Abstract:The proprotein convertase subtilisin/kexin type-9 (PCSK9) plays a central role in cardiovascular disease (CVD) by degrading hepatic low-density lipoprotein receptor (LDLR). As such, loss-of-function (LOF) PCSK9 variants that fail to exit the endoplasmic reticulum (ER) increase hepatic LDLR levels and lower the risk of developing CVD. The retention of misfolded protein in the ER can cause ER stress and activate the unfolded protein response (UPR). In this study, we investigated whether a variety of LOF PCSK9 va… Show more
“…To block Smad3 expression, siGENOME SMARTpool siRNA targeted against Smad3 (UCAAGAGCCUGGUCAAGAA, GAGUUCGCCUUCAAUAUGA, GGACGCAGGUUCUC-CAAAC, GGACGAGGUCUGCGUGAAU) was purchased from Dharmacon (SO-2782081G). 100 nM of siRNA targeted against Smad3 or scrambled siRNA control was transfected using RNAiMAX (ThermoFisher Scientific) as described previously [26].…”
Section: Methodsmentioning
confidence: 99%
“…Whole cell and tissue lysates were prepared using SDS lysis buffer containing a protease inhibitor cocktail (Roche), and protein concentration was measured as described previously [26]. The samples were resolved using SDS-PAGE and subsequently transferred to nitrocellulose membranes.…”
Objective
Growth differentiation factors (GDFs) and bone-morphogenic proteins (BMPs) are members of the transforming growth factor β (TGFβ) 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 TGFβ 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.
“…To block Smad3 expression, siGENOME SMARTpool siRNA targeted against Smad3 (UCAAGAGCCUGGUCAAGAA, GAGUUCGCCUUCAAUAUGA, GGACGCAGGUUCUC-CAAAC, GGACGAGGUCUGCGUGAAU) was purchased from Dharmacon (SO-2782081G). 100 nM of siRNA targeted against Smad3 or scrambled siRNA control was transfected using RNAiMAX (ThermoFisher Scientific) as described previously [26].…”
Section: Methodsmentioning
confidence: 99%
“…Whole cell and tissue lysates were prepared using SDS lysis buffer containing a protease inhibitor cocktail (Roche), and protein concentration was measured as described previously [26]. The samples were resolved using SDS-PAGE and subsequently transferred to nitrocellulose membranes.…”
Objective
Growth differentiation factors (GDFs) and bone-morphogenic proteins (BMPs) are members of the transforming growth factor β (TGFβ) 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 TGFβ 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.
“…3B and C). Our research group has also used the TBCD assay described in this report to demonstrate changes in cell death in HK2 cells transfected with mammalian expression plasmids [2]. Fig.…”
“…It was until 2015, Poirier et al (2015) demonstrated that GRP94, an ER chaperone specifically interacts with PCSK9 and blocks its interaction with LDLR. Interestingly, it was later identified that GRP94 chaperones PCSK mutant pro-forms in ER and alleviate the potential toxic ER-stress ( Lebeau et al, 2018 ). The underlying mechanism was further delineated to GRP94 interaction with mutant PCSK9 which prevents the latter’s binding to GRP78, an ER luminal chaperone that signals proteotoxic stress in ER to major ER stress sensors ( Lebeau et al, 2018 ).…”
Section: Deregulation Of Er Homeostasis and Activation Of Upr By Ldlrmentioning
confidence: 99%
“…Interestingly, it was later identified that GRP94 chaperones PCSK mutant pro-forms in ER and alleviate the potential toxic ER-stress ( Lebeau et al, 2018 ). The underlying mechanism was further delineated to GRP94 interaction with mutant PCSK9 which prevents the latter’s binding to GRP78, an ER luminal chaperone that signals proteotoxic stress in ER to major ER stress sensors ( Lebeau et al, 2018 ). In a way, contradicting to the blocker role of GRP94 in PCSK9-LDLR interactions, PCSK9 is also reported to act as a chaperone for LDLR.…”
Section: Deregulation Of Er Homeostasis and Activation Of Upr By Ldlrmentioning
Familial hypercholesterolemia (FH) is an autosomal genetic disease characterized by high serum low-density lipoprotein (LDL) content leading to premature coronary artery disease. The main genetic and molecular causes of FH are mutations in low-density lipoprotein receptor gene (
LDLR
) resulting in the non-clearance of LDL from the blood by hepatocytes and consequently the formation of plaques. LDLR is synthesized and glycosylated in the endoplasmic reticulum (ER) and then transported to the plasma membrane via Golgi. It is estimated that more than 50% of reported FH-causing mutations in LDLR result in misfolded proteins that are transport-defective and hence retained in ER. ER accumulation of misfolded proteins causes ER-stress and activates unfolded protein response (UPR). UPR aids protein folding, blocks further protein synthesis, and eliminates misfolded proteins via ER-associated degradation (ERAD) to alleviate ER stress. Various studies demonstrated that ER-retained LDLR mutants are subjected to ERAD. Interestingly, chemical chaperones and genetic or pharmacological inhibition of ERAD have been reported to rescue the transport defective mutant LDLR alleles from ERAD and restore their ER-Golgi transport resulting in the expression of functional plasma membrane LDLR. This suggests the possibility of pharmacological modulation of proteostasis in the ER as a therapeutic strategy for FH. In this review, we picture a detailed analysis of UPR and the ERAD processes activated by ER-retained LDLR mutants associated with FH. In addition, we discuss and critically evaluate the potential role of chemical chaperones and ERAD modulators in the therapeutic management of FH.
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