Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M413992200 on January 13, 2005 Originally published In Press as doi:10.1074/jbc.M413992200 on January 7, 2005 Originally published In Press as doi:10.1074/jbc.M413992200 on January 7, 2005 Originally published In Press as doi:10.1074/jbc.M413992200 on December 28, 2004

J. Biol. Chem., Vol. 280, Issue 9, 7786-7792, March 4, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/9/7786    most recent
M413992200v4
M413992200v3
M413992200v2
M413992200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vlahopoulos, S.
Right arrow Articles by Schwartz, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vlahopoulos, S.
Right arrow Articles by Schwartz, R. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Recruitment of the Androgen Receptor via Serum Response Factor Facilitates Expression of a Myogenic Gene*

Spiros Vlahopoulos{ddagger}§, Warren E. Zimmer||, Guido Jenster**, Narasimhaswamy S. Belaguli{ddagger}, Steven P. Balk{ddagger}{ddagger}, Albert O. Brinkmann§§, Rainer B. Lanz{ddagger}, Vassilis C. Zoumpourlis§, and Robert J. Schwartz{ddagger}

From the {ddagger}Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, the ||Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, Alabama 36688, the **Department of Urology, Erasmus MC, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands, the {ddagger}{ddagger}Cancer Biology Program, Hematology-Oncology Division, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, the §§Department of Reproduction & Development, Erasmus MC, The Netherlands, and the §Unit of Biomedical Applications, Institute of Biological Research and Biotechnology, Hellenic Research Foundation, 48 Vasileos Constantinou Avenue, Athens 11635, Greece

Received for publication, December 13, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Androgen receptor (AR) induced precocious myogenesis in culture and myogenic specified gene activity. Increased levels of AR expression in replicating C2C12 myoblasts stimulated fusion into post-differentiated multinucleated myotubes and the appearance of skeletal {alpha}-actin transcripts, even in the absence of ligand. Furthermore, AR activated the skeletal {alpha}-actin promoter, which lacks GRE sites, in co-transfected C2C12 cells. AR co-activation of the skeletal {alpha}-actin promoter required co-expressed full-length serum response factor (SRF). In vitro, AR associated with SRF and was recruited by SRF to a {alpha}-actin promoter SRF binding site. Our data suggest that AR is capable of activating myogenic genes devoid of consensus AR binding sites via its recruitment by the myogenic enriched transcription factor, SRF.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Steroid androgens play an important role in determining lean body mass and muscle strength. For example, men made hypogonadal lose lean body mass and muscle strength (1), whereas in older men, decrements in circulating testosterone levels correlates well with reduced musculature (2). In addition, treatment of hypogonadal men or older men with androgens elicited increased muscle strength and lean body mass (3, 4). Androgens used by healthy young athletes and weightlifters (5) maximize muscle mass and strength. Testosterone increased maximal voluntary strength in a dose-dependent manner (6, 7) and was shown to be a limiting factor in muscle strength development (8), during training-induced muscle hypertrophy (9). Androgens used as a treatment for cachexia also increased fat-free muscle mass and maximum voluntary strength (10). Thus, there is strong evidence that androgens increase muscle mass and strength, both under pathological and physiological conditions.

Most studies conducted to date have focused on the positive role of androgens on skeletal muscle protein synthesis (11–13). The molecular basis of myogenic enhancement by androgens is not well understood (6, 7, 14). The androgen receptor (AR)1 (15) is a member of the zinc finger class of transcription regulators, and the steroid and thyroid hormone receptor superfamily (16). AR binds androgens by a discrete domain at the 250 C-terminal amino acids of the primary structure (17, 18) and upon binding undergoes phosphorylation (19) and a conformational change. Proteins with the capacity to retain the receptor in the cytoplasm are dissociated and AR nuclear localization signals become exposed allowing for nuclear entry and transcription activation (20). Nuclear hormone receptors are divided in two families, in regard to the DNA sequences they recognize. Estrogen, thyroid, and retinoid acid receptors recognize estrogen response element-like (AGGTCA) sequences, whereas androgen, glucocorticoid, and progesterone receptors recognize GRE-like (AGAACA) sequences (21–23). To date, there are no known direct AR DNA targets that drive myogenic specified genes (6). AR, as well as other members of the nuclear receptor superfamily, is modulated by coregulatory proteins (24). Steroid receptors have been shown to interact with other DNA-binding proteins, resulting in modulation of steroid receptor transcriptional activity. AR has been found to interact with a number of transcription factors including AP-1 (25), Smad3 (26, 27), nuclear factor {kappa}B (NF{kappa}B) (28, 29), sex-determining region Y (30), and the Ets family of transcription factors (31).

A feature of a large number of myogenic expressed genes is their dependence upon high affinity binding sites for serum response factor (SRF). SRF shares a highly conserved DNA-binding/dimerization domain of 90 amino acids, termed the MADS box. SRF serves as a versatile protein that binds to its cognate sites in a multitude of promoters to integrate intracellular signals and assists as a docking surface for the binding of accessory factors that may confer the regulation of specific gene programs (32–37). SRF is a key regulator of immediate early gene expression, which frequently results in mitogenesis, and also of myogenic terminal differentiation. Many muscle-specific genes, including skeletal, cardiac, and smooth muscle {alpha}-actins contain combinations of at least three or more strong and weak affinity serum response elements (SRE) that bind SRF in a highly cooperative manner (37–39). Accessory factors may then play essential roles in either facilitating or impeding SRF binding on multi-SRE muscle gene promoters; thus, stimulating or repressing transcription of SRF-dependent gene targets. Recently, Kim et al. (40) showed that the steroid receptors, RXR and/or RAR, physically associated with SRF and repressed SRF-dependent c-fos transcriptional activity. We asked if AR played a similar role in myogenesis, and whether it also functioned as an SRF co-factor. Herein, AR activated the skeletal {alpha}-actin promoter, which lacks GRE sites, in co-transfected C2C12 cells and provided androgen regulation over the {alpha}Sk-actin promoter. AR was observed to associate with SRF and together were recruited to SRF DNA binding elements (SRE). Thus, AR was capable of activating a myogenic gene devoid of consensus AR binding sites via its recruitment by SRF. AR also induced precocious muscle differentiation in culture and acted as a potent activator of myogenic gene activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—C2C12 mouse myoblasts were purchased from American Tissue Culture Collections. Myoblasts were maintained at 37 °C, 5% CO2 in a humidified incubator, in Dulbecco's modified Eagle's medium, supplemented with 20% neonatal calf serum. CV1 green monkey kidney fibroblasts were purchased from American Tissue Culture Collections and maintained at 37 °C, 5% CO2 in a humidified incubator, in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum.

