Here, we investigated the effect of CF on the expression of two bona fide regulators of circulating low-density lipoprotein cholesterol (LDLc) levels; the proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low-density lipoprotein receptor (LDLR). Following the observation that CF reduced circulating PCSK9 levels and increased hepatic LDLR expression, additional CF-derived analogs with increased potency for PCSK9 inhibition compared to CF itself were developed. The PCSK9-lowering effect of CF was subsequently confirmed in a cohort of healthy volunteers. Mechanistically, we demonstrate that CF increases hepatic endoplasmic reticulum (ER) Ca2+ levels to block transcriptional activation of the sterol regulatory element-binding protein 2 (SREBP2) responsible for the regulation of PCSK9, thereby increasing the expression of the LDLR and clearance of LDLc. Our findings highlight ER Ca2+ as a master regulator of cholesterol metabolism and identify a mechanism by which CF may protect against CVD.

Extensive diet-induced atherosclerosis in scavenger receptor class B type 1-deficient mice is associated with substantial leukocytosis and elevated vascular cell adhesion molecule-1 expression in coronary artery endothelium.

High levels of low density lipoprotein (LDL) cholesterol and low levels of high density lipoprotein (HDL) cholesterol are risk factors for cardiovascular disease. Mice that lack genes involved in the clearance of LDL from the bloodstream, such as the LDL receptor and apolipoprotein E, are widely used models of experimental atherosclerosis. Conversely, mice that lack the HDL receptor, scavenger receptor class B type I, and therefore have disrupted HDL functionality, also develop diet-inducible atherosclerosis but are a seldom-used disease model. In this study, we compared atherosclerosis and associated phenotypes in scavenger receptor class B type I knockout mice with those of wild type, LDL receptor knockout, and apolipoprotein E knockout mice after 20 weeks of being fed an atherogenic diet containing sodium cholate. We found that while scavenger receptor class B type I knockout mice had substantially lower plasma cholesterol than LDL receptor and apolipoprotein E knockout mice, they developed atherosclerotic plaques with similar sizes and compositions in their aortic sinuses, and more extensive atherosclerosis in their descending aortas and coronary arteries. This was associated with elevated tumor necrosis factor alpha levels in scavenger receptor class B type I knockout mice compared to wild type and LDL receptor knockout mice, and lymphocytosis, monocytosis, and elevated vascular cell adhesion molecule expression in coronary artery endothelial cells compared to the other mice examined. We conclude that extensive atherosclerosis in arteries that are not generally susceptible to atherosclerosis in scavenger receptor class B type I knockout mice is driven by factors in addition to hypercholesterolemia, including inflammation, dysregulation of the immune system and increased sensitivity of endothelial cells in arteries that are normally resistant to atherosclerosis. Scavenger receptor class B type I knockout mice fed a cholate containing atherogenic diet may prove to be a useful model to study mechanisms of atherosclerosis and evaluate treatments that rely on intact LDL clearance pathways.

Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLR cleaved by furin.

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9-/- mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK103↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.

Calcium as a reliable marker for the quantitative assessment of endoplasmic reticulum stress in live cells.

Calcium (Ca2+) is an essential mineral of endoplasmic reticulum (ER) luminal biochemistry because of the Ca2+ dependence of ER-resident chaperones charged with folding de novo proteins that transit this cellular compartment. ER Ca2+ depletion reduces the ability of chaperones to properly fold the proteins entering the ER, thus leading to an accumulation of misfolded proteins and the onset of a state known as ER stress. However, not all conditions that cause ER stress do so in a manner dependent on ER Ca2+ depletion. Agents such as tunicamycin inhibit the glycosylation of de novo polypeptides, a key step in the maturation process of newly synthesized proteins. Despite this established effect of tunicamycin, our understanding of how such conditions modulate ER Ca2+ levels is still limited. In the present study, we report that a variety of ER stress-inducing agents that have not been known to directly alter ER Ca2+ homeostasis can also cause a marked reduction in ER Ca2+ levels. Consistent with these observations, protecting against ER stress using small chemical chaperones, such as 4-phenylbutyrate and tauroursodeoxycholic acid, also attenuated ER Ca2+ depletion caused by these agents. We also describe a novel high-throughput and low-cost assay for the rapid quantification of ER stress using ER Ca2+ levels as a surrogate marker. This report builds on our understanding of ER Ca2+ levels in the context of ER stress and also provides the scientific community with a new, reliable tool to study this important cellular process in vitro.

Glucose-regulated protein (GRP78) is an important cell surface receptor for viral invasion, cancers, and neurological disorders.

The 78 kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum (ER)-resident molecular chaperone. GRP78 is a member of the 70 kDa heat shock family of proteins involved in correcting and clearing misfolded proteins in the ER. In response to cellular stress, GRP78 escapes from the ER and moves to the plasma membrane where it (a) functions as a receptor for many ligands, and (b) behaves as an autoantigen for autoantibodies that contribute to human disease and cancer. Cell surface GRP78 (csGRP78) associates with the major histocompatibility complex class I (MHC-I), and is the port of entry for several viruses, including the predictive binding of the novel SARS-CoV-2. Furthermore, csGRP78 is found in association with partners as diverse as the teratocarcinoma-derived growth factor 1 (Cripto), the melanocortin-4 receptor (MC4R) and the DnaJ-like protein MTJ-1. CsGRP78 also serves as a receptor for a large variety of ligands including activated α2 -macroglobulin (α2 M*), plasminogen kringle 5 (K5), microplasminogen, the voltage-dependent anion channel (VDAC), tissue factor (TF), and the prostate apoptosis response-4 protein (Par-4). In this review, we discuss the mechanisms involved in the translocation of GRP78 from the ER to the cell surface, and the role of secreted GRP78 and its autoantibodies in cancer and neurological disorders.

The loss-of-function PCSK9Q152H variant increases ER chaperones GRP78 and GRP94 and protects against liver injury.

Individuals harboring the loss-of-function (LOF) proprotein convertase subtilisin/kexin type 9 Gln152His variation (PCSK9Q152H) have low circulating low-density lipoprotein cholesterol levels and are therefore protected against cardiovascular disease (CVD). This uncleavable form of proPCSK9, however, is retained in the endoplasmic reticulum (ER) of liver hepatocytes, where it would be expected to contribute to ER storage disease (ERSD), a heritable condition known to cause systemic ER stress and liver injury. Here, we examined liver function in members of several French-Canadian families known to carry the PCSK9Q152H variation. We report that PCSK9Q152H carriers exhibited marked hypocholesterolemia and normal liver function despite their lifelong state of ER PCSK9 retention. Mechanistically, hepatic overexpression of PCSK9Q152H using adeno-associated viruses in male mice greatly increased the stability of key ER stress-response chaperones in liver hepatocytes and unexpectedly protected against ER stress and liver injury rather than inducing them. Our findings show that ER retention of PCSK9 not only reduced CVD risk in patients but may also protect against ERSD and other ER stress-driven conditions of the liver. In summary, we have uncovered a cochaperone function for PCSK9Q152H that explains its hepatoprotective effects and generated a translational mouse model for further mechanistic insights into this clinically relevant LOF PCSK9 variant.

TDAG51 (T-Cell Death-Associated Gene 51) Is a Key Modulator of Vascular Calcification and Osteogenic Transdifferentiation of Arterial Smooth Muscle Cells.

OBJECTIVE: Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. METHODS AND RESULTS: Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. CONCLUSIONS: Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.

GDF10 blocks hepatic PPARγ activation to protect against diet-induced liver injury.

OBJECTIVE: Growth differentiation factors (GDFs) and bone-morphogenic proteins (BMPs) are members of the transforming growth factor ß (TGFß) superfamily and are known to play a central role in the growth and differentiation of developing tissues. Accumulating evidence, however, demonstrates that many of these factors, such as BMP-2 and -4, as well as GDF15, also regulate lipid metabolism. GDF10 is a divergent member of the TGFß superfamily with a unique structure and is abundantly expressed in brain and adipose tissue; it is also secreted by the latter into the circulation. Although previous studies have demonstrated that overexpression of GDF10 reduces adiposity in mice, the role of circulating GDF10 on other tissues known to regulate lipid, like the liver, has not yet been examined. METHODS: Accordingly, GDF10-/- mice and age-matched GDF10+/+ control mice were fed either normal control diet (NCD) or high-fat diet (HFD) for 12 weeks and examined for changes in liver lipid homeostasis. Additional studies were also carried out in primary and immortalized human hepatocytes treated with recombinant human (rh)GDF10. RESULTS: Here, we show that circulating GDF10 levels are increased in conditions of diet-induced hepatic steatosis and, in turn, that secreted GDF10 can prevent excessive lipid accumulation in hepatocytes. We also report that GDF10-/- mice develop an obese phenotype as well as increased liver triglyceride accumulation when fed a NCD. Furthermore, HFD-fed GDF10-/- mice develop increased steatosis, endoplasmic reticulum (ER) stress, fibrosis, and injury of the liver compared to HFD-fed GDF10+/+ mice. To explain these observations, studies in cultured hepatocytes led to the observation that GDF10 attenuates nuclear peroxisome proliferator-activated receptor γ (PPARγ) activity; a transcription factor known to induce de novo lipogenesis. CONCLUSION: Our work delineates a hepatoprotective role of GDF10 as an adipokine capable of regulating hepatic lipid levels by blocking de novo lipogenesis to protect against ER stress and liver injury.

4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

Endoplasmic reticulum stress plays an important role in cardiovascular disease (CVD) and atherosclerosis. We aimed to assess the ability of 4-phenylbutyrate (4-PBA), a small chemical chaperone administered via drinking water, to reduce atherosclerotic lesion size in chow-fed apolipoprotein (Apo) e-/- mice and to identify mechanisms that contribute to its antiatherogenic effect. Chow-fed 17-wk-old female Apoe-/- mice treated with 4-PBA-supplemented drinking water for 5 wk exhibited smaller lesions as well as increased plasma levels of heat shock protein (HSP) 25, the mouse homolog of human HSP27, compared with controls. In addition, 4-PBA inhibited cell death and increased HSP27 expression as measured by real-time PCR and immunoblotting, as well as induced nuclear localization of its transcription factor, heat shock factor 1, in human monocyte/macrophage (THP-1) cells. Furthermore, HSP27 small interfering RNA diminished the protective effect of 4-PBA on THP-1 macrophage attachment and differentiation. In summary, drinking water containing 4-PBA attenuated early lesion growth in Apoe-/- mice fed a chow diet and increased expression of HSP25 and HSP27 in macrophages and HSP25 in the circulation of Apoe-/- mice. Given that increased expression of HSP27 is inversely correlated with CVD risk, our findings suggest that 4-PBA protects against the early stages of atherogenesis in part by enhancing HSP27 levels, leading to inhibition of both macrophage cell death and monocyte-macrophage differentiation.-Lynn, E. G., Lhoták, S., Lebeau, P., Byun, J. H., Chen, J., Platko, K., Shi, C., O'Brien, E. R., Austin, R. C. 4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

Diet-induced hepatic steatosis abrogates cell-surface LDLR by inducing de novo PCSK9 expression in mice.

The worldwide prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing rapidly. Although this condition is generally benign, accumulating evidence now suggests that patients with NAFLD are also at increased risk of cardiovascular disease (CVD); the leading cause of death in developed nations. Despite the well-established role of the liver as a central regulator of circulating low-density lipoprotein (LDL) cholesterol levels, a known driver of CVD, the mechanism(s) by which hepatic steatosis contributes to CVD remains elusive. Interestingly, a recent study has shown that circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) levels correlate positively with liver steatosis grade. Given that PCSK9 degrades the LDL receptor (LDLR) and prevents the removal of LDL from the blood into the liver, in the present study we examined the effect of hepatic steatosis on LDLR expression and circulating LDL cholesterol levels. We now report that in a manner consistent with findings in patients, diet-induced steatosis increases circulating PCSK9 levels as a result of de novo expression in mice. We also report the finding that steatosis abrogates hepatic LDLR expression and increases circulating LDL levels in a PCSK9-dependent manner. These findings provide important mechanistic insights as to how hepatic steatosis modulates lipid regulatory genes, including PCSK9 and the LDLR, and also highlights a novel mechanism by which liver disease may contribute to CVD.

Cell surface expression of 78-kDa glucose-regulated protein (GRP78) mediates diabetic nephropathy.

The 78-kDa glucose-regulated protein (GRP78) is a well-established endoplasmic reticulum (ER)-resident chaperone that maintains protein homeostasis and regulates the unfolded protein response. Under conditions of ER stress, GRP78 is also expressed at the cell surface and implicated in tumorigenesis, immunity, and cellular signaling events. The role of cell surface-associated GRP78 (csGRP78) in the pathogenesis of diabetic nephropathy has not yet been defined. Here we explored the role of csGRP78 in regulating high glucose (HG)-induced profibrotic AKT Ser/Thr kinase (AKT) signaling and up-regulation of extracellular matrix proteins. Using primary kidney mesangial cells, we show that HG treatment, but not the osmotic control mannitol, induces csGRP78 expression through an ER stress-dependent mechanism. We found that csGRP78, known to be located on the outer membrane leaflet, interacts with the transmembrane protein integrin ß1 and activates focal adhesion kinase and downstream PI3K/AKT signaling. Localization of GRP78 at the cell surface and its interaction with integrin ß1 were also required for extracellular matrix protein synthesis in response to HG. Surprisingly, both the N and C termini of csGRP78 were necessary for this profibrotic response. Increased localization of GRP78 at the plasma membrane was also found in the glomerular mesangial area of type 1 diabetic mice in two different models (streptozotocin-induced and Akita). In freshly isolated glomeruli from Akita mice, csGRP78 co-localized with the mesangial cell surface marker α8-integrin. In conclusion, our work reveals a role for csGRP78 in HG-induced profibrotic responses in mesangial cells, informing a potential approach to treating diabetic nephropathy.

Anti-GRP78 autoantibodies induce endothelial cell activation and accelerate the development of atherosclerotic lesions.

The 78-kDa glucose-regulated protein (GRP78) is an ER molecular chaperone that aids in protein folding and secretion. However, pathological conditions that cause ER stress can promote the relocalization of GRP78 to the cell surface (csGRP78), where it acts as a signaling receptor to promote cancer progression. csGRP78 also possesses antigenic properties, leading to the production of anti-GRP78 autoantibodies, which contribute to tumor growth. In contrast, the presence and role of anti-GRP78 autoantibodies in atherosclerosis is unknown. Here, we show that atherosclerotic-prone ApoE-/- mice develop circulating anti-GRP78 autoantibodies that bind to csGRP78 on lesion-resident endothelial cells. Moreover, GRP78-immunized ApoE-/- mice exhibit a marked increase in circulating anti-GRP78 autoantibody titers that correlated with accelerated lesion growth. Mechanistically, engagement of anti-GRP78 autoantibodies with csGRP78 on human endothelial cells activated NF-κB, thereby inducing the expression of ICAM-1 and VCAM-1, a process blocked by NF-κB inhibitors. Disrupting the autoantibody/csGRP78 complex with enoxaparin, a low-molecular-weight heparin, reduced the expression of adhesion molecules and attenuated lesion growth. In conclusion, anti-GRP78 autoantibodies play a crucial role in atherosclerosis development, and disruption of the interaction between anti-GRP78 autoantibodies and csGRP78 represents a therapeutic strategy.

Sialidase down-regulation reduces non-HDL cholesterol, inhibits leukocyte transmigration, and attenuates atherosclerosis in ApoE knockout mice.

Atherosclerosis is a complex disease that involves alterations in lipoprotein metabolism and inflammation. Protein and lipid glycosylation events, such as sialylation, contribute to the development of atherosclerosis and are regulated by specific glycosidases, including sialidases. To evaluate the effect of the sialidase neuraminidase 1 (NEU1) on atherogenesis, here we generated apolipoprotein E (ApoE)-deficient mice that express hypomorphic levels of NEU1 (Neu1hypoApoe-/-). We found that the hypomorphic NEU1 expression in male Apoe-/- mice reduces serum levels of very-low-density lipoprotein (VLDL) and LDL cholesterol, diminishes infiltration of inflammatory cells into lesions, and decreases aortic sinus atherosclerosis. Transplantation of Apoe-/- bone marrow (BM) into Neu1hypoApoe-/- mice significantly increased atherosclerotic lesion development and had no effect on serum lipoprotein levels. Moreover, Neu1hypoApoe-/- mice exhibited a reduction in circulating monocyte and neutrophil levels and had reduced hyaluronic acid and P-selectin adhesion capability on monocytes/neutrophils and T cells. Consistent with these findings, administration of a sialidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, had a significant anti-atherogenic effect in the Apoe-/- mice. In summary, the reduction in NEU1 expression or function decreases atherosclerosis in mice via its significant effects on lipid metabolism and inflammatory processes. We conclude that NEU1 may represent a promising target for managing atherosclerosis.

MAPping the kinase landscape of macrophage activation.

ARL11 is a tumor suppressor gene with established pro-apoptotic properties, but its function beyond this role is poorly understood. A new analysis of macrophage activation has identified ARL11 as a novel regulator of a mitogen-activated protein kinase (MAPK). These findings expand on the function of ARL11 beyond its tumor suppressor activity and highlight a novel role as a regulator of macrophage activation and inflammatory response.

Thrombotic characteristics of extracellular vesicles derived from prostate cancer cells.

BACKGROUND: Prostate cancer (PC) patients in advanced stages of the disease have high risk of blood coagulation complications. The procoagulant molecule Tissue factor (TF), and the fibrinolysis inhibitor plasminogen activator inhibitor-1 PAI-1 play important role in this complication. Extracellular vesicles (EV) shed from cancer cells may contribute to the regulation of TF and PAI-1. The procoagulant activity of EV can be associated with the oncogenic and metastatic characteristics of their cells. METHODS: We have expressed EGFRvIII in DU145 cells to assess the role of this oncogene in the procoagulant activity of EV. The intercellular exchange of TF via EV was assessed by downregulating its expression in DU145 cells using shRNA vector, and determining the transfer of TF via EV enriched with the protein. Two PC cell lines with different metastatic potential were used to assess the correlation between the procoagulant activity of EV and the metastatic potential of PC cells. Photometric assays were used to determine FXa-activity and thrombin generation as indicators for the procoagulant activity of EV. Double-tagged proteinase-activated receptor 1(PAR-1) expressed in CHO cells to assess its activation by EV. RESULTS: The expression of EGFRvIII in DU145 cells led to increased mRNA levels for TF and PAI-1, but the increase in these proteins expression was detected mostly in the EV. EV with enhanced levels of TF protein conferred higher TF procoagulant activity on the acceptor cells by intercellular exchange of this protein. Procoagulant activity of EV, assessed by FXa activity, and thrombin generation, was correlated with the oncogenic and metastatic potential of PC cells. The ability of EV to generate thrombin led to the activation of PAR-1, which was evident by the truncation of tagged-PAR-1. CONCLUSION: The active oncogene EGFRvIII increases the concentration of TF and PAI-1 in EV. The procoagulant activity of EV is associated with the oncogenic and metastatic characteristics of their PC cells. Also, EV may contribute to the high procoagulant activity in the tumour microenvironment by the intercellular exchange of TF. Finally, through the generation of thrombin, EV can activate PAR-1, which evidently contributes to cancer progression, linking the coagulation system to tumor progression.

Pharmacologic inhibition of S1P attenuates ATF6 expression, causes ER stress and contributes to apoptotic cell death.

Mammalian cells express unique transcription factors embedded in the endoplasmic reticulum (ER) membrane, such as the sterol regulatory element-binding proteins (SREBPs), that promote de novo lipogenesis. Upon their release from the ER, the SREBPs require proteolytic activation in the Golgi by site-1-protease (S1P). As such, inhibition of S1P, using compounds such as PF-429242 (PF), reduces cholesterol synthesis and may represent a new strategy for the management of dyslipidemia. In addition to the SREBPs, the unfolded protein response (UPR) transducer, known as the activating transcription factor 6 (ATF6), is another ER membrane-bound transcription factor that requires S1P-mediated activation. ATF6 regulates ER protein folding capacity by promoting the expression of ER chaperones such as the 78-kDa glucose-regulated protein (GRP78). ER-resident chaperones like GRP78 prevent and/or resolve ER polypeptide accumulation and subsequent ER stress-induced UPR activation by folding nascent polypeptides. Here we report that pharmacological inhibition of S1P reduced the expression of ATF6 and GRP78 and induced the activation of UPR transducers inositol-requiring enzyme-1α (IRE1α) and protein kinase RNA-like ER kinase (PERK). As a consequence, S1P inhibition also increased the susceptibility of cells to ER stress-induced cell death. Our findings suggest that S1P plays a crucial role in the regulation of ER folding capacity and also identifies a compensatory cross-talk between UPR transducers in order to maintain adequate ER chaperone expression and activity.

Endoplasmic Reticulum Chaperone GRP78 Protects Heart From Ischemia/Reperfusion Injury Through Akt Activation.

RATIONALE: Restoration of coronary artery blood flow is the most effective means of ameliorating myocardial damage triggered by ischemic heart disease. However, coronary reperfusion elicits an increment of additional injury to the myocardium. Accumulating evidence indicates that the unfolded protein response (UPR) in cardiomyocytes is activated by ischemia/reperfusion (I/R) injury. Xbp1s (spliced X-box binding protein 1), the most highly conserved branch of the unfolded protein response, is protective in response to cardiac I/R injury. GRP78 (78 kDa glucose-regulated protein), a master regulator of the UPR and an Xbp1s target, is upregulated after I/R. However, its role in the protective response of Xbp1s during I/R remains largely undefined. OBJECTIVE: To elucidate the role of GRP78 in the cardiomyocyte response to I/R using both in vitro and in vivo approaches. METHODS AND RESULTS: Simulated I/R injury to cultured neonatal rat ventricular myocytes induced apoptotic cell death and strong activation of the UPR and GRP78. Overexpression of GRP78 in neonatal rat ventricular myocytes significantly protected myocytes from I/R-induced cell death. Furthermore, cardiomyocyte-specific overexpression of GRP78 ameliorated I/R damage to the heart in vivo. Exploration of underlying mechanisms revealed that GRP78 mitigates cellular damage by suppressing the accumulation of reactive oxygen species. We go on to show that the GRP78-mediated cytoprotective response involves plasma membrane translocation of GRP78 and interaction with PI3 kinase, culminating in stimulation of Akt. This response is required as inhibition of the Akt pathway significantly blunted the antioxidant activity and cardioprotective effects of GRP78. CONCLUSIONS: I/R induction of GRP78 in cardiomyocytes stimulates Akt signaling and protects against oxidative stress, which together protect cells from I/R damage.