Circ-sh3rf3/GATA-4/miR-29a regulatory axis in fibroblast-myofibroblast differentiation and myocardial fibrosis.

The transdifferentiation from cardiac fibroblasts to myofibroblasts is an important event in the initiation of cardiac fibrosis. However, the underlying mechanism is not fully understood. Circ-sh3rf3 (circular RNA SH3 domain containing Ring Finger 3) is a novel circular RNA which was induced in hypertrophied ventricles by isoproterenol hydrochloride, and our work has established that it is a potential regulator in cardiac hypertrophy, but whether circ-sh3rf3 plays a role in cardiac fibrosis remains unclear, especially in the conversion of cardiac fibroblasts into myofibroblasts. Here, we found that circ-sh3rf3 was down-regulated in isoproterenol-treated rat cardiac fibroblasts and cardiomyocytes as well as during fibroblast differentiation into myofibroblasts. We further confirmed that circ-sh3rf3 could interact with GATA-4 proteins and reduce the expression of GATA-4, which in turn abolishes GATA-4 repression of miR-29a expression and thus up-regulates miR-29a expression, thereby inhibiting fibroblast-myofibroblast differentiation and myocardial fibrosis. Our work has established a novel Circ-sh3rf3/GATA-4/miR-29a regulatory cascade in fibroblast-myofibroblast differentiation and myocardial fibrosis, which provides a new therapeutic target for myocardial fibrosis.

miR-29a attenuates cardiac hypertrophy through inhibition of PPARδ expression.

Although cardiac hypertrophy is widely recognized as a risk factor that leads to cardiac dysfunction and, ultimately, heart failure, the complex mechanisms underlying cardiac hypertrophy remain incompletely characterized. The nuclear receptor peroxisome proliferator-activated receptor δ (PPARδ) is involved in the regulation of cardiac lipid metabolism. Here, we describe a novel PPARδ-dependent molecular cascade involving microRNA-29a (miR-29a) and atrial natriuretic factor (ANF), which is reactivated in cardiac hypertrophy. In addition, we identify a novel role of miR-29a, in which it has a cardioprotective function in isoproterenol hydrochloride-induced cardiac hypertrophy by targeting PPARδ and downregulating ANF. Finally, we provide evidence that miR-29a reduces the isoproterenol hydrochloride-induced cardiac hypertrophy response, thereby underlining the potential clinical relevance of miR-29a in which it may serve as a potent therapeutic target for heart hypertrophy treatment.

EGF is required for cardiac differentiation of P19CL6 cells through interaction with GATA-4 in a time- and dose-dependent manner.

The regulation of cardiac differentiation is critical for maintaining normal cardiac development and function. The precise mechanisms whereby cardiac differentiation is regulated remain uncertain. Here, we have identified a GATA-4 target, EGF, which is essential for cardiogenesis and regulates cardiac differentiation in a dose- and time-dependent manner. Moreover, EGF demonstrates functional interaction with GATA-4 in inducing the cardiac differentiation of P19CL6 cells in a time- and dose-dependent manner. Biochemically, GATA-4 forms a complex with STAT3 to bind to the EGF promoter in response to EGF stimulation and cooperatively activate the EGF promoter. Functionally, the cooperation during EGF activation results in the subsequent activation of cyclin D1 expression, which partly accounts for the lack of additional induction of cardiac differentiation by the GATA-4/STAT3 complex. Thus, we propose a model in which the regulatory cascade of cardiac differentiation involves GATA-4, EGF, and cyclin D1.

EGF Protects Cells Against Dox-Induced Growth Arrest Through Activating Cyclin D1 Expression.

It has been reported that the antitumor drug doxorubicin (Dox) exerts its toxic effects via GATA-4 depletion and that over-expression of GATA-4 reverses Dox-induced toxicity and apoptosis; however, the precise mechanisms remain unclear. In this study, we observed, for the first time, that EGF protects cells against Dox-mediated growth arrest, G2/M-phase arrest, and apoptosis. Additionally, EGF expression was down-regulated in Dox-treated cells and up-regulated in GATA-4 over-expressing cells. Utilizing real-time PCR and western blotting analysis, we found that the expression of the cell cycle-associated protein cyclin D1 was inhibited in GATA-4-silenced cells and Dox-treated cells and was enhanced in GATA-4 over-expressing cells and EGF-treated cells. Furthermore, EGF treatment reversed the inhibited expression of cyclin D1 that was mediated by GATA-4 RNAi or Dox. Our results indicate that EGF, as a downstream target of Dox, may be involved in Dox-induced toxicity as well as in the protective role of GATA-4 against toxicity induced by Dox via regulating cyclin D1 expression, which elucidates a new molecular mechanism of Dox toxicity with important clinical implications.

Insulin induces C2C12 cell proliferation and apoptosis through regulation of cyclin D1 and BAD expression.

Insulin is a secreted peptide hormone identified in human pancreas to promote glucose utilization. Insulin has been observed to induce cell proliferation and myogenesis in C2C12 cells. The precise mechanisms underlying the proliferation of C2C12 cells induced by insulin remain unclear. In this study, we observed for the first time that 10 nM insulin treatment promotes C2C12 cell proliferation. Additionally, 50 and 100 nM insulin treatment induces C2C12 cell apoptosis. By utilizing real-time PCR and Western blotting analysis, we found that the mRNA levels of cyclinD1 and BAD are induced upon 10 and 50 nM/100 nM insulin treatment, respectively. The similar results were observed in C2C12 cells expressing GATA-6 or PPARα. Our results identify for the first time the downstream targets of insulin, cyclin D1, and BAD, elucidate a new molecular mechanism of insulin in promoting cell proliferation and apoptosis.

Insulin induces proliferation and cardiac differentiation of P19CL6 cells in a dose-dependent manner.

Insulin is a peptide hormone produced by beta cells of the pancreas. The roles of insulin in energy metabolism have been well studied, with most of the attention focused on glucose utilization, but the roles of insulin in cell proliferation and differentiation remain unclear. In this study, we observed for the first time that 10 nmol/L insulin treatment induces cell proliferation and cardiac differentiation of P19CL6 cells, whereas 50 and 100 nmol/L insulin treatment induces P19CL6 cell apoptosis and blocks cardiac differentiation of P19CL6 cells. By using real-time polymerase chain reaction (PCR) and Western blotting analysis, we found that the mRNA levels of cyclin D1 and α myosin heavy chain (α-MHC) are induced upon 10 nmol/L insulin stimulation and inhibited upon 50/100 nmol/L insulin treatment, whereas the mRNA levels of BCL-2-antagonist of cell death (BAD) exists a reverse trend. The similar results were observed in P19CL6 cells expressing GATA-6 or peroxisome proliferator-activated receptor α (PPARα). Our results identified the downstream targets of insulin, cyclin D1, BAD, α-MHC, and GATA-4, elucidate a novel molecular mechanism of insulin in promoting cell proliferation and differentiation.

miR-200b targets GATA-4 during cell growth and differentiation.

GATA-4 is an important transcription factor involved in several developmental processes of the heart, such as cardiac myocyte proliferation, differentiation and survival. The precise mechanisms underlying the regulation of GATA-4 remain unclear, this is especially true for the mechanisms that mediate the post-transcriptional regulation of GATA-4. Here, we demonstrate that miR-200b, a member of the miR-200 family, is a critical regulator of GATA-4. Overexpression of miR-200b leads to the downregulation of GATA-4 mRNA and a decrease in GATA-4 protein levels. Moreover, miR-200b not only inhibits cell growth and differentiation but also reverses the growth response mediated by GATA-4, whereas depletion of miR-200b leads to a slight reversal of the anti-growth response achieved by knocking down endogenous GATA-4. More importantly, the cell cycle-associated gene cyclin D1, which is a downstream target of GATA-4, is also regulated by miR-200b. Thus, miR-200b targets GATA-4 to downregulate the expression of cyclin D1 and myosin heavy chain (MHC), thereby regulating cell growth and differentiation.

SUMOylation of AhR modulates its activity and stability through inhibiting its ubiquitination.

Aryl hydrocarbon receptor (AhR) is a transcription factor that belongs to the basic helix-loop-helix (bHLH) Per-Arnt-Sim homology domain (PAS) family. AhR can be activated by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (2, 3, 7, 8-TCDD) and once activated, it promotes the abnormal expression of cytochrome P450, leading to several diseases, including cancer. In this study, we showed that AhR is subjected to post-translational modification by SUMOylation and this modification could be reversed by SENP1. Two SUMOylation sites were identified, one in the bHLH domain (K63) and the other in the TAD domain (K510) of AhR. Substitution of either K63 or K510 with arginine resulted in reduced SUMOylation for AhR. Treatment of MCF-7 cells with TCDD led to a reduced level of SUMOylated AhR in a time-dependent manner, and this occurred mainly in the nucleus. SUMOylation of AhR enhanced its stability through inhibiting its ubiquitination. Moreover, SUMOylation also repressed the transactivation activity of AhR and this could be reversed by TCDD. These results suggested that SUMOylation of AhR might play an important role in the regulation of its function, and TCDD may activate the transcriptional activity of AhR through downregulating its SUMOylation.

GSK-3ß-mediated activation of NLRP3 inflammasome leads to pyroptosis and apoptosis of rat cardiomyocytes and fibroblasts.

We previously demonstrated that GSK-3ß mediates NLRP3 inflammasome activation and IL-1ß production in cardiac fibroblasts (CFs) after myocardial infarction (MI). In this study, we show how GSK-3ß-mediated activation of the NLRP3 inflammasome/caspase-1/IL-1ß pathway leads to apoptosis and pyroptosis of cardiomyocytes (CMs) and CFs. Administration of lipopolysaccharide (LPS)/ATP to primary newborn rat cardiac fibroblasts (RCFs) led to increase in proteins of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, IL-1ß, and IL-18. Additionally, the expression of caspase-3 and N-terminal fragments of gasdermin D (N-GSDMD) and the Bax/Bcl-2 ratio increased. Administration of the GSK-3ß inhibitor SB216763 reduced the levels of apoptosis- and pyroptosis-related proteins regulated by NLRP3 inflammasome activation in RCFs. Next, we transferred the culture supernatant of LPS/ATP-treated RCFs to in vitro primary newborn rat cardiomyocytes (RCMs). The results showed that SB216763 attenuate the upregulation of the ratios of Bax/Bcl-2 and the expression of caspase-3 and N-GSDMD in RCMs. Direct stimulation of RCMs and H9c2 cells with recombinant rat IL-1ß increased the p-GSK-3ß/GSK-3ß and Bax/Bcl-2 ratios and the expression of caspase-3 and N-GSDMD, while both SB216763 and TLR1 (an IL-1ß receptor inhibitor) markedly reduced these effects, as assessed using propidium iodide positive staining and the lactate dehydrogenase release assay. The caspase-11 inhibitor wedelolactone decreased the expression level of N-GSDMD but did not alter the p-GSK-3ß/GSK-3ß ratio. Lastly, we established a Sprague-Dawley rat MI model to confirm that SB216763 diminished the increase in caspase-3 and N-GSDMD expression and the Bax/Bcl-2 ratio in the ischemic area. These data demonstrate that GSK-3ß regulates apoptosis and pyroptosis of RCMs and RCFs due to NLRP3 inflammasome activation in RCFs.

A GRIP-1-EZH2 switch binding to GATA-4 is linked to the genesis of rhabdomyosarcoma through miR-29a.

Terminal differentiation failure is an important cause of rhabdomyosarcoma genesis, however, little is known about the epigenetic regulation of aberrant myogenic differentiation. Here, we show that GATA-4 recruits polycomb group proteins such as EZH2 to negatively regulate miR-29a in undifferentiated C2C12 myoblast cells, whereas recruitment of GRIP-1 to GATA-4 proteins displaces EZH2, resulting in the activation of miR-29a during myogenic differentiation of C2C12 cells. Moreover, in poorly differentiated rhabdomyosarcoma cells, EZH2 still binds to the miR-29a promoter with GATA-4 to mediate transcriptional repression of miR-29a. Interestingly, once re-differentiation of rhabdomyosarcoma cells toward skeletal muscle, EZH2 was dispelled from miR-29a promoter which is similar to that in myogenic differentiation of C2C12 cells. Eventually, this expression of miR-29a results in limited rhabdomyosarcoma cell proliferation and promotes myogenic differentiation. We thus establish that GATA-4 can function as a molecular switch in the up- and downregulation of miR-29a expression. We also demonstrate that GATA-4 acts as a tumor suppressor in rhabdomyosarcoma partly via miR-29a, which thus provides a potential therapeutic target for rhabdomyosarcoma.

Circular RNA expression in isoproterenol hydrochloride-induced cardiac hypertrophy.

Circular RNA (circRNA) is a novel class of noncoding RNAs, and the roles of circRNAs in the development of cardiac hypertrophy remain to be explored. Here, we investigate the potential roles of circRNAs in cardiac hypertrophy. By circRNA sequencing in left ventricular specimens collected from 8-week-old mice with isoproterenol hydrochloride-induced cardiac hypertrophy, we found 401 out of 3323 total circRNAs were dysregulated in the hypertrophic hearts compared with the controls. Of these, 303 circRNAs were upregulated and 98 were downregulated. Moreover, the GO and KEGG analyses revealed that the majority of parental gene of differentially expressed circRNAs were not only related to biological process such as metabolic process and response to stimulus, but also related to pathway such as circulatory system and cardiovascular diseases. On the other hand, total 1974 miRNAs were predicted to binding to these differentially expressed circRNAs, and the possible target mRNAs of those miRNAs were also predicted and analyzed in terms of functional annotation. Finally, we identified that ANF and miR-23a are downstream targets of circRNA wwp1, suggesting that circRNA wwp1 exerts inhibitory roles of cardiac hypertrophy via down-regulation of ANF and miR-23a, which underlying the potential mechanisms whereby circRNA regulates cardiac hypertrophy.

Induction of the CLOCK gene by E2-ERα signaling promotes the proliferation of breast cancer cells.

Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.

Transcription factor GATA-6 recruits PPARα to cooperatively activate Glut4 gene expression.

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear hormone receptor that regulates energy metabolism, but its precise mechanisms remain unknown. Here, we demonstrate that the PPARα agonist fenofibrate activated expression of the glucose transporter Glut4. Moreover, PPARα was associated with the Glut4 promoter through GATA sites upon fenofibrate stimulation in cardiomyocytes. This occupancy is achieved through an interaction between amino acids 1-136 of PPARα with amino acids 276-443 of the cardiac transcription factor GATA-6. In addition, the interaction of PPARα with GATA-6 activated Glut4 gene expression, improved glucose consumption, and enhanced activity of mitochondrial citrate synthase in C2C12 myoblasts; both mutants of PPARα (1-101 aa) and GATA-6 (227-331 aa) were unable to cooperate in Glut4 activation. Thus, GATA-6 is an important component of the transcription network required for energy metabolism mediated by PPARα, and these findings provide a molecular basis for understanding the role of GATA-6 proteins in muscle development and disease.

Improvement of myocardial lipid accumulation and prevention of PGC-1α induction by fenofibrate.

We recently demonstrated that fenofibrate induces the activities of citrate synthase and NADH oxidase in cardiac mitochondria. To further determine the molecular mechanisms underlying fenofibrate action, 8-week-old mice were administered fenofibrate (100 mg/kg/day) for 7 and 14 days, and the expression of genes involved in cardiac mitochondrial function, such as nuclear respiratory factor 1 transcript variant 2 (NRF-1-L) and 6 (NRF-1-S), mitochondrial outer membrane protein 40 (Tom40), lipoic acid synthetase (Lias), cytochrome b, medium-chain acyl-coenzyme A dehydrogenase (MCAD) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) was determined. Expression of PGC-1α, a key regulator of the entire fatty acid oxidation system, was significantly downregulated after 14 days of fenofibrate administration. Moreover, ventricular triglycerides were also accumulated following 14 days of fenofibrate administration. Thus, fenofibrate functions to improve myocardial lipid accumulation and to prevent PGC-1α induction, which is crucial for understanding the molecular mechanisms underlying fenofibrate action on the heart.

Effects of Doxorubicin and Fenofibrate on the activities of NADH oxidase and citrate synthase in mice.

Doxorubicin (Dox) has widely been used as an anticancer drug, but its use is limited by serious toxicity to the heart, kidney and liver. Mitochondrial dysfunction is one of the potential mechanisms of toxicity but not fully understood. Fenofibrate, one of the peroxisome proliferator-activated receptor-alpha (PPARα) ligands, is involved in lipid metabolism which takes place primarily in the mitochondria, so mitochondrial function may be affected by fenofibrate. Therefore, we investigated the effects of DOX and fenofibrate on activities of both mitochondrial citrate synthase and NADH oxidase, which are marker enzymes in the tricarboxylic acid (TCA) cycle and a measure of the complex I-III-IV activity in electron transport chain, respectively. Dox (15 mg/kg) and/or fenofibrate (100 mg/kg/day) were administered to mice for 3 or 14 days, and the activities of citrate synthase and NADH oxidase were measured. Our study showed that Dox significantly inhibits the activity of citrate synthase while fenofibrate induces the activity. Similar to citrate synthase, NADH oxidase activity was also induced by fenofibrate except in spleen but inhibited by Dox except in the heart and liver. Furthermore, fenofibrate not only protects citrate synthase activity from Dox-induced toxicity in the ventricle but also significantly rescues NADH oxidase activity in the kidney. These results reveal the actions of fenofibrate and Dox on the mitochondria, and the underlying mechanism may be related to the toxicity of Dox, which has clinical implications in the side effects of Dox treatment by modulation of mitochondrial function.

Cooperative activation of atrial naturetic peptide promoter by dHAND and MEF2C.

An intricate array of cell-specific multiprotein complexes participate in programs of cell-specific gene expression through combinatorial interaction with different transcription factors and cofactors. The dHAND basic helix-loop-helix (bHLH) transcription factor, which is essential for heart development and extra embryonic structures, is thought to regulate cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted transcription factors such as GATA4 and NKX2.5. Here, we determine that dHAND also interacts with the myocyte enhancer binding factor-2c (MEF2C) protein, which belongs to MADS-box transcription factors and is essential for heart development. dHAND and MEF2C synergistically activated expression of the atrial naturetic peptide gene (ANP) in transfected HeLa cells. GST-pulldown and immunoprecipitation assay demonstrate that full-length MEF2C protein is able to interact with dHAND in vitro and in vivo, just like MEF2A and bHLH transcription factors MyoD in skeletal muscle cells. In addition, electrophoretic mobility shift assays (EMSAs) demonstrate that MEF2C and dHAND do not influence each other's DNA binding activity. Using chromatin immunoprecipitation (ChIP) analysis in H9c2 cells we show that dHAND interact with MEF2C to form protein complex and bind A/T sequence in promoter of ANP. Taken together with previous observations, these results suggest the existence of large multiprotein transcriptional complex with core DNA binding proteins that physically interact with other transcriptional factors to form favorable conformation to potentiate transcription.

Cooperative interaction between the basic helix-loop-helix transcription factor dHAND and myocyte enhancer factor 2C regulates myocardial gene expression.

Cardiac-restricted transcription factors dHAND and myocyte enhancer factor 2C are expressed in the developing heart and activate several cardiac promoters. However, their regulatory mechanisms are still to be understood. To elucidate their exact regulatory functions, we have developed an RNA interference strategy to specifically inhibit dHAND and myocyte enhancer factor 2C protein production in H9c2 cells, which are derived from rat embryonic heart. Expression of endogenous cardiac genes atrial natriuretic peptide and alpha-myosin heavy chain was down-regulated in H9c2 cells lacking both dHAND and myocyte enhancer factor 2C, indicating that these factors are required for the maintenance of the cardiac genetic program. Consistent with these, expression of atrial natriuretic peptide and alpha-myosin heavy chain was up-regulated in H9c2 cells, which overexpressed dHAND and myocyte enhancer factor 2C. In addition, dHAND and myocyte enhancer factor 2C interact to synergistically activate atrial natriuretic peptide and alpha-myosin heavy chain transcription. Furthermore, chromatin immunoprecipitation analysis in H9c2 cells treated with phenylephrine showed that dHAND and myocyte enhancer factor 2C protein complex bind to the A/T sequence on atrial natriuretic peptide promoter. Taken together, these results not only suggest that the complex cis-trans interaction of dHAND, myocyte enhancer factor 2C, and the target gene may fine-tune gene expression in cardiac myocytes but also provide a molecular paradigm to elucidate the mechanisms of action of dHAND and myocyte enhancer factor 2C in the developing heart.