help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-1265
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nishida, H.
Right arrow Articles by Yamada, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nishida, H.
Right arrow Articles by Yamada, G.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*TESTOSTERONE
Endocrinology Vol. 149, No. 5 2090-2097
Copyright © 2008 by The Endocrine Society

Positive Regulation of Steroidogenic Acute Regulatory Protein Gene Expression through the Interaction between Dlx and GATA-4 for Testicular Steroidogenesis

Hisayo Nishida, Shinichi Miyagawa, Maxence Vieux-Rochas, Monica Morini, Yukiko Ogino, Kentaro Suzuki, Naomi Nakagata, Hueng-Sik Choi, Giovanni Levi and Gen Yamada

Center for Animal Resources and Development (H.N., S.M., Y.O., K.S., N.N., G.Y.), Graduate School of Medical and Pharmaceutical Sciences and the Global COE Research Program, Kumamoto University, Kumamoto 860-0811, Japan; Centre National de la Recherche Scientifique Unité Mixte de Recherche 5166-MNHN (M.V.-R., G.L.), Evolution des Régulations Endocriniennes, 75231 Paris, Cedex 05, France; Istituto Nazionale per la Ricerca sul Cancro (M.M.), 16132 Genova, Italy; and Hormone Research Center (H.-S.C.), School of Biological Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea

Address all correspondence and requests for reprints to: Gen Yamada, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan. E-mail: gensan{at}gpo.kumamoto-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Split hand/foot malformation (SHFM) is syndromic ectrodactyly often associated with mental retardation and/or craniofacial defects. Several clinical reports previously described urogenital dysplasia such as micropenis, hypospadias, and small testis in SHFM patients. Genetic lesions in the Dlx5 and Dlx6 (Dlx5/6) locus are associated with the human genetic disorder SHFM type 1. Although Dlx5/6 are expressed in the testis, their possible function of Dlx5/6 during testis differentiation has not been described. In this study, we show that Dlx5/6 are expressed in the fetal Leydig cells during testis development. We examined the effect of Dlx5 expression on the promoter activation of the steroidogenic acute regulatory protein (StAR) gene, which is essential for gonadal and adrenal steroidogenesis, in a Leydig cell line. Dlx5 efficiently activates the StAR promoter when GATA-4, another transcription factor essential for testicular steroidogenesis, was coexpressed. The transcriptional activation required the GATA-4-recognition element in the StAR promoter region and Dlx5 can physically interact with GATA-4. Furthermore, we herein show that the double inactivation of Dlx5 and Dlx6 in the mouse leads to decreased testosterone level and abnormal masculinization phenotype. These results suggest that Dlx5 and Dlx6 participate in the control of steroidogenesis during testis development. The findings of this study may open the way to analyze human congenital birth defects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS PLAY A crucial role in initiating and maintaining the expression of male sexual characteristics in mammals. Testosterone is synthesized and secreted by Leydig cells and subsequently converted to 5{alpha}-dihydrotestosterone, the biologically more potent androgen. During embryogenesis, these androgens participate in the control of the male organs development through binding to the androgen receptor, resulting in the masculinization of the external genitalia, Wolffian duct derivatives, and prostate (1, 2, 3, 4). In humans and mice, any defects along the pathway of testosterone production result in congenital disorders such as micropenis, hypospadias and cryptorchism (5, 6).

Steroidogenic acute regulatory protein (StAR) mediates the transport of cholesterol from the outer to the inner mitochondrial membrane, which is the rate-limiting step of steroid hormone biosynthesis in all steroidogenic cells (7, 8). In human, mutations of the StAR gene are associated with lipoid congenital adrenal hyperplasia characterized by impaired adrenal and gonadal steroid synthesis and male pseudohermaphroditism (9). Targeted disruption of StAR in the mouse results in a phenocopy of human lipoid congenital adrenal hyperplasia (10, 11). These findings clearly demonstrate the crucial role of StAR for the regulation of steroidogenesis. StAR expression is known to be influenced by trophic hormones, GnRH, thyroid hormone, growth factors, prostaglandins, and steroids (7, 8). The transcriptional regulation of the StAR gene is mediated by multiple upstream DNA elements with recognition motifs for sequence-specific transcription factors such as steroidogenic factor 1 (12, 13, 14, 15), CCAAT/enhancer binding proteins (14, 16, 17), Sp1 (specificity protein-1) (15, 18), cAMP response element-binding protein/cAMP response element modulator (19, 20), and Yin Yang 1 (21, 22). The zinc finger-containing transcription factor GATA-4 has also been suggested as a potent activator of the mouse StAR promoter (16, 17, 18). A recent report suggests that GATA-4 is required cell autonomously for the proper differentiation of fetal Leydig cells (23).

Dlx genes, the vertebrate homologs of Drosophila Distal-less, play critical roles in the regulation of distal appendage formation (24, 25, 26). Among the Dlx family of homeodomain transcription factors, Dlx5 and Dlx6 (Dlx5/6) are involved in the regulation of the subcortical forebrain, branchial arches, and limb development (27, 28). Split hand/foot malformation (SHFM) is characterized by a profound median cleft of the hands and/or feet, typically associated with the absence or fusion of the remaining fingers (29). In humans, five different loci are associated with different forms of SHFM; in particular, SHFM type 1 maps to chromosome 7q21.3-q22 that includes Dlx5/6 (30). Mice carrying the double-targeted inactivation of Dlx5/6 show a limb phenotype strongly reminiscent SHFM1 reinforcing the notion that these genes are important for the control of limb development (27, 31). Intriguingly, analyses of some clinical cases have reported masculinization defects such as micropenis, hypospadias, and small testis in SHFM patients (32, 33, 34). However, whether such phenotypes constitute one of the symptoms of SHFM or they reflect just phenotype variations of patients remains unclear. These congenital defects appear to result from fetal testicular dysfunctions, suggesting a role of SHFM genes in the differentiation of male sexual characteristics. Although Dlx5 is expressed in the mouse testis (35, 36), the function of Dlx5/6 during testis development is not known.

This study addressed the roles of the Dlx5 and Dlx6 during testicular steroidogenesis. Dlx5 can interact with GATA-4 and enhance the GATA-4-mediated StAR promoter activity in the Leydig cell line, mouse Leydig tumor cell line (mLTC-1). Furthermore, the embryonic mutant for Dlx5/6 genes showed defects in testosterone production and male sexual differentiation. Altogether, these results indicate that Dlx5/6 genes are involved in the proper testicular steroidogenesis and fetal masculinization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse embryos and cell lines
The Dlx5/6 mutant allele used in this study was described previously (31). Experiments using mice were performed according to the guidelines of the Center for Animal Resources and Development, Kumamoto University. The embryonic stage was determined by the day of an appearance of vaginal plug as 0.5 d postcoitum (dpc). mLTC-1 cell line (37) was maintained in RPMI 1640 medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and penicillin-streptomycin (Invitrogen) at 37 C in 5% CO2. COS-7 cells were grown in DMEM (Invitrogen) supplemented with 10% FBS and penicillin-streptomycin at 37 C in 5% CO2.

Plasmids
The –1514/+25- and –966/+25-bp DNA fragments of the StAR promoter (12) were obtained by PCR using mouse genomic DNA and were inserted into the pGL3 basic vector (Promega, Madison, WI). A mutation in the GATA-4 binding site of the StAR promoter was introduced as described previously (16). Mouse Dlx5 cDNA fragment was kindly provided by H. Shibuya (Tokyo Medical and Dental University, Tokyo, Japan). The cDNA fragment of mouse GATA-4 was obtained by RT-PCR. These cDNA fragments were inserted into the pCAGGS vector (38), the pCMV-TNT vector (Promega), pBIND vector (Promega), or pACT vector (Promega). The fragments of Dlx5 deletion mutants were constructed by PCR and cloned into the pCMV-Myc vector (CLONTECH Laboratories, Palo Alto, CA) or the pCMV-TNT vector. The following oligonucleotides were used for PCR: –1514/+25-bp fragment F, 5'-AGT CAT CTC TCC AGC CCA ACA CGT-3'; –966/+25-bp fragment F, 5'-ACC ACA GGG ATC ACA TAC CTG CA-3'; –1514/+25- and –966/+25-bp fragments R, 5'-TCA AGG TCC TGA GTC CTG CAG CT-3'; GATA-4 F, 5'-TCA GAG CTT GGG GCG ATG TAC CAA-3'; GATA-4 R, 5'-TAC GCG GTG ATT ATG TCC CCA TGA CT-3'; Dlx5{Delta}N and Dlx5HD F, 5'-TGA ATT CGG ATG AAA CCA AAG AAA GTT CGT AAA CCC-3'; Dlx5{Delta}C and Dlx5N F, 5'-TGA ATT CGG ATG ACA GGA GTG TTT GAC AGA-3'; Dlx5C F, 5'-TGA ATT CGG ATG AAA AAC GGG GAG ATG-3'; Dlx5{Delta}N and Dlx5C R, 5'-TAA CTC GAG CAA GAG AAA GTA GCC-3'; Dlx5{Delta}C and Dlx5HD R, 5'-ATG GTA CCG ATCT TCT TGA TCT TGG ATC TTT TG-3'; Dlx5N R, 5'-ATG GTA CCT GGT TTA CCA TTC ACC ATC CT-3'.

Luciferase assay
mLTC-1 cells were plated at 105 cells/well in 24-well plates 24 h before transfection. The cells were transfected in triplicate with the relevant combination of plasmids using FuGENE HD (Roche, Basel, Switzerland) according to the manufacturer’s protocol. mLTC-1 cells were transfected with several expression plasmids (400 ng), the luciferase reporter plasmid (200 ng), and pRL-SV40 plasmid (80 ng) encoding Renilla luciferase as an internal control. For the mammalian two-hybrid assay, mLTC-1 cells were transfected with pG5-luc vector (300 ng), Dlx5/pBIND (300 ng), and GATA-4/pACT (300 ng). The cells were harvested 24 h after transfection, and the luciferase activity in cell lysates was determined using the dual-luciferase reporter assay system (Promega). The values were normalized to the Renilla luciferase activity. Three independent experiments were performed. Error bars represent the SD. The statistical comparisons among the experimental groups were assessed by ANOVA. When F ratios were significant (P < 0.05), Scheffé post hoc tests between two groups were done, and P < 0.05 was considered statistically significant differences.

In situ hybridization for gene expression analysis
In situ hybridization was performed on 8-µm sections prepared from paraformaldehyde (PFA)-fixed, paraffin-embedded embryos. Sections were deparaffinized, rehydrated, incubated in 1 µg/ml Proteinase K for 7 min at 37 C, and refixed with 4% PFA for 10 min at room temperature. After washing in PBS containing 0.1% Tween 20, overnight hybridization was performed in a buffer (50% formamide, 5x saline sodium citrate, 50 µg/ml yeast RNA, 1% sodium dodecyl sulfate, 50 µg/ml heparin) with 1 µg/ml probe at 68 C. The following probes were used for section in situ hybridization: Dlx5 (kindly provided by H. Shibuya), Dlx6 (kindly provided by Z. Zhao, Yale University School of Medicine), Insl3, StAR, Dhh (kindly provided by A. McMahon, Harvard University), Ptch1 (kindly provided by J. Motoyama, Brain Science Institute, Riken), PDGFR{alpha} (kindly provided by P. Soriano, Fred Hutchinson Cancer Research Center), and Arx (kindly provided by V. Broccoli, California University). Slides were washed in 5x saline sodium citrate for 1 h at 68 C, 140 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, 25 mM Tris-HCl (pH 7.5) for 5 min at room temperature before incubating for 2 h with blocking solution (10% blocking reagent (Roche) in 100 mM maleate buffer (pH 7.5) and 25% heated FBS in 140 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, 25 mM Tris-HCl (pH 7.5). Antidigoxigenin antibody (Roche) in a blocking solution was added to the slides and incubated overnight at 4 C. After washing, the sections were equilibrated in 100 mM NaCl, 50 mM MgCl2, 0.1% Tween 20, and 100 mM Tris-HCl (pH 9.5) including 2 mM levamisole (Sigma-Aldrich, St. Louis, MO) and incubated in color solution (3.5 µg nitro blue tetrazolium (Roche) and 1.75 µg 5-bromo-4-chloro-3-indolyl phosphate (Roche) per milliliter of 100 mM NaCl, 50 mM MgCl2, 0.1% Tween 20, 100 mM Tris-HCl (pH 9.5) buffer.

Immunochemistry
The mouse embryos were fixed in 4% PFA and embedded in paraffin. Sections for histological analyses were prepared by standard procedures. After deparaffinization, sections were treated for antigen retrieval [microwave treatment 5 min in 10 mM citrate buffer (pH 6.0)] and incubated with 3% H2O2 in PBS for 10 min to inactivate endogenous peroxidases before incubating for 1 h with blocking solution (2% FBS in PBS). Anti-P450 side chain cleavage enzyme (P450scc) polyclonal antibody (American Research Products, Belmont, MA) or anti-Müllerian inhibiting substance polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking solution was added to the slides and incubated for 1 h at room temperature. After washing, the sections were incubated with Alexa 546 goat antirabbit IgG (Invitrogen) or horseradish peroxidase-rabbit antigoat IgG (Zymed, South San Francisco, CA) for 1 h at room temperature. As necessary, the sections were subsequently incubated with 3,3-diaminobenzidine tetrahydrochloride containing H2O2.

Immunoprecipitation
COS-7 cells were plated on 60-mm dishes. On the following day, cells were transfected with the expression plasmids using TransFast (Invitrogen). After 24 h incubation, cells were solubilized in lysis buffer [150 mM NaCl, 0.5% Nonidet P-40, 10 mM Tris (pH 7.5)] with Complete miniprotease inhibitor cocktail (Roche), and cell lysates were cleared by centrifugation. The lysate proteins were immunoprecipitated for 1 h at 4 C with anti-GATA-4 (G-4) antibody (Santa Cruz) or anti-Dlx5 (C-20) (Santa Cruz), followed by precipitation with protein G-Sepharose (GE Healthcare, Buckinghamshire, UK) for 1 h at 4 C. After washing four times with lysate buffer, the immunocomplexes were analyzed by standard SDS-PAGE and Western blotting using anti-Dlx5 antibody, anti-GATA-4 antibody and anti-Myc antibody (Upstate Biotechnology, Lake Placid, NY). The signals were detected with the ECL kit (GE Healthcare).

RNA isolation and real-time quantitative PCR
Total RNA was isolated by ISOGEN (Nippongene, Toyama, Japan) from fetal mouse testis and reverse transcribed by using SuperScript III reverse transcriptase (Invitrogen). Real-time PCR was performed using 7500 real-time PCR system (Applied Biosystems, Foster City, CA) with SYBR Green master mix according to the manufacturer’s instructions. The sets of primer sequences were designed as follows: Dlx5 forward, 5'-AGC TAC CTG GAG AAC TCG GCT T-3'; Dlx5 reverse, 5'-GAT TGA GCT GGC TGC GCT-3'; Dlx6 forward, 5'-GTA TGC CTC CCA ACA GCT ACA TG-3'; Dlx6 reverse, 5'-GTG TCC TGG TGT GGT GAG GAA TA-3'; StAR forward, 5'-GGA GAT GCC GGA GCA GAG T-3'; StAR reverse, 5'-GCC AGT GGA TGA AGC ACC AT-3'; GAPDH forward, 5'-AAC GAC CCC TTC ATT GAC CTC-3'; GAPDH reverse, 5'-CCT TGA CTG TGC CGT TGA ATT-3'. Relative RNA equivalents for each sample were obtained by the standardization of the GAPDH mRNA levels. The error bars represent the SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The expression patterns of Dlx5 and Dlx6 genes in fetal testis
In mammals, the testis contains several different cell types such as germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells. Because little is known about what sort of testicular cells express Dlx5/6 mRNAs, the expression patterns of Dlx5/6 mRNAs were examined by section in situ hybridization expression analysis. During testis development, P450scc is specifically expressed in the fetal Leydig cells (39, 40) (Fig. 1AGo). At 16.5 day postcoitum (dpc), the cells expressing Dlx5/6 mRNAs were detected in the interstitial region of testis in which fetal Leydig cells are located (Fig. 1AGo). These cells showed the morphological feature of fetal Leydig cell with a large round nucleus, which was also observed in P450scc-expressing cells (Fig. 1AGo, boxed areas). To confirm whether Dlx5/6 are certainly expressed in the fetal Leydig cells, we performed double staining using section in situ hybridization (Dlx5/6) and immunohistochemistry (P450scc). As shown in Fig. 1BGo, the expressions of Dlx5/6 and P450scc were overlapped in the interstitial region of testis at 16.5 dpc. Hence, we concluded that Dlx5/6 were expressed in the fetal Leydig cells. During mouse normal development, fetal Leydig cells appear shortly after testicular differentiation around 12.5 dpc (41, 42). Subsequently the testosterone synthesis occurs from around 13.5 dpc increasing until 18.5 dpc (42). Therefore, the embryonic time course of Dlx5/6 mRNA expression was examined by quantitative RT-PCR in a period ranging from 12.5 to 18.5 dpc. These results showed that the Dlx5/6 expression levels also increased in this developmental period (Fig. 1CGo). These spatial and temporal expression patterns of Dlx5/6 suggested that these genes may be involved in testis differentiation and steroidogenesis.


Figure 1
View larger version (55K):
[in this window]
[in a new window]

  		FIG. 1. Dlx5 and Dlx6 are expressed in fetal testis. A, The expression of Dlx5, Dlx6 and P450scc in mouse testis at 16.5 dpc were detected by section in situ hybridization analysis. Dlx5/6 were expressed in interstitial region of mouse testis in which fetal Leydig cells are localized. The morphological feature of fetal Leydig cell with a round nucleus was observed in the Dlx5/6 expressing cells (shown in the boxed areas). The dotted lines demarcate the testis cords (asterisks). B, Double-staining for Dlx5, Dlx6 (purple) and P450scc (red) in fetal testis. Note that the expression of Dlx5/6 overlapped with that of P450scc. C, The quantification of Dlx5/6 mRNA expression in testis from 12.5 to 18.5 dpc, using semiquantitative RT-PCR and real-time quantitative PCR analyses. The expression of Dlx5/6 mRNA in 12.5 dpc testis was designated as the basal level (1.0).

 
Dlx5 enhances the GATA-4-mediated promoter activation of StAR gene
Because Dlx5 transactivates target gene promoters by binding to homeodomain responsive elements (43, 44, 45), we searched for homeodomain binding sites on the promoter region of genes associated with fetal Leydig cell differentiation. Several putative homeodomain binding sites were identified in the previously published StAR promoter sequence (GenBank accession no. AC122752) (12). To assess whether Dlx5 regulates the StAR promoter activity through these sites, transient transfection assays of StAR promoter/luciferase reporter constructs were performed using a mouse Leydig cell line, mLTC-1 (37). We examined the effect of Dlx5 expression on the two upstream DNA fragments of StAR gene promoter: –1514 to +25- and –966 to +25-bp fragments (12), which contain four and no homeodomain binding sites, respectively (Fig. 2AGo, upper panel). mLTC-1 cells were transfected with these reporter constructs in either the presence or absence of the Dlx5 expression vector. Regardless of the presence of four putative homeodomain binding sites between –1514 to –966 bp, there were no significant differences of StAR promoter activity between –1514 to +25- and –966 to +25-bp StAR promoter constructs when Dlx5 was overexpressed (Fig. 2AGo). Therefore, these homeodomain binding sites were not sufficient for the regulation of the StAR promoter in the current experimental conditions. In addition, similar results were obtained using a –4450 to +25-bp StAR promoter construct in the same conditions (data not shown). It has been reported that homeodomain transcription factors can activate the target genes through interactions with other transcriptional factors (46). We then analyzed the possibility that Dlx5 could regulate StAR promoter activity by interacting with other transcription factors.


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 2. Dlx5 enhances the GATA-4-mediated StAR promoter activity. A, Mouse StAR promoter (–1514/+25 bp and –966/+25 bp)/luciferase constructs are schematically illustrated in the upper panel. Homeodomain binding sites are indicated by arrowheads. mLTC-1 cells were transfected with these reporter constructs and pRL-SV40, together with empty vector, Dlx5, and GATA-4 expression vector as indicated. The activity of firefly luciferase was normalized with Renilla luciferase. B, Nucleotide substitutions were introduced at the GATA-4 binding site (TTATCT to TTagT). mLTC-1 cells were transfected with wild-type (WT) or mutant StAR promoter/luciferase reporter constructs together with a control vector, Dlx5, and GATA-4 expression vector as indicated. *, P < 0.001.

 
GATA-4 transactivates the StAR promoter in steroidogenic cells (16, 17, 18) and is known to interact with homeodomain protein (47). Previous studies revealed a GATA binding element at position –66/–61 in the mouse StAR promoter (16, 48, 49). Indeed, the overexpression of GATA-4 significantly activated the StAR promoter in mLTC-1 cells (Fig. 2AGo). Notably, cotransfection of Dlx5 with GATA-4 effectively enhanced GATA-4-mediated StAR promoter activity in mLTC-1 cells (Fig. 2AGo). To examine whether this augmentative effect of Dlx5 to StAR promoter activity is dependent on the binding of GATA-4 to DNA, a mutation was introduced into the GATA-4 binding site within the StAR promoter region. Nucleotide substitutions at the GATA-4 binding site [TTATCT to TTAagT (16)] resulted in the dramatic loss of the augmentative effect of Dlx5 on the GATA-4-mediated StAR promoter activation (Fig. 2BGo). These data suggested that Dlx5 regulates the StAR gene promoter through the GATA-4 binding site.

Dlx5 protein can physically interact with GATA-4 protein
We hypothesized that the protein-protein interaction between Dlx5 and GATA-4 can cooperatively activate the StAR promoter in mLTC-1 cells. To examine this possibility, an immunoprecipitation analysis was performed using COS-7 cells. Dlx5 protein was coimmunoprecipitated with GATA-4 protein when they were coexpressed (Fig. 3AGo, left panel). Furthermore, GATA-4 protein was also coimmunoprecipitated with Dlx5 protein when they were coexpressed (Fig. 3AGo, right panel). To further analyze the extent of such interaction in mLTC-1 cells, a mammalian two-hybrid assay was performed in which the expression of the firefly luciferase gene was driven by five GAL4 binding sites. The full-length Dlx5 was fused with GAL4 DNA binding domain (Dlx5/pBIND) and the full-length GATA-4 was fused with VP16 transactivation domain (GATA-4/pACT). The cotransfection of these two constructs into mLTC-1 cells prominently enhanced the luciferase reporter activity, thus confirming the interaction between Dlx5 and GATA-4 (Fig. 3BGo). To elucidate the potential binding domains of Dlx5 that can interact with GATA-4, various mutant forms of Dlx5 were constructed (Fig. 3CGo, upper panel) and subjected to an immunoprecipitation assay. The results showed that Dlx5{Delta}N [134–289 amino acids (aa)], Dlx5{Delta}C (1–195 aa), and Dlx5N (1–134 aa) could interact with GATA-4, whereas Dlx5C (196–289 aa) could not (Fig. 3CGo, lower left panel). Although the Dlx5HD (135–195 aa) expression level was relatively low, Dlx5HD still interacted with GATA-4 (see the arrowheads in Fig. 3CGo, lower right panels). These data indicated that either the N-terminal (1–134 aa) or homeodomain region (135–195 aa) of Dlx5 was required to interact with GATA-4 protein, albeit not the C-terminal region (196–289 aa).


Figure 3
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 3. Protein-protein interaction between Dlx5 and GATA-4. A, COS-7 cells were transfected with empty vector, Dlx5, and GATA-4 expression vector as indicated. After 24 h incubation, cell lysates were subjected to immunoprecipitation (IP). Immunocomplexes were analyzed by standard SDS-PAGE and Western blotting methods, using respective antibodies. B, A mammalian two-hybrid assay was performed using the expression vectors encoding Dlx5-GAL4 (Dlx5/pBIND) and/or GATA-4-VP16 (GATA-4/pACT). mLTC-1 cells were transfected with the indicated constructs and then were assayed to determine their luciferase activity. C, Schematic representation of various Dlx5 deletion mutants used to identify the interaction domains. The results from the lower panel are presented schematically. The binding of the Dlx5 derivatives to GATA-4 are presented as follows: +, binding; –, no binding. Series of plasmids that express Myc-tagged Dlx5 deletion mutants and GATA-4 expression vector were transfected into COS-7 cells and analyzed as previously indicated. Two different exposure conditions were shown (left panel, short exposure; right panel, long exposure; arrowheads, the Dlx5HD protein).

 
The activation domains of Dlx5 for the cooperative function with GATA-4
To analyze the cooperative effects of the interaction between Dlx5 and GATA-4 on the StAR promoter activation, several Dlx5 deletion constructs were used (Fig. 4AGo). Similar to the case with the full-length Dlx5 expression vector, Dlx5{Delta}N and Dlx5{Delta}C expression constructs enhanced the GATA-4-mediated StAR promoter activity, whereas neither the Dlx5N nor Dlx5C expression constructs did (Fig. 4BGo). The Dlx5HD was less effective for the enhancement of the GATA-4-mediated StAR promoter activity (Fig. 4BGo), although Dlx5HD could interact with GATA-4 (Fig. 3CGo). This low reporter activity by Dlx5HD is possibly caused by the low level of Dlx5HD expression due to its stability (Fig. 3CGo). These results suggested that the homeodomain of Dlx5 would be necessary for the cooperative activating functions with GATA-4 on the StAR promoter, and either the N- or C-terminal region of Dlx5 was required for the full promoter activation. Taken together, Dlx5 could interact with GATA-4 through its homeodomain and enhance the StAR promoter activity by either an N- or C-terminal activation domain.


Figure 4
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 4. Differential ability of Dlx5 mutant proteins for GATA-4-mediated StAR promoter activation. A, Schematic representation of Dlx5 deletion mutants used to analyze the domain for the regulation of StAR promoter activity. B, mLTC-1 cells were transiently transfected with StAR promoter/luciferase construct (–966/+25 bp) and pRL-SV40, together with empty vector, Dlx5 deletion mutants and GATA-4 expression vectors as indicated. *, P = 0.001; **, P < 0.001 vs. GATA-4 only overexpressed groups.

 
The defect of masculinization in Dlx5 and Dlx6 double-knockout (DKO) embryos
The above in vitro data showed that Dlx5 enhances the promoter activity of StAR gene through protein-protein interaction with GATA-4. To examine the function of Dlx5 in vivo, the phenotypes of Dlx5/6 DKO embryos were investigated as a genetic tool for loss-of-function studies. Dlx5/6 DKO embryos die shortly after birth; therefore, the fetal testes of Dlx5/6 DKO embryos from 12.5 to 18.5 dpc were analyzed. The histological analyses indicated that fetal testis of Dlx5/6 DKO embryos displayed grossly normal testicular histology (data not shown). The expression of several marker genes for Sertoli and fetal Leydig cells did not show significant alterations in comparison with control embryos (data not shown). These data suggest that Dlx5/6 may not be directly involved in testicular histogenesis.

The expression of StAR mRNA is first detected in the interstitial region at 12.5 dpc and subsequently detected in the Leydig cells (50). Dlx5/6 were also expressed in the fetal Leydig cells (Fig. 1BGo). A section in situ hybridization analysis revealed the expression of StAR mRNA in the testes of Dlx5/6 DKO embryos to be relatively lower than that of the control (about 40% of the DKO samples examined, Fig. 5AGo), although there were some phenotypic variations. Intriguingly, Dlx5/6 DKO embryos showed decreased intratesticular testosterone levels, compared with those of controls at 18.5 dpc (Fig. 5BGo). To evaluate Dlx5/6 DKO embryos for the phenotypes of androgen-dependent organogenesis, the distance between the anus and genital of mice [anogenital distance (AGD)] was measured. The AGD is one of the frequently used androgen-dependent markers in teratology and a reliable parameter of external masculinization in mice. The AGD was significantly decreased in Dlx5/6 DKO embryos in comparison with that of controls at 18.5 dpc (Fig. 5CGo). These results indicated that Dlx5/6 genes possess regulatory roles on the physiological testosterone production for the masculinization processes during the late embryonic stage.


Figure 5
View larger version (60K):
[in this window]
[in a new window]

  		FIG. 5. Testicular phenotypes of Dlx5/6 DKO embryos. A, StAR mRNA expression pattern of control and Dlx5/6 DKO embryos in the fetal testis at 18.5 dpc. The dotted lines demarcate the testis cords (asterisks). The boxed areas indicate the magnified area. B, Measurement of intratesticular testosterone using the CLIA method. An asterisk means statistical significance from the control by Student’s t test. *, P < 0.01. C, The arrowheads indicate the distance between the AGD of mice. Statistical significance was confirmed with the control by Student’s t test, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Steroid hormones are crucial regulators for embryonic development and tissue homeostasis in adult organisms. Androgens, in particular, play essential roles in controlling the genesis and maintenance of male sexual characteristics and in assuring male reproductive health (51). The StAR protein is a sterol transfer protein required for the biosynthesis of steroid hormones and its expression is regulated by multiple transcriptional factors (10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). The current study showed that Dlx5 is a novel activator of the StAR promoter and Dlx5/6 positively regulate androgen production in the differentiating fetal testis. These observations provide a new insight on the function of Dlx5/6 for regulating steroidogenesis.

Protein-protein interactions are known to be essential for specifying the transcriptional activities of homeodomain proteins (46). Several factors are known to modify the Dlx5-dependent transcriptional activity. Dlxin-1 and GRIP1 function as coactivators to enhance the transcriptional activation of the Dlx5 target genes by interacting with the N-terminal region of Dlx5 (52, 53). The homeodomain transcription factors Msx1 and Msx2, the vertebrate homologs of the Drosophila muscle segment homeobox gene, form a heterodimeric complex with Dlx5 through a protein-protein interaction involving their homeodomains (54). This heterodimerization counteracts their transcriptional activities by inhibiting their capacity to bind to target sequences (54). The current study demonstrated the existence of a protein-protein interaction between Dlx5 and GATA-4. Because Dlx5-dependent promoter activation of the StAR gene required GATA-4 as the coupling factor that binds to DNA, this suggests that Dlx5 regulates the StAR promoter transcriptional activation by interacting with GATA-4. This would be the first report that Dlx5 can function as a coactivator to regulate the downstream target gene expression.

Such a novel property of Dlx5 raises a question of how Dlx5 enhances the promoter activity. The Dlx5 protein has two proline-rich regions upstream and downstream of its homeodomain. It has been proposed that proline-rich domains are implicated in the regulation of transcriptional activation (55). In fact, the N-terminal proline-rich region of Dlx5 functions as an activation domain when fused to the yeast Gal4 DNA binding domain (53). In this study, a luciferase assay using various Dlx5 deletion mutants indicated that HD region of Dlx5 is indispensable for the StAR promoter regulation, and either the N- or C-terminal region of Dlx5 protein is required for the activation of the StAR promoter when coexpressed with GATA-4. Previous reports revealed that p300 functions as a coactivator of GATA-4 (56), and it augments the GATA-4-dependent StAR promoter activity (18). The proline-rich activation domains have been also suggested to interact with p300 and the basal transcriptional component TFIID (TATA binding protein) (57, 58). It is possible that the N- and C-terminal proline-rich regions of Dlx5 protein function as a transcriptional activation domain by recruiting these factors. Taken together, Dlx5 activates the StAR promoter by interacting with GATA-4 and both N- and C-terminal proline-rich regions of Dlx5 function as the activation domain for StAR gene regulation.

Almost all expressing cells for Dlx5/6 and P450scc overlapped in the interstitial region of fetal testis (Fig. 1BGo), thus indicating that Dlx5/6 are expressed in the fetal Leydig cells. Although Dlx5/6 are expressed from 12.5 dpc when fetal Leydig cells start to express the P450scc (39), Dlx5/6 may not be involved in induction of differentiation in the fetal Leydig cells because Dlx5/6 DKO mice exhibit rather normal fetal Leydig cell histogenesis. In fact, the expression of insulin-like factor 3, a maker of the mature fetal Leydig cells, was not altered in Dlx5/6 DKO testis compared with that of control (data not shown). Therefore, Dlx5/6 would be involved in neither the induction nor the differentiation of fetal Leydig cells during testis development. The contribution of Dlx5/6 to the adult Leydig cell differentiation remained unelucidated due to the neonatal lethality of Dlx5/6 DKO mice. Interestingly, Dlx5/6 mRNAs were expressed in the adult ovary but not the fetal ovary (data not shown). It is generally known that steroidogenesis does not occur in the fetal ovary, although steroid progenitor cells exist. These observations indicate that Dlx5/6 appears to thus play a role in steroidogenesis but not in cell differentiation.

This study suggested that the loss of Dlx5/6 genes caused decreased testosterone production in Dlx5/6 DKO embryos at 18.5 dpc. In addition, the amounts of serum testosterone also exhibited a tendency to decrease in Dlx5/6 DKO embryos compared with those in control (data not shown). The Dlx5/6 DKO embryos exhibit the complete absence of calvaria and dysmorphogenesis of nasal, maxillary, and mandibular structures (27). Therefore, they die shortly after birth due to cerebral trauma during delivery and massive postnatal blood loss (27). These observations indicate that the reduction of testosterone production measured in the fetal testes could not be due to embryonic systematic defects. Consistent with above results, AGD, a well-known masculinization marker, was decreased in Dlx5/6 DKO embryos. These facts also indicated that the loss of Dlx5/6 reduced functional testosterone production, resulting in the masculinization disorder.

It has been known that Dlx genes possess some functional compensation (27). In the current study, the overlapping expression patterns of Dlx5/6 are shown in the fetal Leydig cells (Fig. 1BGo). Each Dlx5 or Dlx6 single mutant displayed no significant defects of testis differentiation and masculinization (Levi, G., unpublished data). In contrast, Dlx5/6 DKO embryos showed the reduced testosterone production and abnormal masculinization. These results suggest that Dlx5/6 possess redundant functions in the developing testis.

In summary, we demonstrated that an interaction between the homeobox transcription factor Dlx5 and zinc-finger protein GATA-4 is involved in StAR gene regulation and that Dlx5/6 have regulatory roles for the testicular steroidogenesis during embryogenesis. Further studies on the role of Dlx in the Leydig cell function and testicular steroidogenesis will offer a better understanding of human congenital malformations of the male reproductive organs.


   Acknowledgments
 
We thank Drs. Mitsuo Oshimura, Giorgio R. Merlo, Marc Ekker, Kenta Sumiyama, Tomohiko Wakayama, Matthew P. Hardy, Barry T. Hinton, Tetsuya Taga, Shoen Kume, Shinji Fukuda, Tetsu Yoshida, Nobuaki Shiraki, and Sawako Fujikawa for encouragement and help. We also express our appreciation to Shiho Kitagawa for her valuable assistance.


   Footnotes
 
This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas: General Promotion of Cancer Research in Japan, a Grant-in-Aid for Scientific Research on Priority Areas: Mechanisms of Sex Differentiation, the Global COE Research Program, and a Grant for Child Health and Development (17-2) from the Ministry of Health, Labor, and Welfare. G.L. is supported by the European Consortium CRESCENDO, "Nuclear Receptors in development and aging."

Disclosure Statement: The authors have nothing to disclosure.

First Published Online February 14, 2008

Abbreviations: aa, Amino acids; AGD, anogenital distance; DKO, double knockout; dpc, days postcoitum; FBS, fetal bovine serum; mLTC, mouse Leydig tumor cell; PFA, paraformaldehyde; P450scc, P450 side chain cleavage enzyme; SHFM, split hand/foot malformation; StAR, steroidogenic acute regulatory protein.

Received September 13, 2007.

Accepted for publication February 5, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yamada G, Suzuki K, Haraguchi R, Miyagawa S, Satoh Y, Kamimura M, Nakagata N, Kataoka H, Kuroiwa A, Chen Y 2006 Molecular genetic cascades for external genitalia formation: an emerging organogenesis program. Dev Dyn 235:1738–1752[CrossRef][Medline]
  2. Wilson JD, Leihy MW, Shaw G, Renfree MB 2002 Androgen physiology: unsolved problems at the millennium. Mol Cell Endocrinol 198:1–5[CrossRef][Medline]
  3. Zhou Q, Nie R, Prins GS, Saunders PT, Katzenellenbogen BS, Hess RA 2002 Localization of androgen and estrogen receptors in adult male mouse reproductive tract. J Androl 23:870–881[Abstract/Free Full Text]
  4. Val P, Swain A 2005 Mechanisms of disease: normal and abnormal gonadal development and sex determination in mammals. Nat Clin Pract 2:616–627[CrossRef]
  5. Ge RS, Chen GR, Tanrikut C, Hardy MP 2007 Phthalate ester toxicity in Leydig cells: developmental timing and dosage considerations. Repro Toxicol 23:366–373[CrossRef][Medline]
  6. Holmes NM, Miller WL, Baskin LS 2004 Lack of defects in androgen production in children with hypospadias. J Clin Endocrinol Metab 89:2811–2816[Abstract/Free Full Text]
  7. Stocco DM, Wang X, Jo Y, Manna PR 2005 Multiple signaling pathways regulating steroidogenesis and steroidogenic acute regulatory protein expression: more complicated than we thought. Mol Endocrinol 19:2647–2659[Abstract/Free Full Text]
  8. Manna PR, Wang XJ, Stocco DM 2003 Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression. Steroids 68:1125–1134[CrossRef][Medline]
  9. Bose HS, Sugawara T, Strauss 3rd JF, Miller WL 1996 The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. International Congenital Lipoid Adrenal Hyperplasia Consortium. N Engl J Med 335:1870–1878[Abstract/Free Full Text]
  10. Caron KM, Soo SC, Wetsel WC, Stocco DM, Clark BJ, Parker KL 1997 Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia. Pro Natl Acad Sci USA 94:11540–11545[Abstract/Free Full Text]
  11. Hasegawa T, Zhao L, Caron KM, Majdic G, Suzuki T, Shizawa S, Sasano H, Parker KL 2000 Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice. Mol Endocrinol 14:1462–1471[Abstract/Free Full Text]
  12. Caron KM, Ikeda Y, Soo SC, Stocco DM, Parker KL, Clark BJ 1997 Characterization of the promoter region of the mouse gene encoding the steroidogenic acute regulatory protein. Mol Endocrinol 11:138–147[Abstract/Free Full Text]
  13. Sugawara T, Kiriakidou M, McAllister JM, Holt JA, Arakane F, Strauss 3rd JF 1997 Regulation of expression of the steroidogenic acute regulatory protein (StAR) gene: a central role for steroidogenic factor 1. Steroids 62:5–9[CrossRef][Medline]
  14. Reinhart AJ, Williams SC, Clark BJ, Stocco DM 1999 SF-1 (steroidogenic factor-1) and C/EBPβ (CCAAT/enhancer binding protein-β) cooperate to regulate the murine StAR (steroidogenic acute regulatory) promoter. Mol Endocrinol 13:729–741[Abstract/Free Full Text]
  15. Sugawara T, Saito M, Fujimoto S 2000 Sp1 and SF-1 interact and cooperate in the regulation of human steroidogenic acute regulatory protein gene expression. Endocrinology 141:2895–2903[Abstract/Free Full Text]
  16. Silverman E, Eimerl S, Orly J 1999 CCAAT enhancer-binding protein β and GATA-4 binding regions within the promoter of the steroidogenic acute regulatory protein (StAR) gene are required for transcription in rat ovarian cells. J Bio Chem 274:17987–17996[Abstract/Free Full Text]
  17. LaVoie HA, Singh D, Hui YY 2004 Concerted regulation of the porcine steroidogenic acute regulatory protein gene promoter activity by follicle-stimulating hormone and insulin-like growth factor I in granulosa cells involves GATA-4 and CCAAT/enhancer binding protein β. Endocrinology 145:3122–3134[Abstract/Free Full Text]
  18. Silverman E, Yivgi-Ohana N, Sher N, Bell M, Eimerl S, Orly J 2006 Transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene: GATA-4 and CCAAT/enhancer-binding protein β confer synergistic responsiveness in hormone-treated rat granulosa and HEK293 cell models. Mol Cell Endocrinol 252:92–101[CrossRef][Medline]
  19. Manna PR, Eubank DW, Lalli E, Sassone-Corsi P, Stocco DM 2003 Transcriptional regulation of the mouse steroidogenic acute regulatory protein gene by the cAMP response-element binding protein and steroidogenic factor 1. J Mol Endocrinol 30:381–397[Abstract]
  20. Manna PR, Dyson MT, Eubank DW, Clark BJ, Lalli E, Sassone-Corsi P, Zeleznik AJ, Stocco DM 2002 Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family. Mol Endocrinol 16:184–199[Abstract/Free Full Text]
  21. Nackley AC, Shea-Eaton W, Lopez D, McLean MP 2002 Repression of the steroidogenic acute regulatory gene by the multifunctional transcription factor Yin Yang 1. Endocrinology 143:1085–1096[Abstract/Free Full Text]
  22. Liu Q, Merkler KA, Zhang X, McLean MP 2007 Prostaglandin F2{alpha} suppresses rat steroidogenic acute regulatory protein expression via induction of Yin Yang 1 protein and recruitment of histone deacetylase 1 protein. Endocrinology 148:5209–5219[CrossRef][Medline]
  23. Bielinska M, Seehra A, Toppari J, Heikinheimo M, Wilson DB 2007 GATA-4 is required for sex steroidogenic cell development in the fetal mouse. Dev Dyn 236:203–213[CrossRef][Medline]
  24. Zerucha T, Ekker M 2000 Distal-less-related homeobox genes of vertebrates: evolution, function, and regulation. Biochem Cell Biol 78:593–601[CrossRef][Medline]
  25. Merlo GR, Zerega B, Paleari L, Trombino S, Mantero S, Levi G 2000 Multiple functions of Dlx genes. Int J Dev Biol 44:619–626[Medline]
  26. Park BK, Sperber SM, Choudhury A, Ghanem N, Hatch GT, Sharpe PT, Thomas BL, Ekker M 2004 Intergenic enhancers with distinct activities regulate Dlx gene expression in the mesenchyme of the branchial arches. Dev Biol 268:532–545[CrossRef][Medline]
  27. Robledo RF, Rajan L, Li X, Lufkin T 2002 The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development. Genes Dev 16:1089–1101[Abstract/Free Full Text]
  28. Beverdam A, Merlo GR, Paleari L, Mantero S, Genova F, Barbieri O, Janvier P, Levi G 2002 Jaw transformation with gain of symmetry after Dlx5/Dlx6 inactivation: mirror of the past? Genesis 34:221–227[CrossRef][Medline]
  29. Basel D, Kilpatrick MW, Tsipouras P 2006 The expanding panorama of split hand foot malformation. Am J Med Genet 140:1359–1365
  30. Crackower MA, Scherer SW, Rommens JM, Hui CC, Poorkaj P, Soder S, Cobben JM, Hudgins L, Evans JP, Tsui LC 1996 Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3-q22.1 and analysis of a candidate gene for its expression during limb development. Hum Mol Genet 5:571–579[Abstract/Free Full Text]
  31. Merlo GR, Paleari L, Mantero S, Genova F, Beverdam A, Palmisano GL, Barbieri O, Levi G 2002 Mouse model of split hand/foot malformation type I. Genesis 33:97–101[CrossRef][Medline]
  32. Garcia-Ortiz JE, Banda-Espinoza F, Zenteno JC, Galvan-Uriarte LM, Ruiz-Flores P, Garcia-Cruz D 2005 Split hand malformation, hypospadias, microphthalmia, distinctive face and short stature in two brothers suggest a new syndrome. Am J Med Genet 135:21–27
  33. Giltay JC, Wittebol-Post D, van Bokhoven H, Kastrop PM, Lock MT 2002 Split hand/split foot, iris/choroid coloboma, hypospadias and subfertility: a new developmental malformation syndrome? Clin Dysmorphol 11:231–235[CrossRef][Medline]
  34. Suzuki K, Haraguchi R, Ogata T, Barbieri O, Alegria O, Vieux-Rochas M, Nakagata N, Ito M, Mills A, Kurita T, Levi G, Yamada G 2007 Abnormal urethra formation in mouse models of Split-hand/split-foot malformation type 1 and type 4. Eur J Hum Genet
  35. Miyama K, Yamada G, Yamamoto TS, Takagi C, Miyado K, Sakai M, Ueno N, Shibuya H 1999 A BMP-inducible gene, dlx5, regulates osteoblast differentiation and mesoderm induction. Dev Biol 208:123–133[CrossRef][Medline]
  36. Kimura MI, Kazuki Y, Kashiwagi A, Kai Y, Abe S, Barbieri O, Levi G, Oshimura M 2004 Dlx5, the mouse homologue of the human-imprinted DLX5 gene, is biallelically expressed in the mouse brain. J Hum Genet 49:273–277[CrossRef][Medline]
  37. Rebois RV 1982 Establishment of gonadotropin-responsive murine Leydig tumor cell line. J Cell Biol 94:70–76[Abstract/Free Full Text]
  38. Miyazaki J, Takaki S, Araki K, Tashiro F, Tominaga A, Takatsu K, Yamamura K 1989 Expression vector system based on the chicken β-actin promoter directs efficient production of interleukin-5. Gene 79:269–277[CrossRef][Medline]
  39. Yao HH, Whoriskey W, Capel B 2002 Desert Hedgehog/Patched 1 signaling specifies fetal Leydig cell fate in testis organogenesis. Genes Dev 16:1433–1440[Abstract/Free Full Text]
  40. Rouiller V, Gangnerau MN, Vayssiere JL, Picon R 1990 Cholesterol side-chain cleavage activity in rat fetal gonads: a limiting step for ovarian steroidogenesis. Mol Cell Endocrinol 72:111–120[CrossRef][Medline]
  41. Brennan J, Capel B 2004 One tissue, two fates: molecular genetic events that underlie testis versus ovary development. Nat Rev Genet 5:509–521[Medline]
  42. O’Shaughnessy PJ, Baker PJ, Johnston H 2006 The foetal Leydig cell—differentiation, function and regulation. Int J Andrology 29:90–95; discussion 105–108[CrossRef][Medline]
  43. Roca H, Phimphilai M, Gopalakrishnan R, Xiao G, Franceschi RT 2005 Cooperative interactions between RUNX2 and homeodomain protein-binding sites are critical for the osteoblast-specific expression of the bone sialoprotein gene. J Biol Chem 280:30845–30855[Abstract/Free Full Text]
  44. Lee MH, Kim YJ, Yoon WJ, Kim JI, Kim BG, Hwang YS, Wozney JM, Chi XZ, Bae SC, Choi KY, Cho JY, Choi JY, Ryoo HM 2005 Dlx5 specifically regulates Runx2 type II expression by binding to homeodomain-response elements in the Runx2 distal promoter. J Biol Chem 280:35579–35587[Abstract/Free Full Text]
  45. Benson MD, Bargeon JL, Xiao G, Thomas PE, Kim A, Cui Y, Franceschi RT 2000 Identification of a homeodomain binding element in the bone sialoprotein gene promoter that is required for its osteoblast-selective expression. J Biol Chem 275:13907–13917[Abstract/Free Full Text]
  46. Mann RS, Affolter M 1998 Hox proteins meet more partners. Curr Opin Genet Dev 8:423–429[CrossRef][Medline]
  47. Sepulveda JL, Belaguli N, Nigam V, Chen CY, Nemer M, Schwartz RJ 1998 GATA-4 and Nkx-2.5 coactivate Nkx-2 DNA binding targets: role for regulating early cardiac gene expression. Mol Cell Biol 18:3405–3415[Abstract/Free Full Text]
  48. Tremblay JJ, Hamel F, Viger RS 2002 Protein kinase A-dependent cooperation between GATA and CCAAT/enhancer-binding protein transcription factors regulates steroidogenic acute regulatory protein promoter activity. Endocrinology 143:3935–3945[Abstract/Free Full Text]
  49. Tremblay JJ, Viger RS 2001 GATA factors differentially activate multiple gonadal promoters through conserved GATA regulatory elements. Endocrinology 142:977–986[Abstract/Free Full Text]
  50. Clark BJ, Soo SC, Caron KM, Ikeda Y, Parker KL, Stocco DM 1995 Hormonal and developmental regulation of the steroidogenic acute regulatory protein. Mol Endocrinol 9:1346–1355[Abstract/Free Full Text]
  51. Matsumoto T, Takeyama K, Sato T, Kato S 2005 Study of androgen receptor functions by genetic models. J Biochem (Tokyo) 138:105–110[Abstract/Free Full Text]
  52. Yu G, Zerucha T, Ekker M, Rubenstein JL 2001 Evidence that GRIP, a PDZ-domain protein which is expressed in the embryonic forebrain, co-activates transcription with DLX homeodomain proteins. Dev Brain Res 130:217–230[CrossRef][Medline]
  53. Masuda Y, Sasaki A, Shibuya H, Ueno N, Ikeda K, Watanabe K 2001 Dlxin-1, a novel protein that binds Dlx5 and regulates its transcriptional function. J Biol Chem 276:5331–5338[Abstract/Free Full Text]
  54. Zhang H, Hu G, Wang H, Sciavolino P, Iler N, Shen MM, Abate-Shen C 1997 Heterodimerization of Msx and Dlx homeoproteins results in functional antagonism. Mol Cell Biol 17:2920–2932[Abstract]
  55. Mermod N, O’Neill EA, Kelly TJ, Tjian R 1989 The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain. Cell 58:741–753[CrossRef][Medline]
  56. Dai YS, Markham BE 2001 p300 Functions as a coactivator of transcription factor GATA-4. J Biol Chem 276:37178–37185[Abstract/Free Full Text]
  57. de Caestecker MP, Yahata T, Wang D, Parks WT, Huang S, Hill CS, Shioda T, Roberts AB, Lechleider RJ 2000 The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain. J Biol Chem 275:2115–2122[Abstract/Free Full Text]
  58. Tanese N, Pugh BF, Tjian R 1991 Coactivators for a proline-rich activator purified from the multisubunit human TFIID complex. Genes Dev 5:2212–2224[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Exp. Biol. Med.Home page
H. A. LaVoie and S. R. King
Transcriptional Regulation of Steroidogenic Genes: STARD1, CYP11A1 and HSD3B
Experimental Biology and Medicine, August 1, 2009; 234(8): 880 - 907.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
L. J Martin and J. J Tremblay
The nuclear receptors NUR77 and SF1 play additive roles with c-JUN through distinct elements on the mouse Star promoter
J. Mol. Endocrinol., February 1, 2009; 42(2): 119 - 129.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
B. El-Asmar, X. C Giner, and J. J Tremblay
Transcriptional cooperation between NF-{kappa}B p50 and CCAAT/enhancer binding protein {beta} regulates Nur77 transcription in Leydig cells
J. Mol. Endocrinol., February 1, 2009; 42(2): 131 - 138.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
K. Suzuki, Y. Yamaguchi, M. Villacorte, K. Mihara, M. Akiyama, H. Shimizu, M. M. Taketo, N. Nakagata, T. Tsukiyama, T. P. Yamaguchi, et al.
Embryonic hair follicle fate change by augmented {beta}-catenin through Shh and Bmp signaling
Development, February 1, 2009; 136(3): 367 - 372.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
L. J. Martin, N. Boucher, C. Brousseau, and J. J. Tremblay
The Orphan Nuclear Receptor NUR77 Regulates Hormone-Induced StAR Transcription in Leydig Cells through Cooperation with Ca2+/Calmodulin-Dependent Protein Kinase I
Mol. Endocrinol., September 1, 2008; 22(9): 2021 - 2037.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nishida, H.
Right arrow Articles by Yamada, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nishida, H.
Right arrow Articles by Yamada, G.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*TESTOSTERONE

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals