Role Of Bcl2-associated Athanogene 3 Research Articles

BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

BackgroundDiabetic vascular remodeling is the most important pathological basis of diabetic cardiovascular complications. The accumulation of advanced glycation end products (AGEs) caused by elevated blood glucose promotes the proliferation and migration of vascular smooth muscle cells (VSMCs), leading to arterial wall thickening and ultimately vascular remodeling. Therefore, the excessive proliferation and migration of VSMCs is considered as an important therapeutic target for vascular remodeling in diabetes mellitus. However, due to the lack of breakthrough in experiments, there is currently no effective treatment for the excessive proliferation and migration of VSMCs in diabetic patients. Bcl-2-associated athanogene 3 (BAG3) protein is a multifunctional protein highly expressed in skeletal muscle and myocardium. Previous research has confirmed that BAG3 can not only regulate cell survival and apoptosis, but also affect cell proliferation and migration. Since the excessive proliferation and migration of VSMCs is an important pathogenesis of vascular remodeling in diabetes, the role of BAG3 in the excessive proliferation and migration of VSMCs and its molecular mechanism deserve further investigation.MethodsIn this study, BAG3 gene was manipulated in smooth muscle to acquire SM22αCre; BAG3FL/FL mice and streptozotocin (STZ) was used to simulate diabetes. Expression of proteins and aortic thickness of mice were detected by immunofluorescence, ultrasound and hematoxylin-eosin (HE) staining. Using human aorta smooth muscle cell line (HASMC), cell viability was measured by CCK-8 and proliferation was measured by colony formation experiment. Migration was detected by transwell, scratch experiments and Phalloidin staining. Western Blot was used to detect protein expression and Co-Immunoprecipitation (Co-IP) was used to detect protein interaction.ResultsIn diabetic vascular remodeling, AGEs could promote the interaction between BAG3 and signal transducer and activator of transcription 3 (STAT3), leading to the enhanced interaction between STAT3 and Janus kinase 2 (JAK2) and reduced interaction between STAT3 and extracellular signal-regulated kinase 1/2 (ERK1/2), resulting in accumulated p-STAT3(705) and reduced p-STAT3(727). Subsequently, the expression of matrix metallopeptidase 2 (MMP2) is upregulated, thus promoting the migration of VSMCs.ConclusionsBAG3 upregulates the expression of MMP2 by increasing p-STAT3(705) and decreasing p-STAT3(727) levels, thereby promoting vascular remodeling in diabetes. This provides a new orientation for the prevention and treatment of diabetic vascular remodeling.

The role of BAG3 in dilated cardiomyopathy and its association with Charcot-Marie-Tooth disease type 2.

Bcl2-associated athanogene 3 (BAG3) is a multifunctional cochaperone responsible for protein quality control within cells. BAG3 interacts with chaperones HSPB8 and Hsp70 to transport misfolded proteins to the Microtubule Organizing Center (MTOC) and degrade them in autophagosomes in a process known as Chaperone Assisted Selective Autophagy (CASA). Mutations in the second conserved IPV motif of BAG3 are known to cause Dilated Cardiomyopathy (DCM) by inhibiting adequate removal of non-native proteins. The proline 209 to leucine (P209L) BAG3 mutant in particular causes the aggregation of BAG3 and misfolded proteins as well as the sequestration of essential chaperones. The exact mechanisms of protein aggregation in DCM are unknown. However, the similar presence of insoluble protein aggregates in Charcot-Marie-Tooth disease type 2 (CMT2) induced by the proline 182 to leucine (P182L) HSPB1 mutant points to a possible avenue for future research: IPV motif. In this review, we summarize the molecular mechanisms of CASA and the currently known pathological effects of mutated BAG3 in DCM. Additionally, we will provide insight on the importance of the IPV motif in protein aggregation by analyzing a potential association between DCM and CMT2.

BAG3 protects chondrocytes against lumbar facet joint osteoarthritis by regulating autophagy and apoptosis.

Bcl2-associated athanogene3 (BAG3) protein, mainly induced by stressful stimuli, has been confirmed to participate in apoptosis and autophagy. In recent studies, BAG3 has gradually become a key molecule in tumors. However, the role of BAG3 in the progression of lumbar facet joint osteoarthritis (FJOA) and whether it can regulate chondrocyte apoptosis and autophagy are still unknown. In both human and FJOA rat models, we observed an upregulation of BAG3 and apoptosis and autophagy-related proteins compared with healthy tissues. Then, we established the chondrocytes injury model in vitro by using IL-1β to stimulate human SW1353 cells. Western blot analysis data showed significant expression of BAG3, apoptosis, and autophagy-related proteins in SW1353 cells. Finally, by knocking down and overexpressing BAG3, we discovered possible anti-apoptotic and autophagy-promoted effects of BAG3 in FJOA through various experimental methods. This study demonstrated that BAG3 actively participates in regulating chondrocyte apoptosis and autophagy in FJOA and may be a highly interesting targetfor pharmacological interventions.

The role of BAG3 in health and disease: A "Magic BAG of Tricks".

The multi-domain structure of Bcl-2-associated athanogene 3 (BAG3) facilitates its interaction with many different proteins that participate in regulating a variety of biological pathways. After revisiting the BAG3 literature published over the past ten years with Citespace software, we classified the BAG3 research into several clusters, including cancer, cardiomyopathy, neurodegeneration, and viral propagation. We then highlighted recent key findings in each cluster. To gain greater insight into the roles of BAG3, we analyzed five different published mass spectrometry data sets of proteins that co-immunoprecipitate with BAG3. These data gave us insight into universal, as well as cell-type-specific BAG3 interactors in cancer cells, cardiomyocytes, and neurons. Finally, we mapped variable BAG3 SNPs and also mutation data from previous publications to further explore the link between the domains and function of BAG3. We believe this review will provide a better understanding of BAG3 and direct future studies towards understanding BAG3 function in physiological and pathological conditions.

Abstract MP101: Functional Maintenance of the Sarcomere Requires Bag3-dependent Autophagy

Bcl2-associated athanogene-3 (BAG3) is a pro-autophagy co-chaperone highly expressed in the heart. Clinical studies show BAG3 haploinsufficiency and mutations are associated with heart failure (HF). However, these studies are largely observational and fail to go beyond the observed phenotype and study mechanism. One study in neonatal myocytes suggests a role for BAG3 in structural maintenance of the sarcomere. However, the structural and functional significance of BAG3 in adult myocytes is not known. We found that myofilament BAG3 expression decreases in human heart failure and is associated with impaired myofilament force-generating capacity (F max ). To assess whether rescuing BAG3 levels could restore function, we used a mouse model of HF and treated with BAG3 gene therapy via AAV9. Myofilament function was assessed in skinned cardiomyocytes by force-calcium relationship. HF mice experienced a reduction in F max , but this was fully restored to sham levels by BAG3 gene therapy.To explore mechanism, we used mass spectrometry to identify the BAG3-interactome at the myofilament and found heat shock proteins (HSP) 70 and B8 among the top hits. Immunofluorescence further showed that HSP70, B8, and BAG3 each localized to the sarcomere z-disk. This BAG3-Hsp complex had previously been described to promote ubiquitin-dependent autophagy in skeletal muscle. Notably, in both human HF samples and in the mouse HF model, myofilament ubiquitin levels increased significantly. However, BAG3 gene therapy in HF reduces ubiquitin levels and restores autophagy flux. This suggests that BAG3 serves a role in proteostasis for the sarcomere, which may explain the functional effect of BAG3 gene therapy.To further explore the impact of the BAG3/HSP complex on myofilament function, we used a mouse model with the P209L BAG3 mutation, which had previously been described to disrupt client processing by the complex. We found cardiomyocytes from P209L mice had significantly reduced F max and elevated myofilament ubiquitin levels, suggesting BAG3-dependent autophagy is required to maintain function. Together, our data identify a functional role for BAG3 at the sarcomere and indicate BAG3-mediated autophagy is an important mechanism for maintaining myofilament proteostasis.

Abstract 909: Determining the Role of the Co-chaperone BAG3 at the Cardiac Myofilament

Bcl-2-associated athanogene 3 (BAG3) is an adaptor protein often associated with protein quality control. In the heart, decreased BAG3 expression is linked to dilated cardiomyopathy. Mutations to BAG3 can result in myofibrillar disarray, indicating a possible role at the sarcomere. One previous study found BAG3 has a role in maintaining sarcomere structure, however this was in neonatal myocytes. Whether BAG3 has any role in the myofilament of the adult myocyte is unknown. To determine if BAG3 is present in the cardiac sarcomere and identify the myofilament-BAG3 interactome, we used co-immunoprecipitation of the myofilament fraction from mouse LV and mass spectrometry. This revealed a robust interaction of BAG3 with the myofilament. Furthermore, BAG3 interacted with a heat shock protein (HSPB8) known to assist in the removal of protein aggregates, which are abundant after MI. To determine the mechanistic role of BAG3, we overexpressed the protein in mice hearts using adeno-associated virus 9. At baseline, overexpression caused a slight decrease in skinned myocyte calcium sensitivity compared to WT. This effect may be due to BAG3’s interaction with β-adrenergic receptors, which can cause PKA phosphorylation of thin filament proteins known to reduce calcium sensitivity. Preliminary data support this. We then exposed these mice to left anterior descending coronary artery ligation to induce MI. Compared to shams, MI decreased maximum calcium activated force (F max ) in the WT mice, but BAG3 overexpression restored F max to near sham levels. We also studied the functional impact of a known disease-causing mutation discovered in humans, P209L. Transgenic overexpression of P209L BAG3 had no effect on F max , calcium sensitivity, or cooperativity, suggesting the mutation may not interfere with domains in BAG3 required for its myofilament role. This work is the first to show a functional role for BAG3 in the adult myofilament. However, our findings reveal a complex interaction in which the effect of altered BAG3 expression varies with context. The P209L mutation findings suggest that not all BAG3 mutations have shared phenotypes, a conclusion supported by recent clinical work. Further research is necessary to understand BAG3’s myofilament role in health and disease.

BAG3 contributes to HGF-mediated cell proliferation, migration, and invasion via the Egr1 pathway in gastric cancer.

Bcl2-associated athanogene 3 (BAG3) is elevated in several types of cancers. However, the role of BAG3 in progression of gastric cancer is unknown. Therefore, the present study aims to find out the role of BAG3 in hepatocyte growth factor (HGF)-mediated tumor progression and the molecular mechanisms by which HGF regulates BAG3 expression. BAG3 mRNA and protein were measured using reverse transcription polymerase chain reaction and Western blot in the 2 human gastric cancer cell lines, NUGC3 and MKN28, treated with or without HGF. The effects of BAG3 knockdown on cell proliferation, cell invasion, and apoptosis were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the in vitro 2-chamber invasion assay, and flow cytometry in BAG3 short hairpin RNA (shRNA)-transfected cells and control cells. The signaling pathways involved in BAG3 that are regulated by HGF were analyzed. The chromatin immunoprecipitation assay was used to determine binding of Egr1 to the BAG3 promoter. BAG3 mRNA and protein levels were increased following treatment with HGF. HGF-mediated BAG3 upregulation increased cell proliferation and cell invasion; however, it decreased apoptosis. HGF-mediated BAG3 upregulation is regulated by an ERK and Egr1-dependent pathway. BAG3 may have an important role in HGF-mediated cell proliferation and metastasis in gastric cancer through an ERK and Egr1-dependent pathway. This pathway may provide novel therapeutic targets and provide information for further identification of other targets of therapeutic significance in gastric cancer.

Regulation of angiotensin II-induced B-cell lymphoma-2-associated athanogene 3 expression in vascular smooth muscle cells.

Previous studies have demonstrated that angiotensin II (Ang II) is involved in the process of atherosclerosis and vascular restenosis through its proinflammatory effect. Bcl‑2‑associated athanogene 3 (BAG3) had been suggested to be associated with proliferation, migration and invasion in many types of tumor. However, the role of BAG3 among the proliferative process of vascular smooth muscle cells (VSMCs) induced by Ang II, to the best of our knowledge, remains to be investigated. The present study demonstrated that in growth‑arrested VSMCs, Ang II‑induced VSMC proliferation, accompanied by increased BAG3 mRNA and protein expression levels in a dose‑ and time‑dependent manner. BAG3 expression levels were measured in VSMCs treated in the presence or absence of Ang II. The proliferation of VSMCs was assessed using manual cell counting and Cell Counting kit‑8 assays. mRNA and protein expression levels of BAG3, Toll‑like receptor 4 (TLR4), proliferating cell nuclear antigen, nuclear factor (NF)‑κB p65, smooth muscle protein22α and phosphorylated NF‑κB p65 were assessed by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. In non‑transfected or scramble short hairpin RNA (shRNA)‑transfected VSMCs cells, Ang II significantly induced VSMC proliferation. However, this Ang II‑induce proliferation was attenuated when BAG3 was silenced, suggesting that inhibition of BAG3 may somehow reduce proliferation in Ang II‑induced VSMCs. Furthermore, the TLR4/NF‑κB p65 signaling pathway was involved in BAG3 gene upregulation. In conclusion, to the best of our knowledge, the present study demonstrated for the first time that inhibition of BAG3 attenuates cell proliferation. Furthermore, Ang II induced VSMCs proliferation through regulation of BAG3 expression via the TLR4/NF‑κB p65 signaling pathway.

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy.

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2-associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

BAG3 regulates contractility and Ca2 + homeostasis in adult mouse ventricular myocytes

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na+–K+–ATPase and L-type Ca2+ channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca2+ channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca2+]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ content but not Na+/Ca2+ exchange current (INaCa) or SR Ca2+ uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca2+ entry molecules and other important excitation–contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca2+ channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure.