Two Distinct Coactivators, DRIP/Mediator and SRC/p160, Are Differentially Involved in Vitamin D Receptor Transactivation during Keratinocyte Differentiation

Yuko Oda, Carina Sihlbom, Robert J. Chalkley, Lan Huang, Christophe Rachez, Chao-Pei Betty Chang, Alma L. Burlingame, Leonard P. Freedman and Daniel D. Bikle

Departments of Medicine and Endocrinology (Y.O., D.D.B.), University of California San Francisco and Veterans Affairs Medical Center, San Francisco, California 94121; Department of Pharmaceutical Chemistry (C.S., R.J.C., L.H., A.L.B.), University of California San Francisco, San Francisco, California 94143; and Cell Biology Program (C.R., C.-P.B.C., L.P.F.), Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Address all correspondence and requests for reprints to: Yuko Oda, Ph.D., Endocrine Research 111N, Veterans Affairs Medical Center San Francisco, 4150 Clement Street, San Francisco, California 94121. E-mail: y2073{at}itsa.ucsf.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D [1,25-(OH)2D3)], controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the vitamin D receptor (VDR)-interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the VDR. However, the relationship between these two classes of coactivators is not clear. Using glutathione-S-transferase-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP and SRC proteins were expressed in keratinocytes. DRIP205 expression decreased during differentiation, although SRC-3 levels increased. Both DRIP205 and SRC-3 potentiated vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are sequentially involved in vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EPIDERMAL KERATINOCYTES PROVIDE an excellent model for the study of epithelial cell proliferation and differentiation. Keratinocytes undergo differentiation as they migrate to the upper layers of the skin. In cell culture, this process can be reproduced by maintaining epidermal keratinocytes in different calcium concentrations (1). In low calcium (0.03 mM), most keratinocytes are proliferative. Increased calcium (1.2 mM) inhibits proliferation and induces the onset of terminal differentiation accompanied by elevated expression of transglutaminase, involucrin, loricrin, and profilaggrin and cornified envelope formation (2). The active metabolite of vitamin D, 1,25-dihydroxyvitamin D [1,25-(OH)2D3], has profound effects upon this process by potentiating the action of calcium (3). Binding of 1,25-(OH)2D3 to the vitamin D receptor (VDR) is believed to exert this effect by modulating the transcription of target genes, such as the cell cycle inhibitor p21waf1/Cip1 (4), or the cornified envelope protein involucrin (5). Targeted disruption of VDR alters this process both in epidermal and hair follicle keratinocytes (6, 7, 8). VDR, like other nuclear hormone receptors, is modulated in its activity by coactivators and, perhaps, corepressors. In particular, a key complex called VDR-interacting proteins (DRIP) [also known as thyroid hormone receptor-associated protein (TRAP)/Srb/Med containing cofactor complex (SMCC), peroxisome proliferator-activated protein (PBP), activator recruited cofactor (ARC), or human mediator] has been isolated and shown to be required for VDR transactivation (9). In addition, subcomplexes of DRIP have been defined biochemically, as those required for transcription activation in vitro [positive cofactor (PC2) and cofactor required for Sp1 activation (CRSP)]. These coactivators, the subunit compositions of which range in size from seven subunits (CRSP) to at least 18 subunits (negative regulator of activated transcription, SMCC), contain both novel proteins and a subset of proteins homologous to components of yeast mediator (10). Mediator-containing complexes such as DRIP do not have intrinsic histone acetyltransferase (HAT) activity. Their means of enhancing gene transcription has not yet been clearly elucidated, although evidence points to their ability to directly recruit RNA polymerase II (RNA Pol II) to the promoter of regulated genes (11). One subunit, DRIP205/TRAP220, directly binds to VDR and other nuclear receptors through its second nuclear receptor binding motif called NR box, having a conserved LxxLL nuclear receptor binding motif, and is thought to be the main anchor for the complex to VDR (12). A second group of coactivators is the p160 coactivator or the steroid receptor coactivator (SRC) family (reviewed in Ref. 13). The SRC/p160 family includes three members each with multiple names, SRC-1/NcoA-1, SRC-2/TIF2/GRIP1/NcoA-2, and SRC-3/RAC3/pCIP/ACTR/AIB1/TRAM-1. These coactivators also bind to VDR via their NR boxes. The SRC/p160 family recruits other coactivators such as cAMP response element binding protein (CREB)-binding protein (CBP), its homolog p300, and pCAF proteins (13), which likely facilitate transcription through their HAT activity. Importantly, in biochemical purifications, DRIP/mediator and SRC/p160 exist as distinct complexes (12). The coexistence of these two distinct coactivator complexes raises the question of whether they are competitively or cooperatively functioning in transcriptional activation. In this study, we examined their respective roles during keratinocyte differentiation. Our data suggest a model in which both DRIP/mediator and SRC/p160 have important roles during the early stages of differentiation, but subsequently major DRIP components decrease, and SRC/160 has the predominant role in the later stages of differentiation.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To isolate cofactors interacting with VDR, we used a glutathione-S-transferase (GST) fusion protein containing the ligand-binding domain (LBD) of VDR including the activation domain AF-2 essential for binding to the coactivators. Nuclear extracts were prepared from proliferating and differentiated epidermal keratinocytes and from a squamous carcinoma cell line SCC12B2 (SCC). They were incubated with GST-VDR (LBD) affinity beads in the presence or absence of ligand (1 x 10-7 M 1,25-(OH)2D3), using a purification scheme similar to that described in the original isolation of the DRIP complex (14). The bound proteins were eluted and analyzed by SDS-PAGE. The protein complex from proliferating keratinocytes (PKs) included different proteins ranging in molecular mass from 33–250 kDa, which were purified specifically with the ligand (Fig. 1Go, lanes 3 and 4). The protein bands were excised from the sodium dodecyl sulfate (SDS) gel, in-gel digested by trypsin, and subsequently analyzed by mass spectrometry (MS). The proteins were identified by matrix-assisted laser desorption/ionization (MALDI) MS peptide mass mapping and/or peptide sequencing by liquid chromatography-electrospray ionization (nanoLC-ESI) tandem MS (15). The proteins p250–p77 were identified as DRIP250, 240, 205, 150, 130, 100, 92, and 77, respectively (Table 1AGo). The low molecular weight proteins included mammalian homologs of mediator proteins such as mediator 6 and mediator 8, which can assemble with RNA Pol II and Elongin complex capable of stimulating the rate of elongation by RNA Pol II (16). Members of the SRC/p160 family were not observed in the VDR binding complex isolated from PKs using both MS (Table 1Go) and Western analysis (Fig. 3BGo, lane 8). The protein complex from SCC showed a similar subunit profile compared with that of the PKs (Fig. 1Go, lanes 1 and 2). The proteins p250–77 were identified as DRIP subunits DRIP250, 240, 205, 150, 130, 100, 92, and 77 (Table 1BGo). The low molecular weight bands were mammalian homologs of mediator proteins, although different subunits were observed (Table 1Go). Protein p25 is the TATA binding protein-related factor (TRF) proximal protein, which is a component of the RNA Pol II/SRB complex (17). Consistent with this, RNA Pol II appears to be recruited by the DRIP/mediator complex in a VDR- and ligand-dependent manner (11). The DRIP/mediator complex in PKs contains major DRIP subunits including a DRIP205 that directly binds to VDR through its NR boxes. The complex also contains mammalian homologs of yeast mediator proteins (17), indicating that the DRIP/mediator complex may activate transcription (18). The DRIP/mediator complex in SCC contains same major DRIP subunits and mediator proteins, although the composition of the mediator subunit is rather different. However, the existence of mediator 8 or TRF proximal protein, which is a component of the Elongin complex (16) or the RNA Pol II/SRB complex (17), suggests that the DRIP/mediator complex may activate transcription by connecting VDR to the general transcriptional machinery. The TRF proximal protein has been detected in the DRIP subcomplex PC2 and SMCC (18), but not in the DRIP/mediator complex isolated from Namalwa B cells (19). The composition of mediator proteins may be different among different cell types. Several ribosomal proteins were also identified in the low-molecular weight bands from both PKs and SCC, although the biological significance of this observation is not clear.



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Fig. 1. Purification of VDR Binding Proteins from Keratinocytes

Nuclear extracts were prepared from the squamous carcinoma cell line SCC12B2 (SCC) and from primary cultures of PKs and DKs. The extracts were incubated with GST-VDR (LBD) affinity beads in the presence or absence of ligand [1 x 10-7 M 1,25-(OH)2D3) (D3)]. Bound proteins were eluted, separated by SDS-PAGE, and visualized by silver staining. Apparent molecular weights of these bands are shown. These protein bands were excised from SDS gel and analyzed by MS (Table 1Go). A ligand-independent band (*) was observed but was not analyzed. The band between p77 and p60 was GST-VDR leaked from the affinity beads.

 

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Table 1. Identification of VDR Binding Proteins from Keratinocytes by MS

 


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Fig. 3. Western Analysis of DRIP Subunits and SRC Family Members

A, Changes in the expression level of DRIP subunits and SRC family members during keratinocyte proliferation and differentiation. Protein expression of DRIP and SRC was examined. The same amounts of protein in the nuclear extracts from SCC (lanes 1 and 4), PKs (lanes 2 and 5), and DKs (lanes 3 and 6) were subjected to Western analysis. The expression of VDR coactivators, DRIP205, DRIP240, DRIP92, SRC-3, SRC-2, and SRC-1, was detected by incubation with their antibodies. The DRIP levels were compared with SRC levels on the blots where the same amounts of protein samples were loaded (left and right panels). DRIP205 and SRC-3 were observed using both short (S) and long exposure (L) of the same blot; SRC-1 was not detected from any keratinocytes in contrast to positive control cells K562, which showed the expression of SRC-1 (P). B, Western analysis of purified VDR coactivators. The VDR binding complex was purified from keratinocytes using GST-VDR (LBD) affinity beads. The eluted fractions from SCC (lanes 1, 4, and 7), PKs (lanes 2, 5, and 8), and DKs (lanes 3, 6, and 9) were subjected to SDS-PAGE and analyzed by Western blot using an antibody against DRIP205, DRIP240, and SRC-3. Long exposure of DRIP240 on the same blot was also shown in the bottom of the panel of DRIP240.

 
In contrast, differentiated keratinocytes (DKs) showed a ligand-dependent protein pattern with a lack of major DRIP proteins, and the appearance of a p180 band (a doublet) (Fig. 1Go, lanes 5 and 6). Analysis by MS of the p180 band identified tryptic peptides from two proteins, SRC-3 and SRC-2 (Fig. 2AGo). Protein identification was further confirmed by peptide sequencing using tandem MS as shown in representative spectra (Fig. 2Go, B and C). The peptides derived from both SRC-3 and SRC-2 were identified by LC MS/MS as summarized in Table 2Go (A and B, respectively). These results demonstrate that the p180 doublet band is a mixture of the SRC/p160 family members SRC-2 and SRC-3. SRC-1 was not detected in keratinocytes (Table 1Go and Fig. 3AGo). Major DRIP subunits such as DRIP205, 250, 240, 130, and 92 were not identified, although some DRIP proteins, including DRIP150, 100, and 77, remained (Table 1CGo). Several protein bands above p205 were thoroughly analyzed and still remain unknown, although they did not match peptide peaks corresponding to DRIP250, -240, and -205. Absence of DRIP205 and -240 was confirmed by Western analysis of purified VDR binding proteins from DKs (Fig. 3BGo, lanes 3 and 6). These results indicate that the DRIP/mediator complex was the major VDR binding complex in PKs (both normal and SCC), similar to that previously reported in Namalwa B (19) and Hela cells (20). However, upon differentiation, major DRIP subunits decreased and SRC family members became predominant.



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Fig. 2. MS Analysis of the p180 Band from DKs

A, MALDI mass spectrum of the tryptic digest of the p180 band. The spectrum was internally calibrated using trypsin autoproteolysis products (T). Peaks labeled A and B correspond to the proteins SRC-3 and SRC-2, respectively; B, MS/MS spectrum of the tryptic peptide (MH22+ = 710.36) from the liquid chromatography tandem MS (LC-MS/MS) analysis of the p180 band. The sequence was determined as QQVFQGTNSLGLK, a peptide that is derived from SRC-3; C, MS/MS spectrum of the tryptic peptide (MH22+ = 719.37) from the LC-MS/MS analysis of the p180 band; the sequence was determined as FSLSDGTLVAAQTK, a peptide that is derived from SRC-2.

 

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Table 2. Peptide Sequences Identified by LC-MS/MS in the p180 Band from Differentiated Keratinocytes

 
We next examined the protein levels of the DRIP subunits and SRC/p160 family members during keratinocyte differentiation (Fig. 3AGo). Equivalent protein amounts from nuclear extracts of SCC, PKs, and DKs were compared by Western analysis (Fig. 3AGo) (21, 22). DRIP205 was detected as a discrete single band (200 kDa) using our custom-made antibody (Fig. 3AGo, upper left panel). The DRIP205 protein was detected in proliferating cells (lane 2), but decreased after the cells differentiated (lane 3), and was only detected after longer exposure of the same blot (L). DRIP240 and 92 were also decreased by differentiation (Fig. 3AGo). SRC-3 was detected as a single 160-kDa band by a specific monoclonal antibody (Fig. 3AGo, upper right panel). It was expressed at low levels in PKs (lane 5) and increased after the cells differentiated (lane 6). SRC-2 was decreased by differentiation. SRC-1 was not detected in any keratinocytes in contrast to the leukemia cell line K562, which showed SRC-1 expression (P). Major DRIP subunits of DRIP205, DRIP240, DRIP92, and SRC-3 were overexpressed in SCC compared with normal keratinocytes (PK, DK).

The functional requirements for DRIP205 and SRC-3 in the cells at different stages of differentiation were then determined. First, we overexpressed DRIP205 or SRC-3 and examined their effects on vitamin D-induced transactivation by a promoter reporter assay using a vitamin D response element (VDRE) promoter linked with luciferase (12) (Fig. 4Go, A and B). Vitamin D-induced transactivation was significantly potentiated by both DRIP205 and SRC-3 compared with the pcDNA3 vector control in PKs (Fig. 4AGo). In contrast, DKs showed a significant increase in activation by SRC-3 but not by DRIP205 (Fig. 4BGo), even though both SRC-3 and DRIP205 were overexpressed in both PKs and DKs (data not shown). Next, we used the dominant negative construct of DRIP205 (amino acids 527–714; 205 Box Wild), which is derived from the nuclear hormone receptor binding domain containing two LxxLL motifs, to inhibit the function of DRIP205 (Fig. 4Go) (12). A construct that contains an L to A mutation in each NR box in the same construct was also used as a control (205 Box Mut) (Fig. 4Go). When PKs were cotransfected with the dominant negative DRIP205, vitamin D-induced transactivation was significantly inhibited (Fig. 4CGo; 205 Box Wild), but not when transfected by the mutant (Fig. 4CGo; 205 Box Mut). In contrast, when DKs were cotransfected by the dominant negative DRIP205, vitamin D-induced transactivation was not affected (Fig. 4DGo). These results indicate that both DRIP205 and SRC-3 may be involved in vitamin D transactivation in PKs, but only SRC-3 is effective in differentiated cells.



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Fig. 4. Differential Effects of DRIP205 and SRC-3 on Vitamin D Transactivation during Keratinocyte Differentiation

The functional requirements for DRIP205 and SRC-3 were examined by either overexpression (A and B) or dominant negative inhibition (C and D). Both PKs (A and C) and DKs (B and D) were compared. Keratinocytes were transfected with a VDRE construct linked to luciferase. Cells were cotransfected by full-length DRIP205 or SRC-3, dominant-negative DRIP205 (Box Wild) containing LxxLL motif, or the L to A mutant form of this dominant negative DRIP205 (Box Mut). The 1,25-(OH)2D3 (D3)-induced transactivation was measured and compared with vector control (pcDNA3). Data are represented as mean and SD of triplicate measurements. The experiments were repeated at least twice to confirm their reproducibility. All data were analyzed by two-factor ANOVA. Statistical significance compared with pcDNA3 control was shown with asterisks (P < 0.05).

 
Our DRIP/mediator complex did not contain SRC/p160 proteins, although PKs express SRC/p160 proteins. Functional studies showed that both DRIP205 and SRC-3 may be involved in vitamin D-regulated transcription in proliferating cells. Our preliminary data demonstrated that both DRIP and SRC are recruited to a VDRE promoter (24 hydroxylase) in proliferating cells (data not shown), suggesting that both DRIP and SRC may be involved in VDR transactivation in PKs. However, the binding of DRIP/mediator complex to VDR appears to be dominant compared with SRC in PKs. CBP and p300, which are known to be recruited to SRC/p160 complexes, were not detected in our coactivator complex by either MS (Table 1Go) or Western analysis (data not shown). However, CBP and p300 are expressed in keratinocytes (data not shown) and may function in other transcriptional complexes. As differentiation proceeds, major DRIP components decreased, and SRC/p160 family members appeared to play a greater role in VDR transactivation. However, this change is more than just one of substitution. Even when DRIP205 was overexpressed in differentiated cells, it failed to enhance 1,25-(OH)2D3-regulated gene transcription, suggesting that differentiated cells may selectively utilize SRC/p160 coactivators. Alternatively, other DRIP partner subunits missing in differentiated cells may have an important role in VDR transactivation such that the overexpression of only DRIP205 would not be sufficient. The differential expression of DRIP205 has also been reported in rat tissues (23). DRIP205 is widely expressed in embryonic tissues but is attenuated and restricted to reproductive organs in the adult rat. In the adult testes and ovary, its levels fluctuate during the various stages of spermatogenesis and follicle development (23). Targeted disruption of DRIP205 proved to be embryonic lethal, and embryonic fibroblasts showed retarded cell cycle progression (24). Thus, DRIP205 may be involved in transcriptional activation specifically in proliferating cells.

We propose a model (Fig. 5Go) in which both DRIP/mediator and SRC/p160 regulate vitamin D-controlled transcription in a sequential process during differentiation. In the early stages of differentiation, direct activation of the general transcriptional machinery through DRIP/mediator and HAT activity through SRC/p160 may be required to initiate gene transcription by VDR and 1,25-(OH)2D3. Subsequently, major DRIP components decrease, and the SRC/p160 complex becomes dominant, enabling transcription to occur as the cells differentiate. This model could explain how two distinct coactivators are differentially involved in VDR transcription during the differentiation process and how they coordinately function in transcription.



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Fig. 5. Model Showing Selective Utilization of Two Distinct VDR Coactivators of DRIP/Mediator and SRC/p160 in Transcriptional Activation during Keratinocyte Differentiation

Both DRIP/mediator and SRC/p160 may be involved in vitamin D-regulated transcription of undifferentiated proliferating cells (upper). At this stage, transcriptional activation may require the linking of the VDR complex to the general transcription machinery including RNA Pol II and HAT activity through SRC/p160. After the cells are differentiated, DRIP205 apparently is no longer required, and SRC/p160 family members take the dominant role in transcriptional control. Remaining DRIP subunits may also be involved (lower).

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
Normal epidermal keratinocytes were isolated from neonatal human foreskin and grown in serum-free keratinocyte growth medium (KGM) (Clonetics, San Diego, CA) as described previously (1). Second-passage keratinocytes were cultured with KGM containing 0.07 mM calcium for 3 d, and then switched to the same medium containing different concentrations of calcium (0.03 to 1.2 mM) and cultured for different lengths of time before harvest. The human squamous carcinoma cell line SCC12B2 (ATCC, Manassas, VA) was maintained in 10% fetal calf serum in a 1:1 mixture of Ham’s F-12 and DMEM, before being switched to KGM containing 0.07 mM calcium.

Preparation of GST and GST-VDR Beads
GST or GST fusion proteins were prepared using the Bulk GST purification module (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s protocol. Bacterial cultures (BL21) carrying the GST plasmid pGEX2TK or GST-VDR construct (LBD116–427) were grown at 37 C to an OD600 of 0.3, at which time the temperature was reduced to room temperature. Cells were induced by the addition of 0.1 mM isopropyl-D-thiogalactopyranoside at OD600 of 0.6. After 3–4 h, bacteria were collected and resuspended in lysis buffer [PBS containing 0.5 mM phenylmethylsulfonylfluoride (PMSF), 0.5 µg/ml leupeptin, 1 mM dithiothreitol (DTT)], sonicated, and centrifuged. Soluble extracts were incubated with glutathione-Sepharose for 30 min on ice. The beads were washed three times in lysis buffer. The amounts of protein immobilized on beads were estimated by boiling the beads with SDS and analyzed on SDS-PAGE by comparison with various amounts of BSA (Sigma, St. Louis, MO) as standard.

Nuclear Extract
Nuclear extracts were prepared from keratinocytes as described previously (25). Briefly, cells were collected by PBS with a proteinase inhibitor cocktail (Complete mini-EDTA free) (Roche Molecular Biochemicals, Indianapolis, IN) either by scraping or with brief trypsin treatment. Cells were resuspended in hypotonic buffer [10 mM KCl, 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.2 mM PMSF, 0.5 mM DTT, 20 µg/ml leupeptin], lysed using a Dounce homogenizer, and centrifuged to collect the nuclear fraction. Soluble nuclear proteins were extracted by incubation of the nuclear pellet using a high-salt buffer containing up to 0.6 M KCl. The protein concentration of the nuclear extracts was determined by BCA protein assay (Pierce Chemical Co., Rockford, IL).

Purification of VDR Binding Proteins from Keratinocytes
VDR binding proteins were purified using GST-VDR affinity beads as described previously (12). Nuclear extracts were dialyzed to adjust the salt concentration to 150 mM KCl and preincubated with GST beads in binding buffer [10 mM Tris-HCl (pH 7.9), 150 mM KCl, 0.2 mM EDTA, 0.05% Nonidet P-40 containing 0.5 mM PMSF, 1 mM DTT, 20 µg/ml leupeptin, 1000x dilution of protease inhibitor cocktail (Sigma)] to remove nonspecific binding proteins. Meanwhile, GST-VDR beads were preincubated with either vehicle (EtOH) or 1 x 10-7 M 1,25-(OH)2D3 for 30 min. Preadsorbed nuclear extracts were then incubated with GST-VDR (LBD) affinity beads in binding buffer at 4 C overnight in the presence or absence of ligand with 1 mg/ml BSA as carrier protein. After washing the beads six times with binding buffer including 0.1% Nonidet P-40, bound proteins were eluted with 0.2% Sarkosyl in binding buffer. Eluted proteins were separated by SDS-PAGE using a 4–15% gradient gel. The proteins were visualized by silver staining using silver stain plus (Bio-Rad Laboratories, Inc., Hercules, CA) according to the manufacturer’s protocol. For mass spectrometric analysis, eluted protein samples were concentrated using a Microcon 30 column (Millipore Corp., Bedford, MA). The samples were analyzed by SDS-PAGE and visualized by a MS-compatible silver staining protocol (26).

Protein Identification by MS
The protein bands were excised from the SDS gel, digested with trypsin, and identified as described previously (15). Minced gel pieces were subjected to reduction by DTT and alkylation with iodoacetamide. The gel pieces were then rehydrated in trypsin (12.5 ng/µl) and incubated at 37 C for 4 h. Peptides were extracted with 50% acetonitrile, 2% formic acid. The unseparated tryptic digests were desalted using a C18 ZipTip column (Millipore Corp.). The digest (one fifth) was cocrystallized with an equal volume of saturated 2,5-hydroxybenzoic acid in H2O and analyzed by matrix-assisted laser desorption/ionization MS (MALDI-MS) on a Voyager DE-STR (Applied Biosystems, Foster City, CA). The MALDI spectra were internally calibrated using trypsin autoproteolysis products to obtain high mass accuracy (<20 ppm). The monoisotopic peptide masses were submitted for peptide mass mapping using MS-Fit (http://prospector.ucsf.edu) against the public NCBI protein database. Protein identification of selected bands was further confirmed by peptide sequencing using nanoLC-ESI MS/MS using a QSTAR Pulsar (Applied Biosystems/MDS Sciex, Toronto, Ontario, Canada). A Pepmap column (75-mm internal diameter x 150 mm, Dionex, Sunnyvale, CA) was used for HPLC separation, employing a gradient of 0–50% acetonitrile/0.1% formic acid over 50 min. When a peptide was observed above a minimum threshold intensity in the mass spectrum, it was automatically selected for fragmentation by collision-induced dissociation. Fragmentation spectra were initially analyzed against protein databases using Mascot (http://www.matrixscience.com). Some of the uninterpreted collision-induced dissociation spectra by Mascot were submitted to MS-Tag (http:// prospector.ucsf.edu) for protein identification.

Preparation of anti-DRIP205 Antibody
A custom-made antibody against DRIP205 was prepared by rabbit immunization with a keyhole limpet hemocyanin (KLH)-conjugated synthetic peptide (NH2-KNHPMLMNLLKDNPAQDF-COOH) derived from the NR2 box of human DRIP205 (ImmunoVision Technologies, Daily City, CA). The titer and specificity of the antibody were monitored by ELISA and Western analysis. Affinity-purified antibody was prepared from the antiserum using a peptide column.

Western Analysis of Coactivator Proteins
The protein expression of coactivators was detected by Western analysis as described previously (21, 22). Briefly, 20 µg nuclear extracts or purified VDR binding proteins from keratinocytes were separated by SDS-PAGE and electroblotted onto a polyvinylidenedifluoride membrane. After blocking, the blot was incubated with our custom-made polyclonal antibody against DRIP 205, or with a monoclonal antibody raised against the GST fusion protein 605-1294 of human SRC-3 (AIBI, MA1-845; Affinity Bioreagents (ABR), Golden, CO). Antibodies against DRIP240 [TRAP230 (R-19) sc-5375], DRIP92 [TRAP95 (C-19) sc-5363], SRC-2 [GRIP1 (M-343) sc-8996], SRC-1 [SRC-1 (C-20) sc-6096] (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were also used. Subsequently, the blot was incubated with secondary antibody conjugated with horseradish peroxidase (Amersham), and bound antibody was visualized using a chemiluminescence system (SuperSignal ULTRA) (Pierce Chemical Co., Rockford, IL).

Nuclear extract from human chronic myelogenous leukemia cell line K-562 (Santa Cruz Biotechnology, Inc., sc-2131) was used as a positive control for SRC-1 detection.

Promoter Reporter Assay
Human keratinocytes at two different stages in the cell cycle were used for transfection: second-passage human keratinocytes cultured in KGM containing 0.03 mM calcium at the cell density of 30–50% are mainly proliferating cells; keratinocytes maintained in 1.2 mM calcium for 7 d to reach confluence are predominantly differentiated cells. Both cell types were transfected by a promoter reporter construct of either VDREx2 E1B luciferase (12) or the full-length rat 24-hydroxylase promoter containing multiple VDREs (27). For overexpression or dominant negative inhibition, each cDNA construct including control vector pcDNA3, full-length DRIP205 (12), full-length SRC-3 (RAC3), dominant negative of DRIP205 (Box Wild) (12), or the L to A mutant (Box Mut) (12) was cotransfected with the promoter reporter. Proliferating cells (35-mm dish) were transfected using 2 µl Effectene with 300 ng cDNA of promoter reporter, 30 ng cotransfectant, and 6 ng control vector of Renilla luciferase-thymidine kinase promotor (pRL-TK) (Promega Corp., Madison, WI) according to the manufacturer’s protocol (QIAGEN, Valencia, CA). Differentiated cells were transfected by using increased amounts (3-fold) of Effectene and DNA. Transfected cells were treated either with vehicle (EtOH) or 1 x 10-8 M 1,25-(OH)2D3 for 24 h. Cells were lysed using passive lysis buffer (Promega Corp.), and both firefly and Renilla luciferase activities were measured with a dual luciferase reporter assay system (Promega Corp.). Firefly luciferase activity was normalized to the level of Renilla luciferase activity. The results were expressed as the fold induction by 1,25-(OH)2D3 over the vehicle control. Each transfection was carried out in triplicate and repeated at least twice.


    ACKNOWLEDGMENTS
 
We thank Dr. P. N. McDonald for providing the GST-VDR (LBD) construct and Dr. D. Chen for providing the full-length SRC-3 cDNA construct. We thank Dr. Chia-Ling Tu, Dean Ng, and Scott Munson for useful technical advice. We are also grateful to Ms. Sally Pennypacker for cell culture support.


    FOOTNOTES
 
This work was supported by Grants 98A079 (to D.D.B.) from the American Institute of Cancer Research and NIH Grants AR38386 and AR39448 (to D.D.B.) and NIH Grant RR01614 (to A.L.B.).

Abbreviations: CBP, CREB binding protein; DK, differentiated keratinocyte; DRIP, vitamin D receptor-interacting protein; DTT, dithiothreitol; 1,25-(OH)2D3 or D3, 1,25-dihydroxyvitamin D3; GST, glutathione-S-transferase; HAT, histone acetyltransferase; KGM, keratinocyte growth medium; LBD, ligand-binding domain; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; PK, proliferating keratinocyte; PMSF, phenylmethylsulfonylfluoride; RNA Pol II, RNA polymerase II; SMCC, Srb/Med containing cofactor complex; SRC, steroid receptor coactivator; SCC, squamous carcinoma cell; SDS, sodium dodecyl sulfate; SRC, steroid receptor coactivator; TRAP, thyroid hormone receptor-associated protein; TRF, TATA binding protein-related factor; VDR, vitamin D receptor; VDRE, vitamin D response element.

Received for publication February 26, 2003. Accepted for publication July 23, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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