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.

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.

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.

The role of circulating extracellular vesicles in breast cancer classification and molecular subtyping.

Currently, tumor biopsies are used for breast cancer molecular subtyping. Biopsies are associated with various pathological changes and are thought to contribute to the dissemination of tumor cells. Extracellular vesicles shed by tumor cells into circulation exhibit the molecular signature of the parent cells. Herein, we show that proteomic analysis of circulating EV can discriminate BC patients from healthy subjects and indicate stage of the disease. Also, we performed a correlation between the BC molecular subtype using plasma EV and immunohistochemistry of tumor biopsies. Circulating EV may represent a useful, non-invasive tool to study the molecular makeup of BC tumors.

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.

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.

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.

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.

Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles.

Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators.