Min-Ji Song

Histone Deacetylase 3 and 4 Complex Stimulates the Transcriptional Activity of the Mineralocorticoid Receptor.

Histone deacetylases (HDACs) act as corepressors in gene transcription by altering the acetylation of histones, resulting in epigenetic gene silencing. We previously reported that HDAC3 acts as a coactivator of the mineralocorticoid receptor (MR). Although HDAC3 forms complexes with class II HDACs, their potential role in the transcriptional activity of MR is unclear. We hypothesized that HDAC4 of the class II family stimulates the transcriptional activity of MR. The expression of MR target genes was measured by quantitative real-time PCR. MR and RNA polymerase II recruitment to promoters of MR target genes was analyzed by chromatin immunoprecipitation. The association of MR with HDACs was investigated by co-immunoprecipitation. MR acetylation was determined with an anti-acetyl-lysine antibody after immunoprecipitation with an anti-MR antibody. Among the class II HDACs, HDAC4 interacted with both MR and HDAC3 after aldosterone stimulation. The nuclear translocation of HDAC4 was mediated by protein kinase A (PKA) and protein phosphatases (PP). The transcriptional activity of MR was significantly decreased by inhibitors of PKA (H89), PP1/2 (calyculin A), class I HDACs (MS-275), but not class II HDACs (MC1568). MR acetylation was increased by H89, calyculin A, and MS-275, but not by MC1568. Interaction between MR and HDAC3 was significantly decreased by H89, calyculin A, and HDAC4 siRNA. A non-genomic effect of MR via PKA and PP1/2 induced nuclear translocation of HDAC4 to facilitate the interaction between MR and HDAC3. Thus, we have uncovered a crucial role for a class II HDAC in the activation of MR-dependent transcription.

Effects of 3',4'-dihydroxyflavonol on vascular contractions of rat aortic rings

1. 3′,4′‐Dihydroxyflavonol (DiOHF) is an effective vasodilator with anti‐oxidant activity. The aim of the present study was to elucidate the effects of DiOHF on vascular contractions.

Flavone Attenuates Vascular Contractions by Inhibiting RhoA/Rho Kinase Pathway

Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300 microM) or vehicle. We determined the phosphorylation level of the myosin light chain (MLC(20)), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of MLC(20) phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619-or NaF-induced phosphorylation of MYPT1(Thr855) and CPI17(Thr38), the downstream effectors of Rho-kinase. In regards to the Ca(2+)-free solution, flavone inhibited the phosphorylation of MYPT1(Thr855) and CPI17(Thr38), as well as vascular contractions induced by U46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.

A ROLE FOR RHO-KINASE IN Ca2+-INDEPENDENT CONTRACTIONS INDUCED BY PHORBOL-12,13-DIBUTYRATE

1 Phorbol‐12,13‐dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca2+‐depleted solutions. In the present study, we tested the hypothesis that Rho‐kinase plays a role in Ca2+‐independent contractions induced by PDBu in vascular smooth muscles. 2 In Ca2+‐free solution, 0.1 and 1 µmol/L PDBu induced contraction and myosin light chain (MLC20) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs’ solution. Hydroxyfasudil (H1152; 1 µmol/L), an inhibitor of Rho‐kinase, but not ML7 (10 µmol/L), an inhibitor of myosin light chain kinase, inhibited Ca2+‐independent contractions induced by PDBu. 3 In Ca2+‐free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI‐17 (PKC‐potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI‐17 almost completely abolished by 1 µmol/L Ro31‐8220, an inhibitor of PKC. 4 In Ca2+‐free solution, PDBu increased the amount of GTP‐RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31‐8220, but not by the Rho kinase inhibitor H1152. 5 In conclusion, RhoA/Rho‐kinase plays an important role in Ca2+‐independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca2+‐independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP‐RhoA and subsequent phosphorylation of MYPT1 and CPI‐17.

Fluoride induces vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aortas

We hypothesized that fluoride induces vascular contraction through activation of the RhoA/Rho kinase pathway in isolated rat aortas. Rat aortic rings were mounted in organ baths and contracted with sodium fluoride (NaF). We measured the amount of GTP-RhoA as well as vascular tension. We also determined the level of phosphorylation of the myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17kDa (CPI17). In both physiological salt solution and Ca(2+)-free solution, NaF increased vascular tension and MLC(20) phosphorylation in dose-dependent manners. NaF increased not only phosphorylation level of MYPT1(Thr855) and CPI17(Thr38), but also the amount of GTP-RhoA. Both H1152 and Y27632, inhibitors of Rho kinase, but not Ro31-8220, an inhibitor of PKC, attenuated NaF-induced contraction and phosphorylation level of MLC(20), MYPT1(Thr855) and CPI17(Thr38). In conclusion, fluoride induces vascular contraction through activation of the RhoA/Rho kinase pathway.

Histone deacetylase inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats

CG200745 is a novel inhibitor of histone deacetylases (HDACs), initially developed for treatment of various hematological and solid cancers. Because it is water-soluble, it can be administered orally. We hypothesized that the HDAC inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in deoxycorticosterone acetate (DOCA)-induced hypertensive rats. For establishment of hypertension, 40 mg/kg of DOCA was subcutaneously injected four times weekly into Sprague-Dawley rats. All the rats used in this study including those in the sham group had been unilaterally nephrectomized and allowed free access to drinking water containing 1% NaCl. Systolic blood pressure was measured by the tail-cuff method. Blood chemistry including sodium, potassium, glucose, triglyceride, and cholesterol levels was analyzed. Sections of the heart were visualized after trichrome and hematoxylin and eosin stain. The expression of hypertrophic genes such as atrial natriuretic peptide A (Nppa) and atrial natriuretic peptide B (Nppb) in addition to fibrotic genes such as Collagen-1, Collagen-3, connective tissue growth factor (Ctgf), and Fibronectin were measured by quantitative real-time PCR (qRT-PCR). Injection of DOCA increased systolic blood pressure, heart weight, and cardiac fibrosis, which was attenuated by CG200745. Neither DOCA nor CG200745 affected body weight, vascular contraction and relaxation responses, and blood chemistry. Injection of DOCA increased expression of both hypertrophic and fibrotic genes, which was abrogated by CG200745. These results indicate that CG200745 attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats.

Lysine acetyltransferases cyclic adenosine monophosphate response element-binding binding protein and acetyltransferase p300 attenuate transcriptional activity of the mineralocorticoid receptor through its acetylation.

Acetylation of the mineralocorticoid receptor (MR) by inhibition of lysine deacetylases attenuates MRs transcriptional activity. However, the specific lysine acetyltransferases that are responsible for acetylation of the MR remain unknown. We hypothesized that the acetyltransferases cyclic adenosine monophosphate response element‐binding binding protein (CBP) and acetyltransferase p300 (p300) attenuate transcriptional activity of the MR through its acetylation. Expression of MR target genes was measured by quantitative real‐time polymerase chain reaction. Recruitment of MR and RNA polymerase II (Pol II) on promoters of target genes was analysed by chromatin immunoprecipitation. Acetylation of the MR was determined by western blot with an anti‐acetyl‐lysine antibody after immunoprecipitation with an anti‐MR antibody. In human embryonic kidney (HEK) 293 cells, overexpression of CBP or p300, but not p300/CBP‐associated factor, increased MR acetylation and decreased expression of MR target genes. The downregulation of target genes coincided with a decrease in the recruitment of MR and Pol II to specific hormone response elements. These results demonstrate that overexpression of CBP or p300 attenuates the transcriptional activity of the MR through its acetylation in HEK 293 cells. Our data provide strong evidence identifying CBP and p300 as lysine acetyltransferases responsible for the regulation of MR that may provide new therapeutic targets for the treatment of hypertension.

PS 10-13 HISTONE DEACETYLASE INHIBITION AMELIORATES HYPERGLYCEMIA THROUGH DOWN-REGULATION OF GLUCONEOGENESIS

Objective: Diabetes mellitus (DM) is a chronic disease manifested by hyperglycemia and dyslipidemia. Histone deacetylase (HDAC) inhibitors have been emerged as new pharmacological approaches in the control of DM. However, the roles for HDAC inhibition in controlling DM are still elusive. We hypothesized that HDAC inhibition ameliorates hyperglycemia through down-regulation of gluconeogenesis in type II diabetic rats. Design and Method: Male normoglycemic Long-Evans-Tokushima-Otsuka (LETO) and hyperglycemic Otsuka Long-Evans Tokushima Fatty (OLETF) rats aged 14 weeks were administered sodium valproate (VPA, 0.71% w/v) dissolved in drinking water for 20 weeks during which the levels of blood glucose were analyzed at 13 and 19 weeks. Blood chemistry such as glycated hemoglobin HbA1c, triglyceride and cholesterol were analyzed. The expression of gluconeogenic genes such as glucose 6-phosphatase, phosphoenolpyruvate carboxykinase, fructose-1,6-biphosphatase and pyruvate carboxylase was measured by quantitative real-time polymerase chain reaction. Acetylation of forkhead box protein O1 (FOXO1), which is a master transcription factor for gluconeogenic genes, was determined by western blot with an anti-acetyl-lysine antibody after immunoprecipitation with an anti-FOXO1 antibody. Results: The levels of blood glucose, HbA1c, triglyceride and cholesterol as well as body weight, were greater in OLETF rats than those in LETO rats. VPA administration attenuated weight gain, blood glucose, HbA1c, triglyceride and cholesterol, as well as the expression of gluconeogenic genes in OLETF rats but not in LETO rats. VPA increased acetylation of FOXO1, decreasing its transcription activity for gluconeogenic genes. Conclusions: These results suggest that HDAC inhibition ameliorates hyperglycemia through down-regulation of gluconeogenesis in type II diabetic rats.