scholarly journals Prostaglandin F2α Suppresses Rat Steroidogenic Acute Regulatory Protein Expression via Induction of Yin Yang 1 Protein and Recruitment of Histone Deacetylase 1 Protein

Endocrinology ◽  
2007 ◽  
Vol 148 (11) ◽  
pp. 5209-5219 ◽  
Author(s):  
Qiyuan Liu ◽  
Kathleen A. Merkler ◽  
Xiaohui Zhang ◽  
Mark P. McLean
Keyword(s):  
Protein Expression ◽  
Promoter Activity ◽  
Trichostatin A ◽  
Histone H3 ◽  
Prostaglandin F2α ◽  
Star Protein ◽  
Histone Deacetylase 1 ◽  
Yin Yang 1 ◽  
Steroidogenic Acute Regulatory ◽  
H3 Acetylation

Prostaglandin F2α (PGF2α) plays a pivotal role in ovarian luteolysis by inhibiting the expression of steroidogenic acute regulatory (StAR) protein, leading to a decrease in intracellular cholesterol transport and luteal steroid production. Previously we have demonstrated that the transcription factor Yin Yang 1 (YY1) bound to three regions in the StAR promoter in vitro and repressed promoter activity. This study further defined the YY1-mediated PGF2α effect on the inhibition of StAR protein expression through YY1 interaction with a single region in the StAR promoter in vivo. PGF2α consistently suppressed StAR mRNA and protein expression in cultured luteal cells in a dose-dependent manner. PGF2α also enhanced YY1 protein expression and binding to its cis-element in a time-dependent pattern that preceded the decline in StAR protein levels. The StAR promoter region bound by YY1 was also associated with histone deacetylase 1 (HDAC1). PGF2α treatment promoted HDAC1 binding to and suppressed the histone H3 acetylation in this region. On the contrary, YY1 knockdown decreased HDAC1 binding, increased histone H3 acetylation, enhanced StAR protein expression, and negated PGF2α effect on StAR protein expression. Luciferase assays showed that YY1 overexpression inhibited StAR promoter activity and the addition of a HDAC inhibitor, trichostatin A, abrogated the effect of YY1. Trichostatin A-treated luteal cells displayed increased StAR protein expression. These data indicate that PGF2α enhances a direct YY1/StAR promoter interaction and the recruitment of HDAC1 to the promoter, thereby preventing transcriptional activation of the StAR gene.

scholarly journals Inhibition of Cyclooxygenase-2 Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (8) ◽  
pp. 3368-3375 ◽  
Author(s):  
XingJia Wang ◽  
Matthew T. Dyson ◽  
Youngah Jo ◽  
Douglas M. Stocco
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Leydig Cells ◽  
Promoter Activity ◽  
Regulatory Gene ◽  
Steroid Production ◽  
Star Protein ◽  
Cox Inhibitor ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Abstract To study the mechanism for the regulatory effect of arachidonic acid (AA) on steroidogenesis, the role of cyclooxygenase (COX) in steroid production and steroidogenic acute regulatory (StAR) gene expression was investigated. Although stimulation with 0.05 mm dibutyryl cAMP (Bt2cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 μm of the COX inhibitor indomethacin increased StAR protein expression and progesterone production by 5.7-fold and 34.3-fold, respectively. In the presence of indomethacin, the level of Bt2cAMP required for maximal steroidogenesis was reduced from 1.0 mm to 0.25 mm. Similar results were obtained in studies on StAR promoter activity and in Northern blot analyses of StAR mRNA expression, suggesting that inhibition of COX activity enhanced StAR gene transcription. COX2 (an inducible isoform of COX) was constitutively detected in MA-10 cells. Although SC560, a selective COX1 inhibitor, did not affect steroidogenesis, the COX2 inhibitor NS398 significantly enhanced Bt2cAMP-stimulated StAR protein expression and steroid production. Overexpression of the COX2 gene in COS-1 cells significantly inhibited StAR promoter activity. The results of the present study suggest that inhibition of COX2 activity increases the sensitivity of steroidogenesis to cAMP stimulation in MA-10 Leydig cells.

scholarly journals Valproate Potentiates Androgen Biosynthesis in Human Ovarian Theca Cells

Endocrinology ◽  
2004 ◽  
Vol 145 (2) ◽  
pp. 799-808 ◽  
Author(s):  
Velen L. Nelson-DeGrave ◽  
Jessica K. Wickenheisser ◽  
Jennifer E. Cockrell ◽  
Jennifer R. Wood ◽  
Richard S. Legro ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Polycystic Ovary ◽  
Regulatory Protein ◽  
Histone H3 ◽  
Steroidogenic Acute Regulatory Protein ◽  
Ovary Syndrome ◽  
Theca Cells ◽  
Histone H3 Acetylation ◽  
Steroidogenic Acute Regulatory ◽  
H3 Acetylation

Abstract In patients with epilepsy, treatment with valproate (VPA) has been reported to be associated with polycystic ovary syndrome-like symptoms including weight gain, hyperandrogenemia, and hyperinsulinemia. We examined the effect of VPA on androgen biosynthesis in ovarian theca cells isolated from follicles of normal cycling women to determine whether the hyperandrogenemia reported with VPA treatment could be a result of direct effects of VPA on the ovary. In long-term cultures of theca cells treated for 72 h with sodium valproate (30–3000 μm), we observed an increase in basal and forskolin-stimulated dehydroepiandrosterone (DHEA), androstenedione, and 17α-hydroxyprogesterone production compared with control values. In contrast, low doses of VPA treatment (i.e. 30–300 μm) had no effect on basal and forskolin-stimulated progesterone production, whereas higher doses of VPA (1000–3000 μm) inhibited progesterone production. The most pronounced effect of VPA on androgen biosynthesis was observed in the dose range of 300-3000 μm, which represent therapeutic levels in the treatment of epilepsy and bipolar disorder. Western analyses demonstrated that VPA treatment increased both basal and forskolin-stimulated P450c17 and P450scc protein levels, whereas the amount of steroidogenic acute regulatory protein was unaffected. In transient transfection studies, VPA was found to increase P450 17α-hydroxylase and P450 cholesterol side-chain cleavage promoter activity, whereas steroidogenic acute regulatory protein promoter activity was unaffected. Consistent with the ability of VPA to act as a histone deacetylase (HDAC) inhibitor in other cell systems, VPA (500 μm) treatment was observed to increase histone H3 acetylation and P450 17α-hydroxylase mRNA accumulation. The HDAC inhibitor butyric acid (500 μm) similarly increased histone H3 acetylation and DHEA biosynthesis, whereas the VPA derivative valpromide (500 μm), which lacks HDAC inhibitory activity, had no effect on histone acetylation or DHEA biosynthesis. These data suggest that VPA-induced ovarian androgen biosynthesis results from changes in chromatin modifications (histone acetylation) that augment transcription of steroidogenic genes. These studies provide the first biochemical evidence to support a role for VPA in the genesis of polycystic ovary syndrome-like symptoms, and establish a direct link between VPA treatment and increased ovarian androgen biosynthesis.

scholarly journals Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells

2018 ◽  
Vol 19 (12) ◽  
pp. 4071 ◽  
Author(s):  
Mei-Chi Chang ◽  
Yunn-Jy Chen ◽  
Yun-Chia Lian ◽  
Bei-En Chang ◽  
Chih-Chia Huang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Viability ◽  
Butyric Acid ◽  
Root Canal ◽  
Histone H3 ◽  
Rankl Expression ◽  
Alp Activity ◽  
Histone H3 Acetylation ◽  
Apoptosis And Necrosis ◽  
H3 Acetylation

Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2–4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1–4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.

scholarly journals CREM confers cAMP responsiveness in human steroidogenic acute regulatory protein expression in NCI-H295R cells rather than SF-1/Ad4BP

2006 ◽  
Vol 191 (1) ◽  
pp. 327-337 ◽  
Author(s):  
Teruo Sugawara ◽  
Noriaki Sakuragi ◽  
Hisanori Minakami
Keyword(s):  
Promoter Activity ◽  
Activity Level ◽  
Expression Vectors ◽  
Camp Response Element ◽  
Star Protein ◽  
Protein Levels ◽  
Sirna Treatment ◽  
H295r Cells ◽  
Steroidogenic Acute Regulatory ◽  
Camp Induction

Steroidogenic acute regulatory (StAR) protein plays a critical role in steroid hormone synthesis. Tropic hormones induce human StAR gene expression by a cAMP-dependent pathway. Steroidogenic factor-1/adrenal-4-binding protein (SF-1/Ad4BP) plays an important role in the expression of human StAR gene. We investigated the mechanism of cAMP responsiveness in human StAR gene expression in NCI-H295R cells. The StAR promoter activity and protein levels in cells subjected to various treatments were examined. Anti-SF-1/Ad4BP IgG transfection treatment resulted in decreases in the basal StAR promoter activity and StAR protein levels, but did not affect cAMP-stimulated promoter activity and protein levels. The basal and cAMP-stimulated StAR promoter activity levels were reduced in SF-1/Ad4BP mutant (G35E)-transfected cells, but the cAMP induction of StAR promoter activity in response to 1 mM 8-Br-cAMP was not inhibited when G35E SF-1/Ad4BP mutant expression vectors were co-transfected with cAMP-response element-binding (CREB) expression vectors. Although the basal StAR mRNA expression and protein levels were decreased by SF-1/Ad4BP-siRNA treatment, the cAMP-stimulated StAR mRNA expression and protein levels did not change. The basal StAR promoter activity level was not decreased by cAMP-response element modulator (CREM)-siRNA treatment, but the cAMP-stimulated StAR promoter activity level, the magnitude of cAMP induction of StAR promoter, and the cAMP-stimulated StAR protein level were decreased. The cAMP induction of StAR promoter activity in cells was inhibited when S117ACREM mutant expressionvectors were transfected. We conclude that inhibition of the function of SF-1/Ad4BP does not reduce the cAMP induction of StAR promoter activity and protein level. CREM is needed to confer cAMP responsiveness in human StAR protein expression.

scholarly journals Regulation of Steroidogenesis and the Steroidogenic Acute Regulatory Protein by a Member of the cAMP Response-Element Binding Protein Family

2002 ◽  
Vol 16 (1) ◽  
pp. 184-199 ◽  
Author(s):  
Pulak R. Manna ◽  
Matthew T. Dyson ◽  
Darrell W. Eubank ◽  
Barbara J. Clark ◽  
Enzo Lalli ◽  
...  
Keyword(s):  
Family Member ◽  
Promoter Activity ◽  
Binding Protein ◽  
Camp Response Element Binding ◽  
Camp Response Element ◽  
Response Element ◽  
Star Protein ◽  
Element Binding Protein ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Abstract The mitochondrial phosphoprotein, the steroidogenic acute regulatory (StAR) protein, is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells through cAMP-dependent pathways. In many cases transcriptional induction by cAMP is mediated through the interaction of a cAMP response-element binding protein (CREB) family member with a consensus cAMP response element (CRE; 5′-TGACGTCA-3′) found in the promoter of target genes. The present investigation was carried out to determine whether a CRE-binding protein (CREB) family member [CREB/CRE modulator (CREM) family] was involved in the regulation of steroidogenesis and StAR protein expression. Transient expression of wild- type CREB in MA-10 mouse Leydig tumor cells further increased the levels of (Bu)2cAMP-induced progesterone synthesis, StAR promoter activity, StAR mRNA, and StAR protein. These responses were significantly inhibited by transfection with a dominant-negative CREB (A-CREB), or with a CREB mutant that cannot be phosphorylated (CREB-M1), the latter observation indicating the importance of phosphorylation of a CREB/CREM family member in steroidogenesis and StAR expression. The CREB/CREM-responsive region in the mouse StAR gene was located between −110 and −67 bp upstream of the transcriptional start site. An oligonucleotide probe (−96/−67 bp) containing three putative half-sites for 5′-canonical CRE sequences (TGAC) demonstrated the formation of protein-DNA complexes in EMSAs with recombinant CREB protein as well as with nuclear extracts from MA-10 or Y-1 mouse adrenal tumor cells. The predominant binding factor observed with EMSA was found to be the CREM protein as demonstrated using specific antibodies and RT-PCR analyses. The CRE elements identified within the− 96/−67 bp region were tested for cAMP responsiveness by generating mutations in each of the CRE half-sites either alone or in combination. Although each of the CRE sites contribute in part to the CREM response, the CRE2 appears to be the most important site as determined by EMSA and by reporter gene analyses. Binding specificity was further assessed using specific antibodies to CREB/CREM family members, cold competitors, and mutations in the target sites that resulted in either supershift and/or inhibition of these complexes. We also demonstrate that the inducible cAMP early repressor markedly diminished the endogenous effects of CREM on cAMP-induced StAR promoter activity and on StAR mRNA expression. These are the first observations to provide evidence for the functional involvement of a CREB/CREM family member in the acute regulation of trophic hormone-stimulated steroidogenesis and StAR gene expression.

scholarly journals Butyrate Stimulates Histone H3 Acetylation, RANKL Expression, But Inhibited Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells

2018 ◽  
Author(s):  
Mei-Chi Chang ◽  
Yunn-Jy Chen ◽  
Yun-Chia Lian ◽  
Bei-En Chang ◽  
Chi-Chia Huang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Butyric Acid ◽  
Root Canal ◽  
Biological Activities ◽  
Histone H3 ◽  
Immunofluorescent Staining ◽  
Rankl Expression ◽  
Histone H3 Acetylation ◽  
Apoptosis And Necrosis ◽  
H3 Acetylation

Butyric acid as a histone deacetylase (HDAC) inhibitor was produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on matrix and mineralization markers’ expression of MG-63 osteoblasts. Cell viability and proliferation were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/Annexin V flow cytometry. Protein and mRNA expression of OPG, and RANKL were analyzed by western blotting and RT-PCR. OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, MMP-2, osteonectin (SPARC), osteocalcin and osteopontin secretion into culture medium were measured by enzyme-linked immunosorbant assay. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and western blot. We found that butyrate induced morphologic changes of growing MG-63 cells, with bigger and flattened in appearance. Butyrate activated histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell number with no marked increase in apoptosis and necrosis. Butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas sRANKL level was below detection limit. Butyrate stimulated 8-isoprostane, MMP-2 and osteopontin secretion, but not procollagen I, osteonectin, osteocalcin in MG-63 cells. In conclusion, butyric acid generated by periodontal and root canal microorganisms may potentially induce bony destruction and impair bone repair by alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2, and osteopontin secretion, and affect cell proliferation. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis of periodontal and periapical destruction.

scholarly journals Cholesterol sulphate affects production of steroid hormones by reducing steroidogenic acute regulatory protein level in adrenocortical cells

2007 ◽  
Vol 195 (3) ◽  
pp. 451-458 ◽  
Author(s):  
Teruo Sugawara ◽  
Eiji Nomura ◽  
Nobuhiko Hoshi
Keyword(s):  
Gene Expression ◽  
Steroid Hormones ◽  
Protein Level ◽  
Promoter Activity ◽  
Adrenocortical Cells ◽  
Star Protein ◽  
Cell Extracts ◽  
H295r Cells ◽  
Steroidogenic Acute Regulatory ◽  
In Cells

Steroidogenic acute regulatory (StAR) protein plays a crucial role in the intramitochondrial movement of cholesterol, where P450 side chain cleavage enzyme resides. Cholesterol sulphate (CS), which is present ubiquitously in mammalian tissues, is not only a precursor of sulphated adrenal steroids but also an inhibitor of cholesterol biosynthesis. This study was designed to examine the biological roles of CS in steroidogenesis in adrenocortical cells. Human adrenocortical carcinoma H295R cells were cultured with various amounts of CS. To evaluate steroid hormone synthesis, pregnenolone production in cells was assayed. The amount of pregnenolone produced by H295R cells in culture medium, to which over 50 μg/ml CS was added, was significantly (P<0.05) decreased compared with that produced by control cells. Western blot analysis was performed to determine StAR protein level using whole cell extracts from cells. StAR protein level decreased when the concentration of CS in the medium was 50 μg/ml, whereas the level of glyceraldehyde-3-phosphate dehydrogenase did not change. To examine the mechanism by which StAR gene expression is controlled, we performed RT-PCR and measured promoter activity in cells transfected with pGL2 StAR reporter constructs. StAR mRNA level and promoter activity were decreased in cells. The decrease in StAR protein level is a result of the low StAR gene expression level. In conclusion, CS affects the production of steroid hormones by reducing StAR protein level in adrenocortical cells.

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