DNA Plasmids—Expression vectors for hAR (human androgen receptor) mutants have been described previously (17). Vector encoding hAR (human AR) wild type was used via PCR to subclone hAR into pCGN vector (41) for mammalian expression and into Bluescript (Stratagene) for transcription/translation by reticulocyte lysates. The construction of pCGN-hAR used primers: 5' primer, 5'-GGACCTTCTAGAGAAGTGCAGTTAGGGCTGGGA-3' and 3' primer, 5'-GCGTGTGGATCCTCACTGGGTGTGGAAATAGATGG-3'.

Human androgen receptor from plasmid pAR0 (17) was PCR amplified using the above primers. The PCR product was agarose gel-purified, digested with XbaI and BamHI, and ligated to PCGN vector that had been digested with the same enzymes. Construction of Bluescript-hAR used the following primers: 5' primer, 5'-GGACCTTCTAGAATGAGTGCAGTTAGGGCTGGGA-3' and the 3' primer, 5'-GCGTGTGGATCCTTTATTTCACTGGGTGTGGAAATAGATGG-3'.

Oligonucleotide primers were used to amplify hAR from pAR0 and ligate to plasmid pBluescript KS(+), after digestions with BamHI and XbaI. hAR LBD (ligand binding domain) from pcDNA3 was cut with HindIII and XbaI, and ligated into pP(A)LiS(MS-2) that was cut with those two enzymes. This gave rise to pP(A)LiSK-hAR-LBD, which we used for Sp6-driven AR LBD transcription. SRF mutants cloned in the vector pCGN have been described elsewhere (42). pCGN-SRF wild type and pm1 were a kind gift from Ron Prywes. pGEX plasmids encoding fusions of hAR truncations with glutathione S-transferase (GST) have been described elsewhere (43). Plasmid MMTV-Luc was a kind gift from Dr. Ben Peeters (44). Plasmid with the sequence –398 to +25 of the avian skeletal {alpha}-actin promoter cloned upstream of the firefly luciferase gene is described elsewhere (45).

Analysis of Skeletal {alpha}-Actin Expression—C2C12 myoblasts were plated on 100-mm plates at a density of 106 cells/well. Myoblasts were first cultured in growth medium, which was switched after 24 h to differentiation medium. Myogenic cells were either treated with 50 nM of the synthetic androgen R1881 (PerkinElmer Life Sciences)/ml of culture medium or control dilutant. After the indicated times, cells were harvested in TRIzol (Invitrogen) and assayed by standard RNA blotting using a probe specific for the 3'-untranslated region of the skeletal {alpha}-actin gene (46) using previously described conditions (47). The probe mouse {alpha}-skeletal 3'-untranslated region (196 bp) (48) was cut with EcoRI and HindIII, gel purified, and labeled with 10 ng of the "Ready to go" kit (Amersham Biosciences) and [32P]dCTP from ICN.

Transfection Assays—Cells were seeded in 24-well plates at a density of 35,000 cells per well and left overnight. The next day, transfections were carried out with 2.5 µl of Lipofectamine (Invitrogen) according to the manufacturer's instructions. Expression vectors were used at 0.4 µg/well; reporter used at 0.2 µg/well. Cells were harvested in cell lysis buffer (Promega) 48 h after transfection, and assayed using luciferase substrate as described previously (45). For stable transfection, we used human androgen receptor cDNA, subcloned in the cytopmegalovirus-driven vector pCGN (41), which confers a hemagglutinin tag to express AR in stably transfected C2C12 myoblasts (Fig. 1A). For this purpose, we transfected C2C12 cells with either pCGN vector containing the hAR cDNA insert, or pCGN vector devoid of insert. We included a 20-fold lesser amount of vector pCMV-Neo, which encodes for the selectable trait of geneticin ("G418") resistance. After at least 2 weeks in selective medium, we pooled and expanded the surviving cells. C2C12 myoblasts plated at a density of 1 x 106 cells/100-mm tissue culture dish in growth medium were co-transfected with 20 µg of pCGN or pCGN-hAR and 1 µg of pCMVneo selection plasmid in a DNA/Lipofectamine mixture. After overnight incubation, myoblasts were allowed to recover, split 1:20 with fresh growth media, and then challenged in selection media (400 µg/ml G418 from Invitrogen). Cells were selected ~10–14 days following the addition of selection media, and were evaluated by harvesting in SDS sample buffer and analysis by Western blot as follows: 3 x 105 C2C12 myoblasts stably transfected either with pCGN or with pCGN-hAR, growing on 60-mm plates, were taken up in SDS sample buffer. Aliquots were loaded on an SDS-PAGE gel, and transferred onto an Immobilon-P membrane (Millipore). Other aliquots loaded on SDS-PAGE were visualized with Coomassie stain to see comparative protein loads. The membrane was assayed by immunoblotting for the hemagglutinin-tagged hAR, using anti-HA antibody (clone 12CA5 Roche) according to the manufacturer's instructions at the dilution 1:100, and secondary anti-mouse antibody from Bio-Rad (catalog number 170-6516).



View larger version (57K):
[in this window]
[in a new window]
  		FIG. 1.
Stably AR-transfected C2C12 myoblasts caused precocious fusion, myotube formation, and appearance of skeletal {alpha}-actin mRNA. Panel A, myoblasts, stably transfected with empty vector pCGN (c) or pCGNhAR (clones 1, 2, 3) were cultured in charcoal-stripped DMEM with 20% fetal calf serum until 70% confluent, and then brought in charcoal-stripped DMEM with 2% horse serum for 48 h. Clones 1, 2, and 3, demonstrate extensive development of myotubes. Panel B, C2C12 myoblasts were plated on 100-mm plates at a density of 106 cells/well. Cells were then brought in either growth medium (GM) or differentiation medium (DM). Cells were also either left untreated or treated with 50 nM R1881/ml of culture medium. After the indicated times, cells were harvested and assayed by Northern blotting for the 3'-untranslated region of the skeletal {alpha}-actin gene. Panel C, myoblasts stably transfected with vector pCGN hAR were cultured in charcoal-stripped DMEM with 20% fetal calf serum until 70% confluent, and then brought in charcoal-stripped DMEM with 2% horse serum for 6, 12, 18, and 24 h. Rabbit polyclonal anti-AR antibody was used to immunoprecipitate AR from total cell extracts and monoclonal anti-SRF antibody was used for immunoblot.

 
For cell differentiation assay, C2C12 myoblasts were plated on six-well plates at a density of 105 cells/well. After 24 h cells were washed with phosphate-buffered saline and transferred to charcoal-stripped Dulbecco's modified Eagle's medium (DMEM) and 2% horse serum. After 24, 48, 72, and 96 h, cells were viewed by an Olympus IMT 2-F microscope and photographed using an Olympus OM88 camera.

GST Fusions of AR or SRF—These fusions were expressed in Escherichia coli BL21-Gold (DE3) (Stratagene), and bacteria were broken by sonication. After pelleting debris, aliquots of the supernatants were taken to isolate GST fusions using glutathione-Sepharose beads (Amersham Biosciences) according to the manufacturer's instructions. After 3 washes, supernatant aliquots were loaded on an SDS-PAGE gel, and visualized by Coomassie staining. The remainder were used in binding assays, by incubating with 20 µl of reticulocyte-translated 35S-labeled SRF. After five washes with phosphate-buffered saline, pellets were taken up in SDS sample buffer and analyzed by SDS-PAGE via autoradiography to detect 35S-labeled SRF or AR. GST-fused protein loading was analyzed by Coomassie stain, whereas labeled proteins were visualized by loading 10% of input. In vitro transcription-translation of proteins was carried out with TNT (Promega), using the manufacturers' instructions.

Coimmunoprecipitations—For coimmunoprecipitation, C2C12 myoblasts stably transfected with hAR were harvested in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 300 mM KCl, 5% (v/v) glycerol, 0.5% (v/v) Triton X-100, 1 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml aprotinin). Cells were lysed for 1 h at 4 °C, and the C2C12 soluble lysate was mixed with non-immune sera or polyclonal rabbit IgG anti-hAR specific for the C-terminal AR (Santa Cruz Biotechnology, Inc.) or 2 µg of normal rabbit serum IgG bound to 10 µl of protein A-Sepharose beads for 2 h at 4 °C. The beads were then washed three times in immunoprecipitation buffer, washed once in phosphate-buffered saline, eluted in SDS-PAGE sample buffer, and run on 10% SDS-PAGE gels under reducing conditions.

DNA Affinity Precipitations—These precipitations were carried out as described by Chen and Schwartz (33), using 25 µl of reticulocyte-translated protein for each reactant. SRF binding sites used their complementary oligonucleotides, after boiling and overnight return to room temperature, to form double strands: SRE3, biotin-5'-GGACGCTCCTTATACGGCCCGG-3'; and SREmt, biotin-5'-GGACGCTaaTTATACGGCCCGG-3'.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increased Expression of the Human Androgen Receptor in C2C12 Myoblasts Enhanced Terminal Myogenic Differentiation—Human androgen receptor, a member of the family of steroid binding nuclear receptors (16), has DBD (DNA binding domain) and ligand binding domains identical to those of mouse and rat (49), whereas the N-terminal domain and hinge region are also highly conserved. We asked if stably transfected pCGN-hAR would affect differentiation of cultured murine C2C12 myoblasts. hAR myoblasts formed myotubes precociously and to an overwhelming extent when compared with myoblasts transfected with the empty pCGN vector (Fig. 1A). In particular, as shown in Fig. 1A, after 48 h hAR-transfected cell clones had already started to convert to multinucleated myotubes, whereas vector-only cells were just initiating differentiation.

Skeletal {alpha}-actin ({alpha}Sk) is a major component of differentiated striated muscle (46, 50) in the adult vertebrate and appears post-replication and after fusion in terminally differentiated myogenic cultures. In growth media, C2C12 stable hAR transfectants barely induced the appearance of {alpha}Sk mRNA over control myoblasts. However, following a switch to differentiation media, we observed a robust appearance of {alpha}Sk transcripts and myoblast fusion in hAR-transfected C2C12 cells, independent of exogenous androgen ligand, R1881. We have observed that {alpha}SK appeared ~96 h post-platting of myogenic cultures (51), as observed in Fig. 1B. Thus, addition of hAR caused precocious induction of a terminal differentiation marker, {alpha}Sk, within 48 h of plating myoblasts stably transfected with hAR, in contrast to control myoblasts containing empty vector (Fig. 1B). These results revealed a profound role for AR in early myogenesis and indicated that {alpha}Sk is an AR gene target. Fig. 1C shows a 10% PAGE and anti-SRF blot of soluble extracts taken from the stable transfected C2C12 cultures; indicating association of SRF with hAR protein, as SRF expression is induced by the transfer of myoblasts in differentiation medium.

Skeletal Actin Gene Promoter Is Activated by AR in an SRF-dependent Fashion—We determined that there were no consensus DNA recognition sites for AR on the {alpha}Sk-actin promoter (–398 to +25 nucleotides; described in Ref. 45), employing the program TFSEARCH2 set at reduced default settings to a score of 70 (52). Because other closely related members of the steroid family were shown to interact with SRF, we asked if AR might also require cofactors such as SRF to transactivate the {alpha}SK-actin promoter. In proliferating C2C12 cells (low in SRF, Ref. 39), neither expression of SRF or AR alone appreciably increased reporter activity. The presence of the synthetic androgen, R1881, and SRF increased luciferase activity by 7.5-fold (Fig. 2A), when compared with pCGN. However, transient co-transfection experiments with hAR and SRF expression vectors with the {alpha}Sk-actin promoter luciferase reporter gene revealed synergistic co-activation of AR with SRF in prefusion C2C12 myoblasts (Fig. 2A) by increasing promoter activity by 22-fold. The addition of R1881 to SRF and AR cotransfected myoblasts stimulated {alpha}Sk-actin promoter transcription up to 98-fold over pCGN levels, thus, showing dependence for full activation on the addition of exogenous androgens.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 2.
Synergy shared between AR and SRF strongly stimulated a skeletal-actin promoter activity and was dependent upon androgen. Panel A, C2C12 myoblasts were transiently transfected with the Luciferase gene under the control of the {alpha}-skeletal actin promoter (with/without SRE) and the indicated expression vectors. Cells were brought to charcoal-stripped 10% fetal bovine serum/DMEM, and assayed after 48 h. Under those conditions, cotransfection of SRF and AR confers androgen inducibility (filled column, +R1881; white, R1881) to the skeletal actin promoter. This experiment was repeated with two different batches of cells, each time in triplicate. Panel B, AR required full-length SRF to increase skeletal actin promoter activity. SRF mutants capable of sustaining promoter activity were, nevertheless, unable to cooperate with AR. CV1 fibroblasts were transiently transfected with the Luciferase gene under the control of the skeletal actin promoter, and the indicated expression vectors. Cells were brought to charcoal-stripped 2% horse serum, 7.5 µg/ml insulin, DMEM (differentiation medium) for 48 h, and assayed for luciferase activity. SRF abbreviations: Wt, wild type, 1–266 (contains N terminus and MADS box); {Delta}MADS, lacks MADS box; {Delta}N, lacks N terminus; pm, point mutations on DNA binding domain; {Delta}5, lacks the part of transactivation domain that is encoded by the fifth exon. This experiment was repeated in triplicate cultures four times (shown is a representative), with cells from three different batches, with identical results. Error bars = S.D.

 
SRF is a protein of 550 amino acids. 141–222 comprise a DNA binding and dimerization domain (referred to as a MADS box), whereas the C terminus, including amino acids 384–448, has transactivation function (42). We made several truncated SRF proteins to define the domains of SRF that may be required for co-activation with AR. We used cells devoid of appreciable SRF (42) or AR (19) expression: co-activation of promoter activity was observed in co-transfected CV1 fibroblasts (Fig. 2B). Only full-length SRF was able to cooperate with AR in activation of the {alpha}Sk-actin promoter. All mutants of SRF had in the presence of AR lower transactivation potential than alone. Absence of SRE and thereby DNA-bound SRF abrogates induction of the promoter.

Recruitment of Androgen Receptor by SRF on Intact SRE— We next investigated whether SRF could selectively recruit AR protein in the cellular lysate containing SRF binding sites. The SRE3, a site essential for full activity of the skeletal actin promoter, located between –186 to –167 bp (55), was biotinylated and precipitated using avidin-coated magnetic beads. The bound material was eluted and analyzed by SDS-PAGE. We show that AR is specifically recruited by SRE3 only in the presence of SRF (Fig. 3). This demonstrates the possibility of a physical association of hAR with DNA-bound SRF. Our results indicate that SRF and AR are able to establish protein-protein contacts in solution and AR protein is recruited to the SRF DNA binding site; it is unlikely that both proteins interact with each other by nonspecific aggregation.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 3.
hAR protein was recruited to an SRF DNA binding target. Reticulocyte-translated, 35S-labeled AR and SRF were assayed for binding to a biotinylated SRE3 DNA binding site. Translated proteins are shown to the left, results of SRE3 precipitation by paramagnetic avidin beads to the right. This assay was repeated three times with very similar results.

 
Wild-type AR Is Required for Androgen-dependent Skeletal Actin Promoter Activity—We asked if any of the AR protein domains (17) were required for regulating {alpha}Sk-actin reporter activity under differentiation conditions. In these experiments myogenic differentiation induces the appearance of SRF (39), thus, the addition of SRF expression vectors was viewed as being redundant and were not included in the assays. After switching to differentiation medium, Fig. 4A showed that {alpha}Sk-actin promoter activity was stimulated by transient co-transfection of full-length AR and required the presence of the synthetic androgen, R1881. Removal of the ligand-binding domain provided activity similar to the wild type AR, but was insensitive to exogenous androgen. Removal of the AR activation domain (AR-(528–910)) also prevented {alpha}SK promoter activation by steroid. It is interesting to note that co-transfection of C2C12 myoblasts with the MMTV-Luc reporter and wild type and mutated AR expression vectors showed that wild type AR caused robust transactivation dependent on androgen, whereas deletion of the LBD caused MMTV activation independent of exogenous R1881. Other mutations in regions that surround the activation domain or that partially delete the activation domain still allowed for MMTV promoter activation, a promoter containing AR binding sites (44) to wild type AR levels (Fig. 4B). Thus, the loss of androgen-dependent activation of the {alpha}Sk-actin promoter activity by the AR mutants (Fig. 4A) cannot be explained by a deficiency in their transactivation potential, but may be explained by weakened association with SRF. Also, judging from the fact that AR mutants contained intact DNA binding domain, it must be concluded that {alpha}Sk-actin does not behave like a target of direct AR DNA binding, but requires parts of AR protein distinct from those that induce MMTV activity, to work with SRF. Because the AR truncations displayed deficient activation of skeletal {alpha}-actin promoter, at least two AR domains are expected to interact with SRF.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4.
Differential transcriptional activities of AR mutants were revealed by assays with the SRF target, {alpha}SK-actin promoter, and the AR target, MMTV promoter. C2C12 myoblasts were cotransfected with the Luciferase gene under the control of the skeletal actin promoter (panel A) or the MMTV promoter (which contains AR binding sites: panel B), and the indicated expression vectors. The next day cells were brought in charcoal-stripped differentiation medium with (filled) or without (white) the synthetic androgen R1881. 48 h after, cells were harvested and assayed for luciferase activity. AR, a 910-amino acid protein contains transactivation functions in amino acids 1–550, the DNA binding domain at 550–615, and the ligand (steroid:androgen) binding domain at 662–910. (Numbers on the AR mutants refer to the parts of the primary amino acid sequence that each mutant contains.) Experiments of this figure were repeated with two different passages of cells, each time in triplicate.

 
Identification of SRF Interacting Domains in hAR—Which domains of AR protein are necessary for SRF interaction? We used glutathione-coated Sephadex beads to precipitate portions of the AR protein linked to GST (43), by incubating them in the presence of reticulocyte-translated 35S-labeled SRF. Domains with a selectively higher affinity for SRF ought to recruit SRF to the beads. We analyzed the results by taking aliquots of precipitated, washed beads in SDS-PAGE buffer and subsequent electrophoresis. Coomassie stain was used to visualize GST-fused AR domains, whereas AR-recruited, labeled SRF was visualized by autoradiography. Multiple GST-fused AR domains were capable of interaction with SRF (Fig. 5). This hints to multiple contacts between the two proteins, and explains why in transfection experiments (Fig. 4) all tested deletions abolished the synergistic effect of hAR with SRF. This experiment indicates that the C-terminal tip of 55 amino acids (865–919) does not bind SRF and may interfere in AR-SRF binding as revealed by the strong binding of the AR LBD minus the N-terminal tip versus the weaker binding of AR LBD full-length to SRF. At least part of this functional interaction can be ascribed to the capacity for a selective physical association between AR and SRF. Our binding results suggested that amino acids overlapping the DBD, from amino acids 634 to 804, amino acids 505 to 635, and especially amino acids 505 to 566, appear to mediate the protein-protein interactions between AR and SRF. Our results suggest that two separate subdomains of the AR DBD mediate protein-protein interactions with SRF.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 5.
Identification of interacting hAR and SRF domains. GST-hAR mutant recruitment of SRF. Bacterially expressed GST fusions of AR domains were incubated with reticulocyte-translated, 35S-labeled SRF, mixed with glutathione-coated Sepharose beads, and washed five times using phosphate-buffered saline. The pellets were taken up in SDS sample buffer and separated using SDS-PAGE. The gel was then dried and autoradiographed. Shown here is one of two experiments performed with cell lysates of two different batches. Left is the Coomassie-stained gel.

 
When we carried out the converse experiment, 35S-labeled AR was strongly recruited by GST-fused wild type SRF, but to a far lesser extent by SRF mutants (Fig. 6). Because all data indicated that SRF and AR make more than one interdomain contact, we assayed GST-fused SRF (wild type and mutants) for recruitment of 35S-labeled AR LBD or AR-(1–370) (Fig. 7). The binding study indicates that the SRF N-terminal sequences, including the {alpha}1-coil of the MADS box, recruited AR LBD, whereas SRF constructs containing C-terminal sequences bound to AR-(1–370).



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 6.
hAR recruitment by SRF mutants fused to GST. Lanes are as indicated. The autoradiograph shows 35S-labeled full-length hAR recruited by the indicated GST fusions with SRF mutants. This is representative of three experiments, each performed with different reticulocyte lysates, and different bacterial lysates. All experiments yielded identical results. Left is the Coomassie-stained gel. DMADS, lacks MADS box.

 



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 7.
hAR domain 1–370 and LBD were differentially recruited by SRF truncations. 35S-Labeled hAR-(1–370) or hAR LBD recruited by GST-fused SRF truncations. Panel A, hAR-(1–370) recruited by GST-fused SRF truncations. Panel B, hAR LBD assayed for recruitment by GST-fused SRF truncations. Bottom, Coomassie-stained gels. Shown each time is a representative of three experiments with very similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Detailed mechanisms by which the androgen-AR signaling pathway regulates skeletal muscle development required further study at the molecular level. Previously Joubert and Tobin (56) demonstrated that testosterone treatment of rat levator ani muscle resulted in activation of quiescent satellite cells, and increased replication and the fusion of satellite cells with myofibers. In the rodent, the role of AR in exercise-induced muscle growth was proven (57), as antiandrogen significantly reduced skeletal muscle gain.

Lee (58) showed that C2C12 myoblast cells stably transfected with AR suppressed skeletal myoblast cell growth and accelerated myoblast cell differentiation and up-regulated myogenin expression. We also observed that human AR strongly facilitated C2C12 myotube formation and stimulated increased {alpha}Sk-actin mRNA steady state levels within 48 h upon exposure to charcoal-stripped differentiation medium, in the absence of exogenous androgens. The {alpha}Sk-actin promoter was stimulated by AR and SRF; eliminating SRF DNA binding blocked coactivation (Fig. 2B). In proliferating myoblasts reporter gene activity responded to androgens. Thus, sarcomeric contractile protein gene activity was switched to higher levels by either increasing available AR, or ligand, or both. Recruitment of AR by selective association with SRF via SREs appears to be a primary mechanism for promoting myogenesis. The capacity of AR to transactivate in the absence of steroids (Fig. 1B versus Fig. 2A) in stable transfectants and normally AR-deficient cells probably results from insufficient endogenous levels of AR-sequestering proteins that would normally retain stably overexpressed AR in the cytoplasm, and prevent AR contact with SRF. Indeed, low level expression of AR results in strong agonist-dependent activation, while increasing AR leads to hormone-independent activation (59).

We mapped the physical and functional roles of AR domain interactions with SRF (Figs. 5, 6, 7). In vitro, GST-fused AR parts capable of SRF recruitment were regions 505–553 (residues N-terminal from DBD, including part of the first zinc finger) and 634–804 (part of the hinge region and the first two-thirds of LBD), were examined, without excluding potential contributions from other regions. In detail, aa 505–566, aa 505–635, and aa 1–627 (all off them include 505–553) bound SRF, whereas the region at aa 553–635 showed diminished binding. Our data indicated that aa 865–919 (C-terminal one-third of LBD) had a negative effect (in the context of aa 634–919 and 505–919) when it was part of GST-AR. By itself it was also unable to bind SRF (Fig. 5). In contrast, aa 634–804 bound SRF, demonstrating that LBD is another AR domain region displaying high affinity for SRF. We also found that the AR N terminus and DBD (aa 1–627) activated MMTV promoter, as strong as AR full-length (Fig. 4B), but failed to synergize with SRF on the skeletal actin promoter, in comparison with the wild type AR (Fig. 4A). These data showed that the AR ligand binding domain also associates with SRF. In vitro, LBD interaction with SRF was seen both by recruitment of SRF from the GST-LBD (Fig. 5, 634–804), and by recruitment of LBD by the GST-fused SRF (Fig. 7B). Thus, both the hAR LBD and hAR N terminus plus DBD have the capacity to interact with SRF, and that neither association alone can restore the strength of the interaction of SRF with wild type hAR.

In cell culture, full-length SRF was obligatory for AR to influence skeletal actin promoter activity in nonmyogenic, SRF-deficient CV1 cells. If the AR-SRF cooperative effect was because of titration of a common repressor protein by AR, then AR-SRF coactivation should also occur with SRF mutants, and not only SRF wild type. Deletions of the fifth exon, MADS box, or N terminus of SRF, in all cases nullified AR co-stimulatory activity on skeletal actin promoter (Fig. 2B). We also observed that wild type SRF (Fig. 6) bound AR more avidly than any of the SRF mutants. Under closer scrutiny, GST-fused N-terminal SRF sequences interacted with AR LBD, whereas C-terminal sequences interacted with AR-(1–370) (Fig. 7). Thus, these results support the notion that AR and SRF interact through more than one domain and that AR and SRF might actually align antiparallel to each other. This type of association may result in juxtaposition of their transactivation domains, because the AR transactivation domain is in the AR N terminus, and the SRF transactivation domain in the C terminus. Interestingly, AR contacts N- and C-terminal ends of the RAP74 subunit of basal transcription factor TFIIF (60), whereas SRF contacts the central part of RAP74 (61). This might explain part of the AR-SRF synergism as presentation of mutually complementary surfaces to TFIIF, especially because RAP74 was required to relieve SRF-dependent squelching (53).

The AR-SRF interaction we describe here is surprisingly different from the one described before between MEF2 and thyroid receptor (54), two proteins homologous to AR (thyroid receptor) and SRF (MEF2 is a MADS box protein). MEF2 and thyroid receptor interact via their DNA binding domains, whereas SRF and AR interaction involves different parts of the two proteins. The fact that a number of mutations of AR or SRF disrupt the synergistic effect on skeletal actin promoter activity, while not disrupting activation of promoters responding to either SRF or AR alone, argues against an interaction mediated by mutual expression of a third protein. If AR effects on skeletal actin were because of independent action of AR, then truncated AR-(1–627) would still activate skeletal actin promoter activity, at levels comparable with wild type AR, which it did on the AR-dependent MMTV promoter. If in cultured cells AR-SRF interaction was mediated by a third protein, then it would not require both proteins to be full-length; retention of contact surface with that third protein would suffice. This certainly does not exclude the possibility that a large number of other factors and AR coactivators (reviewed Ref. 24) might further influence or modulate the AR-SRF interaction. To conclude, myogenic gene expression, as revealed by the activation of the SRF target gene skeletal {alpha}-actin, was regulated by AR and androgen ligand. As SRF is a transcription factor central to the myogenic process and essential for skeletal {alpha}-actin expression, AR shows the potential to activate a host of SRF-dependent genes.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01 HL50422 and P01 HL49953 (to R. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Inst. of Biosciences and Technology, The Texas A&M University System Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-677-7710; E-mail: rschwartz{at}ibt.tamhsc.edu.

1 The abbreviations used are: AR, androgen receptor; SRF, serum response factor; SRE, serum response element; hAR, human androgen receptor; LBD, ligand binding domain; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; DBD, DNA binding domain; {alpha}Sk, skeletal {alpha}-actin; MMTV, murine mammary tumor virus; aa, amino acid(s). Back

2 TFSEARCH: Searching Transcription Factor Binding Sites, rwcp.or.jp/papia. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Ben Peeters, University of Leuven, B-3000 Leuven, Belgium, for the gift of MMTV-Luc.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Mauras, N., Hayes, V., Welch, S., Rini, A., Helgeson, K., Dokler, M., Veldhuis, J. D., and Urban, R. J. (1998) J. Clin. Endocr. Metab. 83, 1886–1892[Abstract/Free Full Text]
  2. Mastrogiacomo, I., Feghali, G., Foresta, C., and Ruzza, G. (1982) Arch. Androl. 9, 293–296[Medline] [Order article via Infotrieve]
  3. Bhasin, S., Storer, T. W., Berman, N., Yarasheski, K. E., Clevenger, B., Phillips, J., Lee, W. P., Bunnell, T. J., and Casaburi, R. (1997) J. Clin. Endocrinol. Metab. 82, 407–413[Abstract/Free Full Text]
  4. Ferrando, A. A., Sheffield-Moore, M., Yeckel, C. W., Gilkison, C., Jiang, J., Achacosa, A., Lieberman, S. A., Tipton, K., Wolfe, R. R., and Urban, R. J. (2002) Am. J. Physiol. 282, E601–E607
  5. Alen, M., and Hakkinen, K. (1985) Int. J. Sports Med. 6, 24–29[Medline] [Order article via Infotrieve]
  6. Sheffield-Moore, M. (2000) Ann. Med. 32, 181–186[Medline] [Order article via Infotrieve]
  7. Herbst, K. L., and Bhasin, S. (2004) Curr. Opin. Clin. Nutr. Metab. Care 7, 271–277[Medline] [Order article via Infotrieve]
  8. Hakkinen, K., Pakarinen, A., Kraemer, W. J., Newton, R. U., and Alen, M. (2000) J. Gerontol. A Biol. Sci. Med. Sci. 55, B95–B105[Abstract]
  9. Hakkinen, K., Pakarinen, A., Kraemer, W. J., Hakkinen, A., Valkeinen, H., and Alen, M. (2001) J. Appl. Physiol. 91, 569–580[Abstract/Free Full Text]
  10. Basaria, S., Wahlstrom, J. T., and Dobs, A. S. (2001) J. Clin. Endocrinol. Metab. 86, 5108–5117[Abstract/Free Full Text]
  11. Urban, R. J., Bodenburg, Y. H., Gilkison, C., Foxworth, J., Coggan, A. R., Wolfe, R. R., and Ferrando, A. (1995) Am. J. Physiol. 269, E820–E826[Medline] [Order article via Infotrieve]
  12. Sheffield-Moore, M., Urban, R. J., Wolf, S. E., Jiang, J., Catlin, D. H., Herndon, D. N., Wolfe, R. R., and Ferrando, A. A. (1999) J. Clin. Endocrinol. Metab. 84, 2705–2711[Abstract/Free Full Text]
  13. Shahidi, N. T. (2001) Clin. Ther. 23, 1355–1390[CrossRef][Medline] [Order article via Infotrieve]
  14. Evans, N. A. (2004) Am. J. Sports Med. 32, 534–542[Abstract/Free Full Text]
  15. Gronemeyer, H., and Govindan, M. V. (1986) Mol. Cell. Endocrinol. 46, 1–19[CrossRef][Medline] [Order article via Infotrieve]
  16. Evans, R. M. (1988) Science 240, 889–895[Abstract/Free Full Text]
  17. Jenster, G., van der Korput, H. A., van Vroohoven, C., van der Kwast, T. H., Trapman, J., and Brinkmann, A. O. (1991) Mol. Endocrinol. 5, 1396–1404[Abstract/Free Full Text]
  18. Zhou, Z. X., Lane, M. V., Kemppainen, J. A., French, F. S., and Wilson, E. M. (1995) Mol. Endocrinol. 9, 208–218[Abstract/Free Full Text]
  19. Kemppainen, J. A., Lane, M. V., Sar, M., and Wilson, E. M. (1992) J. Biol. Chem. 267, 968–974[Abstract/Free Full Text]
  20. Green, S., and Chambon, P. (1988) Trends Genet. 4, 309–314[CrossRef][Medline] [Order article via Infotrieve]
  21. Laudet, V., Haenni, C., Coll, J., Caetzeflis, F., and Stechelin, D. (1992) EMBO J. 11, 1003–1013[Medline] [Order article via Infotrieve]
  22. Forman, B. M., and Samuels, H. H. (1990) Mol. Endocrinol. 4, 1293–1301[Abstract/Free Full Text]
  23. Freedman, L. P. (1992) Endocr. Rev. 13, 129–145[Abstract/Free Full Text]
  24. McKenna, N. J., Lanz, R. B., and O'Malley, B. W. (1999) Endocr. Rev. 20, 321–344[Abstract/Free Full Text]
  25. Sato, N., Sadar, M. D., Bruchovsky, N., Saatcioglu, F., Rennie, P. S., Sato, S., Lange, P. H., and Gleave, M. E. (1997) J. Biol. Chem. 272, 17485–17494[Abstract/Free Full Text]
  26. Hayes, S. A., Zarnegar, M., Sharma, M., Yang, F., Peehl, D. M., ten Dijke, P., and Sun, Z. (2001) Cancer Res. 61, 2112–2118[Abstract/Free Full Text]
  27. Kang, H.-Y., Lin, H.-K., Hu, Y.-C., Yeh, S., Huang, K.-E., and Chang, C. (2001) Proc. Natl. Acad. Sci. U. S. A. 9, 3018–3023
  28. Palvimo, J. J., Reinikainen, P., Ikonen, T., Kallio, P. J., Moilanen, A., and Janne, O. A. (1996) J. Biol. Chem. 271, 24151–24156[Abstract/Free Full Text]
  29. Aarnisalo, P., Palvimo, J. J., and Janne O. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2122–2127[Abstract/Free Full Text]
  30. Yuan, X., Lu, M. L., Li, T., and Balk, S. P. (2001) J. Biol. Chem. 276, 46647–46654[Abstract/Free Full Text]
  31. Schneikert, J., Peterziel, H., Defossez, P.-A., Klocker, H., de Launoit, Y., and Cato, A. C. B. (1996) J. Biol. Chem. 271, 23907–23913[Abstract/Free Full Text]
  32. Belaguli, N. S., Sepulveda, J. L., Nigam, V., Charron, F., Nemer, M., and Schwartz, R. J. (2000) Mol. Cell. Biol. 20, 7550–7558[Abstract/Free Full Text]
  33. Chen, C. Y., and Schwartz, R. J. (1996) Mol. Cell. Biol. 16, 6372–6384[Abstract]
  34. Chen, C. Y., Croissant, J., Majesky, M., Topouzis, S., McQuinn, T., Frankovsky, M. J., and Schwartz, R. J. (1996) Dev. Genet. 19, 119–130[CrossRef][Medline] [Order article via Infotrieve]
  35. Graham, R., and Gilman, M. (1991) Science 251, 189–192[Abstract/Free Full Text]
  36. Ling, Y., West, A. G., Roberts, E. C., Lakey, J. H., and Sharrocks, A. D. (1998) J. Biol. Chem. 273, 10506–10514[Abstract/Free Full Text]
  37. Mack, C. P., Thompson, M. M., Lawrenz-Smith, S., and Owens, G. K. (2000) Circ. Res. 86, 221–232[Abstract/Free Full Text]
  38. Chang, P. S., Li, L., McAnally, J., and Olson, E. N. (2001) J. Biol. Chem. 276, 17206–17212[Abstract/Free Full Text]
  39. Lee, T. C., Chow, K. L., Fang, P., and Schwartz, R. J. (1991) Mol. Cell. Biol. 11, 5090–5100[Abstract/Free Full Text]
  40. Kim, S. W., Kim, H. J., Jung, D. J., Lee, S. K., Kim, Y. S., Kim, J. H., Kim, T. S., and Lee, J. W. (2001) Oncogene 20, 6638–6642[CrossRef][Medline] [Order article via Infotrieve]
  41. Tanaka, M., and Herr, W. (1990) Cell 60, 375–386[CrossRef][Medline] [Order article via Infotrieve]
  42. Belaguli, N. S., Zhou, W., Trinh, T. H., Majesky, M. W., and Schwartz, R. J. (1999) Mol. Cell. Biol. 19, 4582–4591[Abstract/Free Full Text]
  43. Sun, Z., Pan, J., and Balk, S. P. (1997) Nucleic Acids Res. 25, 3318–3325[Abstract/Free Full Text]
  44. Alen, P., Classens, F., Schoenmakers, E., Swinnen, J. V., Verhoeven, G., Rombauts, W., and Peeters, B. (1999) Mol. Endocrinol. 13, 117–128[Abstract/Free Full Text]
  45. MacLellan, W. R., Lee, T. C., Schwartz, R. J., and Schneider, M. D. (1994) J. Biol. Chem. 269, 16754–16760[Abstract/Free Full Text]
  46. Gunning, P., Ponte, P., Blau, H., and Kedes, L. (1983) Mol. Cell. Biol. 3, 1985–1995[Abstract/Free Full Text]
  47. Virca, G. D., Northemann, W., Shiels, B. R., Widera, G., and Broome, S. (1990) BioTechniques 8, 370–371[Medline] [Order article via Infotrieve]
  48. Leader, D. P., Gall, I., Campbell, P., and Frischauf, A. M. (1986) DNA (N. Y.) 5, 235–238[Medline] [Order article via Infotrieve]
  49. Chang, C. S., Kokontis, J., and Liao, S. T. (1988) Science 240, 324–326[Abstract/Free Full Text]
  50. Schwartz, R. J., and Rothblum, K. (1980) Biochemistry 19, 2506–2514[CrossRef][Medline] [Order article via Infotrieve]
  51. Hayward, L. J., and Schwartz, R. J. (1986) J. Cell Biol. 102, 1485–1493[Abstract/Free Full Text]
  52. Heinemeyer, T., Wingender, E., Reuter, I., Hermjakob, H., Kel, A. E., Kel, O. V., Ignatieva, E. V., Ananko, E. A., Podkolodnaya, O. A., Kolpakov, F. A., Podkolodny, N. L., and Kolchanov, N. A. (1998) Nucleic Acids Res. 26, 364–370
  53. Zhu, H., Joliot, V., and Prywes, R. (1994) J. Biol. Chem. 269, 3489–3497[Abstract/Free Full Text]
  54. Lee, Y., Nadal-Ginard, B., Mahdavi, V., and Izumo, S. (1997) Mol. Cell. Biol. 17, 2745–2755[Abstract]
  55. Croissant, J. D., Kim, J. H., Eichele, G., Goering, L., Lough, J., Prywes, R., and Schwartz, R. J. (1996) Dev. Biol. 177, 250–264[CrossRef][Medline] [Order article via Infotrieve]
  56. Joubert, Y., and Tobin, C. (1995) Dev. Biol. 169, 286–294[CrossRef][Medline] [Order article via Infotrieve]
  57. Inoue, K., Yamasaki, S., Fushiki, T., Okada, Y., and Sugimoto, E. (1994) Eur. J. Appl. Physiol. Occup. Physiol. 69, 88–91[CrossRef][Medline] [Order article via Infotrieve]
  58. Lee, D. K. (2002) Biochem. Biophys. Res. Commun. 294, 408–413[CrossRef][Medline] [Order article via Infotrieve]
  59. Huang, Z. Q., Li, J., and Wong, J. (2002) Mol. Endocrinol. 16, 924–937[Abstract/Free Full Text]
  60. Reid, J., Murray, I., Watt, K., Betney, R., and McEwan, I. J. (2002) J. Biol. Chem. 277, 41247–41253[Abstract/Free Full Text]
  61. Joliot, V., Demma, M., and Prywes, R. (1995) Nature 373, 632–635[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 PhysiologyHome page
M. Sandri
Signaling in Muscle Atrophy and Hypertrophy
Physiology, June 1, 2008; 23(3): 160 - 170.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. H. Hong, H. Sun, C. H. Jin, M. Chapman, J. Hu, W. Chang, K. Burnett, J. Rosen, A. Negro-Vilar, and J. N. Miner
Cell-Specific Activation of the Human Skeletal {alpha}-Actin by Androgens
Endocrinology, March 1, 2008; 149(3): 1103 - 1112.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. V. Heemers, K. M. Regan, S. M. Dehm, and D. J. Tindall
Androgen Induction of the Androgen Receptor Coactivator Four and a Half LIM Domain Protein-2: Evidence for a Role for Serum Response Factor in Prostate Cancer
Cancer Res., November 1, 2007; 67(21): 10592 - 10599.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Jagla, M. Feve, P. Kessler, G. Lapouge, E. Erdmann, S. Serra, J.-P. Bergerat, and J. Ceraline
A Splicing Variant of the Androgen Receptor Detected in a Metastatic Prostate Cancer Exhibits Exclusively Cytoplasmic Actions
Endocrinology, September 1, 2007; 148(9): 4334 - 4343.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
M. Yoshioka, A. Boivin, C. Bolduc, and J. St-Amand
Gender difference of androgen actions on skeletal muscle transcriptome
J. Mol. Endocrinol., August 1, 2007; 39(2): 119 - 133.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
K. Hayashi, S. Nakamura, W. Nishida, and K. Sobue
Bone Morphogenetic Protein-Induced Msx1 and Msx2 Inhibit Myocardin-Dependent Smooth Muscle Gene Transcription
Mol. Cell. Biol., December 15, 2006; 26(24): 9456 - 9470.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/9/7786    most recent
M413992200v4
M413992200v3
M413992200v2
M413992200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vlahopoulos, S.
Right arrow Articles by Schwartz, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vlahopoulos, S.
Right arrow Articles by Schwartz, R. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement