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. Author manuscript; available in PMC: 2014 Jul 29.
Published in final edited form as: J Invest Dermatol. 2010 Jun 3;130(10):2377–2388. doi: 10.1038/jid.2010.148

The Transcriptional Coactivator DRIP/Mediator Complex Is Involved in Vitamin D Receptor Function and Regulates Keratinocyte Proliferation and Differentiation

Yuko Oda 1, Robert J Chalkley 2, Alma L Burlingame 2, Daniel D Bikle 1
PMCID: PMC4114506  NIHMSID: NIHMS607044  PMID: 20520624

Abstract

Mediator is a multisubunit coactivator complex that facilitates transcription of nuclear receptors. We investigated the role of the mediator complex as a coactivator for vitamin D receptor (VDR) in keratinocytes. Using VDR affinity beads, the vitamin D receptor interacting protein (DRIP)/mediator complex was purified from primary keratinocytes, and its subunit composition was determined by mass spectrometry. The complex included core subunits, such as DRIP205/MED1 (MED1), that directly binds to VDR. Additional subunits were identified that are components of the RNA polymerase II complex. The functions of different mediator components were investigated by silencing its subunits. The core subunit MED1 facilitates VDR activity and regulating keratinocyte proliferation and differentiation. A newly described subunit MED21 also has a role in promoting keratinocyte proliferation and differentiation, whereas MED10 has an inhibitory role. Blocking MED1/MED21 expression caused hyperproliferation of keratinocytes, accompanied by increases in mRNA expression of the cell cycle regulator cyclin D1 and/or glioma-associated oncogene homolog. Blocking MED1 or MED21 expression also resulted in defects in calcium-induced keratinocyte differentiation, as indicated by decreased expression of differentiation markers and decreased translocation of E-cadherin to the membrane. These results show that keratinocytes use the transcriptional coactivator mediator to regulate VDR functions and control keratinocyte proliferation and differentiation.

Introduction

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D3), regulates transcription through its binding to the vitamin D receptor (VDR) for subsequent regulation of proliferation and differentiation in a number of cells, including keratinocytes (Bikle, 1996; Haussler et al., 1998). The VDR binds selectively to genomic sites, termed VDR response elements, and activate transcription (Haussler et al., 1998). For example, binding of VDR to 1,25(OH)2D3 induces transcription of 24 hydroxylase (24OHase) through VDR response element sequences in its promoter (Haussler et al., 1998). The expression of the antimicrobial cathelicidin (CAMP) is likewise regulated by 1,25(OH)2D3 and VDR to facilitate the innate immune response in the skin (Schauber et al., 2008). VDR also interacts with Wnt/catenin signaling (Palmer et al., 2001). VDR binds to β-catenin (Shah et al., 2006), modulating the impact of β-catenin on proliferation and differentiation of keratinocytes (Palmer et al., 2008). Several Wnt target genes, which are upregulated in the skin of the activated β-catenin mouse model, including cell cycle regulator cyclin D1, oncogene glioma-associated oncogene homolog (Gli 1; transcription factor and target gene in the Hedgehog pathway), peptidyl arginine deiminase 1 (PADI1), are regulated by 1,25(OH)2D3 and VDR. These genes possess VDR response elements similar to sequences in their promoter, some of which may interact with TCF/Lef response elements also found in the promoters of these genes (Palmer et al., 2008). The 1,25(OH)2D3 independent actions of VDR in keratinocytes have also been shown in in vivo mouse models (Skorija et al., 2005). The most striking example is alopecia, which is observed in VDR null mice, but not in CYP27b1 (the enzyme producing 1,25(OH)2D3) null mice (Bikle et al., 2004). However, similar 1,25(OH)2D3 independent effects of VDR have been observed in in vitro keratinocyte culture systems (Ellison et al., 2007; Hawker et al., 2007).

These VDR actions are mediated by transcriptional regulatory complexes called coactivators. We have previously purified and identified the protein complex of vitamin D receptor interacting proteins (DRIP) from primary keratinocytes, as well as in the squamous carcinoma cell line SCC12B2 (Oda et al., 2003) as VDR coactivators. Later, we found out that DRIP complex belong to the transcriptional coactivator complex called “Mediator” by structural simila­rity. The mediator was first isolated as a complex binding to thyroid hormone receptor (Ito et al., 2000; Ito and Roeder, 2001) and also is used by other nuclear receptors or transcription factors (Ito et al., 2000; Ito and Roeder, 2001; Bourbon et al., 2004) (Blazek et al., 2005; Kornberg, 2005). This complex activates transcription by directly recruiting the RNA polymerase (RNA Pol) II general transcriptional machinery to the promoter of regulated genes.

The mediator is a multiprotein complex. One core subunit, DRIP205/MED1 (referred to as MED1) that directly binds to VDR and anchors the rest of the complex, is required for VDR transactivation in keratinocytes (Oda et al., 2003). MED1 is also involved in peroxisome proliferator activated receptor-induced liver degeneration (Matsumoto et al., 2007) and adipogenesis (Ge et al., 2002, 2008). However, the mediator complex contains other subunits. A multidimensional study using Hela cells showed 32 subunits in the mediator complex (Sato et al., 2004). Structural analysis of yeast mediator complex using electron microscopic studies proposed four different modules referred to as kinase, head, middle, and tail (Chadick and Asturias, 2005). The RNA Pol II binding to the middle module causes a conformational change of the mediator complex to activate transcription. Some subunits were assigned to these modules but not all (Chadick and Asturias, 2005). The mediator complex from primary keratinocytes and SCC cells share core subunits such as MED1 with mediator complexes in immortalized cells such as Namalwa B or Hela cells. However, the presence of the smaller molecular-weight subunits varies. The subunit composition of the keratinocyte mediator complex also differs from previously described mediator complexes thyroid hormone receptor, Srb/Med containing cofactor complex, or activator–recruited cofactor complex. These differences could also emanate from technical differences in the analysis. On the other hand, the mediator complex in immortalized cell lines such as Hela cells may not represent the appropriate regulatory complex critical for control of cell proliferation and differentiation in normal cells because these cells are characterized by their aberrant cell growth and lack of differentiation. Therefore, we reexamined the mediator complex from primary keratinocytes by tandem mass spectrometry (MS) to provide a more accurate identification of the proteins involved.

We identified the core subunit MED1 along with additional subunits not found in our earlier analysis of the mediator complex. The functional relevance of these newly described mediator subunits in VDR function and keratinocyte proliferation and differentiation was evaluated. Silencing of the new subunit MED21 or the core subunit MED1 resulted in significant morphological changes in epidermal keratinocytes and increased their proliferation. This was accompa­nied by increased expression of cyclin D1 and/or Gli 1 and loss of 1,25(OH)2D3 responsiveness. Silencing of MED21 and MED1 also resulted in defects in keratinocyte differentiation, accompanied by loss in formation of the E-cadherin–β-catenin complex in the membrane. However, silencing of MED10 inhibited proliferation and promoted 1,25(OH)2D3 responsiveness, showing differences in the roles of the different subunits of mediator in VDR regulation of keratinocyte proliferation and differentiation.

Results

In a previous study, we purified VDR binding protein complexes from keratinocytes and determined their identities mainly by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping (Oda et al., 2003). Two different coactivator complexes, mediator and steroid receptor coactivator (SRC/p160), were identified at different stages of differentiation. In this study, we examined the mediator complex more comprehensively using tandem MS, which produces sequence information and is more effective for analyzing mixtures of proteins. Nuclear extracts from proliferating human keratinocytes were incubated with glutathione S-transferase (GST)-VDR ligand bind­ing domain beads in the presence or absence of ligand (1,25(OH)2D3). The bound proteins were eluted and sepa­rated by SDS–PAGE. The multiple proteins, ranging in molecular weight from 25 to 250 kDa, were then identified by MS (Figure 1a and b). Specifically, the protein bands were excised from the SDS gel and analyzed by liquid chromatography-electrospray ionization (nanoLC-ESI) tandem MS. The protein identification results are presented in Table 1. The core DRIP/Mediator subunits—DRIP250/MED13, DRIP240/MED12, DRIP205/MED1, DRIP150/MED14, DRIP130/MED23, DRIP100/MED24, DRIP92/MED16, DRIP77/MED17, and DRIP33/MED6 were identified (nomenclature includes the numbering scheme for the mediator complex as it relates to the numbering scheme for the DRIP complex; Bourbon et al., 2004). These proteins were previously observed in Namalwa (Rachez et al., 1999) and SCC cells (Oda et al., 2003). MED1 is a critical subunit, as it directly binds to VDR and activates VDR mediated transcription. Subunits MED10/hNut2, MED21/hSrb7, and MED31/hSoh1 had previously not been found in DRIP complexes from Namalwa and SCC cells (the equivalent subunit name in yeast is shown with MED names; Table 1). MED10 and 21 are reported to be components of the RNA Pol II general complex. They may serve as adaptors and convey regulatory information from promoter sites to the general transcriptional machinery to activate or repress transcription. The function of the core subunit MED1, as well as these newly described components of the VDR binding complex, was then explored.

Figure 1. A purification of vitamin D receptor (VDR) binding proteins from keratinocytes.

Figure 1

Nuclear extracts from proliferating keratinocytes were incubated with GST-VDR (LBD) affinity beads in the absence (a) and presence (b) of ligand (1,25-dihydroxyvitamin 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 the SDS gel and analyzed by tandem mass spectrometry. A ligand-independent binding protein without ligand (a) was not run consecutively with (b) but consistently appeared as a single band (*) and it was not analyzed. The model shows the mediator complex identified in keratinocytes including core mediator subunits and the newly described mediator subunits MED31, MED21, and MED10.

Table 1. Protein identification of the VDR binding protein complex isolated from keratinocytes.

Band Acc no. Num Pep Sequence coverage (%) Protein MW Protein name
p250 Q93074 28 12.8 247335.8 DRIP240/TRAP230/MED12
Q9UHV7 24 12.2 239233.5 DRIP250/TRAP240/MED13
p240 Q93074 66 33.5 247335.8 DRIP240/TRAP230/MED12
Q9UHV7 2 0.9 239233.5 DRIP250/TRAP240/MED13
p205 Q15648 55 34.5 168439.4 DRIP205/TRAP220/MED1
Q93074 2 1.0 247335.8 DRIP240/TRAP230/MED12
Q9UHV7 2 1.0 239233.5 DRIP250/TRAP240/MED13
p150 Q4KMR7 40 30.1 160608 DRIP150/TRAP170/MED14
Q93074 13 5.8 247335.8 DRIP240/TRAP230/MED12
Q15648 4 2.2 168439.4 DRIP205/TRAP220/MED1
p130 Q9ULK4 27 21.5 156475.5 DRIP130/MED23
Q4KMR7 23 16.3 160608 DRIP150/TRAP170/MED14
Q15648 4 2.1 168439.4 DRIP205/TRAP220/MED1
Q93074 2 0.9 247335.8 DRIP240/TRAP230/MED12
p100 O75448 15 18.4 110306.3 DRIP100/TRAP100/MED24
P92 Q9Y2X0 14 17.2 96794.1 DRIP92/TRAP95/MED16
O75448 11 11.7 110306.3 DRIP100/TRAP100/MED24
Q15648 2 1.1 168439.4 DRIP205/TRAP220/MED1
P77 Q9NVC6 15 24.1 72876.8 DRIP77/TRAP80/MED17
P11021 7 12.8 72333.5 GRP78/BiP
Q9UG63 2 3.7 71290.9 ATP-binding cassette subfamily F member 2
P42 P23396 12 43.6 26688.6 40S ribosomal protein S3
P15880 8 22.5 31324.7 40S ribosomal protein S2
O75586 3 11.8 28424.9 DRIP33/MED6
P34 P23396 7 25.1 26688.6 40S ribosomal protein S3
P62701 7 18.3 29466.8 40S ribosomal protein S4
Q96G25 2 5.2 29080.2 MED8
P25 P62249 16 65.5 16314.3 40S ribosomal protein S16
P62269 10 44.7 17718.8 40S ribosomal protein S18
P62829 9 49.3 14865.6 60S ribosomal protein L23
P60866 5 35.5 13372.8 40S ribosomal protein S20
P62244 5 34.6 14708.5 40S ribosomal protein S15a
P39019 4 18.8 15929.4 40S ribosomal protein S19
Q5T8T9 3 18.6 16479.7 MED22/SURF5
Q9Y3C7 3 22.1 15805.3 MED31/hSOH1
P62263 2 14.0 16141.7 40S ribosomal protein S14
Q13503 2 17.4 15564.5 MED21/SURB7
Q9BTT4 2 16.3 15688.1 MED10/hNut2
P16402 2 9.5 22218.9 Histone H1 family member 2, 3, or 4
P07305 2 9.8 20731.9 Histone H1.0
P62807 2 12.8 13775.1 Histone H2B

Abbreviations: DRIP, vitamin D receptor interacting proteins; TRAP, thyroid hormone receptor.

Analysis of p25–p250 bands by tandem mass spectrometry is shown. Identifications are shown by NCBI accession number (Acc no.), number of peptides identified (Num Pep), sequence coverage (%), calculated molecular weight (MW) of the protein, and protein name.

Nomenclature includes the numbering scheme for the mediator (bold letters).

The function of these mediator subunits in VDR activity was examined using small interfering RNA (siRNA)-silencing technology. Keratinocytes were transfected with siRNA for VDR (siVDR), MED1 (siMED1), MED10 (siMED10), MED21 (siMED21), and MED31 (siMED31) to block their expression. The effect of blockage on VDR transactivation was evaluated by comparison to controls transfected with nontarget siRNA (sicontrol). When VDR and MED1 were targeted by siRNA, their mRNA expression (Supplementary Table S1 online) and protein levels (Figure 2a) were significantly reduced. MED10, MED21, and MED31 silencing also led to significant reductions in their mRNA levels (Supplementary Table S1 online). Protein levels of these subunits were not examined because antibodies are not available. The VDR activity was assessed by measuring 1,25(OH)2D3 induced 24OHase and CAMP. Keratinocytes were treated with 1,25(OH)2D3, and mRNA levels of 24OHase, CAMP, and control L19 were measured using quantitative reverse transcriptase PCR (QPCR). VDR silencing decreased VDR transactivation (Figure 2c). When the major mediator subunit MED1 was silenced, 1,25(OH)2D3 induced 24OHase expression signifi­cantly decreased (Figure 2c), indicating a coactivator func­tion for MED1, as expected. In contrast, when MED10 or MED31 was silenced, 1,25(OH)2D3 induction of 24OHase increased compared with control (Figure 2c), suggesting they have a suppressive effect at least for the 24OHase. The same negative function of MED10 was observed when a second VDR target gene, CAMP, was examined. MED10 silencing significantly increased both basal and 1,25(OH)2D3 induced expression (Figure 2d). MED31 and MED21 silencing did not show significant changes. These results indicate that the mediator complex contains functionally distinct subunits.

Figure 2. Mediator is involved in vitamin D receptor (VDR) transcriptional activities as shown by 24 hydroxylase expression.

Figure 2

Keratinocytes were transfected with small interfering RNA (siRNA) to block VDR or mediator subunit expression. Blocking efficiency of VDR (a, upper lanes) and MED1 proteins (a, lower lanes) in keratinocytes transfected with sicontrol (lane 1), siVDR (lane 2), and siMED1 (lane 3) in 0.03 mm calcium are shown (a). Blocking efficiency during keratinocyte differentiation wherein cells are transfected with 0.03 mm calcium and then switched to 1.2 mm calcium and maintained for 3–7 days to induce differentiation are also monitored (b), VDR expression in sicontrol 3 days (lane 4), siVDR 3 days (lane 5), siVDR 7 days (lane 6); and MED1 proteins in sicontrol 3 days (lane 7), siMED1 3 days (lane 8), siMED1 7 days (lane 9) are shown (these samples correspond to data shown in Figure 5a and b). Keratinocytes were then treated with either vehicle (ethanol; open bars) or 1,25(OH)2D3 (closed bars) at 1 × 10−8 m (c, left graph and d) or 1 × 10−9 m (c, right graph). To normalize the data in each experiment, the percentage of induction compared with sicontrol was calculated. Statistical significance evaluated by t-test (**P<0.01, *P<0.05) compared with control is shown with asterisks. The results are representative of two separate experiments.

We noticed that MED1, MED21, and VDR silencing resulted in significant changes in morphology (from cuboidal epithelia to spindle-shaped cells; Figure 3a, phase contrast). To determine whether these morphological changes were accompanied by changes in proliferation, we transfected the cells with the siRNAs under low calcium (0.03 mm) condi­tions and examined proliferation, initially using the cell proliferation assay (XTT). Significant increases in proliferation were observed when MED1, MED21, or VDR were blocked (data not shown). Proliferation was further evaluated using the BrdU assay. BrdU incorporation was visualized histologically (Figure 3a BrdU staining), and the percentage of BrdU-positive cells per total cells was determined (Figure 3b). When VDR, MED1, or MED21 were blocked, the number of BrdU-positive cells increased (Figure 3b). In contrast, MED10 silencing decreased proliferation shown by BrdU incorpora­tion (Figure 3b). The changes in MED10 and MED21 were statistically significant, although the effects were smaller than those of silencing either MED1 or VDR. These results suggested that MED1 and MED21 facilitate VDR function in keratinocyte proliferation, but MED10 has an inhibitory role.

Figure 3. The blockage of mediator subunits resulted in hyperproliferation of keratinocytes.

Figure 3

Keratinocytes were transfected with small interfering RNA (siRNA) to block expression of vitamin D receptor (VDR) or mediator subunits. Silencing of VDR, MED1, MED21 resulted in morphological changes (a, phase contrast). Bar = 20 μm. Proliferation was accessed by BrdU incorporation. BrdU was immunohistochemically visualized (a, BrdU staining; bars = 20μm), and the ratio of BrdU-positive cells (brown) per total cells (blue nuclear counter staining) was calculated using Bioquant (Materials and Methods) (b). The data are shown as mean ± SD of three different preparations of keratinocytes. Statistical significance evaluated by t-test (**P<0.01, *P<0.05) compared with control is shown with asterisks (b).

To explore the mechanism by which silencing of MED1 and MED21 results in hyperproliferation, we investigated their role on Wnt target genes that are also regulated by VDR. Previously, it was reported that 1,25(OH)2D3 and VDR regulate several Wnt target genes including cyclin D1, Gli 1, and PADI1 (Palmer et al., 2008). In this study we evaluated MED1 and MED21 regulation of these genes in human epidermal keratinocytes. We observed that 1,25(OH)2D3 repressed the expression of both cyclin D1 and Gli 1 in human keratinocytes (Figure 4, sicontrol). When VDR was blocked, the basal level of cyclin D1 increased (Figure 4) despite the lack of 1,25(OH)2D3 in the medium suggesting that VDR may have a ligand independent, as well as ligand dependent ability to suppress cyclin D1 expression. The 1,25(OH)2D3 was still able to repress its expression, but to a lesser extent compared with the control group, presumably, as inhibition of VDR expression by siRNA was incomplete. When MED1 or MED21 were blocked, a similar increase in basal expression and decrease in repression by 1,25(OH)2D3 were observed (Figure 4) indicating their roles in the VDR mediated suppression of cyclin D1. Gli 1 was regulated similarly to cyclin D1 by VDR, MED1, and 1,25(OH)2D3 (Figure 4). In contrast to cyclin D1 and Gli 1, other Wnt target genes examined were found to be activated by 1,25(OH)2D3. When VDR was blocked, 1,25(OH)2D3 induced transactivation of PADI1, which is involved in terminal differentiation, was abolished. However, MED1 and MED21 silencing did not change either basal or 1,25(OH)2D3 induced transactivation (Figure 4), indicating that neither MED1 nor MED21 is involved in its regulation. Our results suggest that MED21 and MED1 may be involved in the VDR function of repressing the Wnt target genes cyclin D1, but not in the activation of PADI1.

Figure 4. The mediator complex is involved in regulation of Wnt target genes.

Figure 4

Keratinocytes were transfected with small interfering RNA (siRNA) for vitamin D receptor (VDR) or mediators and treated with either vehicle (EtOH; open bars) or 1,25(OH)2D3 (closed bars). The mRNA levels of cyclin D1, glioma-associated oncogene homolog (Gli 1), peptidyl arginine deiminase 1 (PADI1), and the control gene L19 were measured. To normalize the data in each experiment, the percentage of induction compared with sicontrol was calculated. The data are shown as mean±SD of three different preparations of keratinocytes. Statistical significance (*P<0.05, **P<0.01) compared with sicontrol vehicle treated group is shown with asterisks.

Next we examined the effect of mediator subunits on calcium-induced differentiation. Keratinocytes were transfected with siRNA in low calcium media (0.03 mm) for 2 days to block the expression of VDR, MED1 (Figure 5a) and the newly described subunits MED10, MED21, and MED31 (Figure 5b). They were then switched to high calcium (1.2 mm) and maintained for 3 or 7 days to induce differentiation. We have developed and verified a specific method to extend efficacy of siRNA by using multiple transfections of siRNA up to 7 days of culture. Blocking efficiency stayed stable over time for at least 7 days as shown in Supplementary Table S1 online and Figure 2b. Differentia­tion was evaluated by measuring mRNA levels of various differentiation markers. After 3 days of culture, levels of the early markers keratin 1 and keratin 10 were increased (shown by closed circles in inserts in the K1 and K10 graphs, Figure 5a and b). When MED1 (shown by squares in Figure 5a) or MED21 (shown by squares in Figure 5b) were blocked, the induction of K1 and K10 were significantly inhibited. MED10 and MED31 showed partial effects (Figure 5b inserts in K1 and K10 graphs). The expression of K1 and K10 further increased by 7 days in control cells. Silencing of MED1 suppressed this induction (Figure 5a). MED10, 21, and 31 silencing had effects on K1, but not on K10 (Figure 5b 7 days). In contrast, the mid-differentiation markers involucrin and transglutaminase were less affected (Figure 5a and b). Late differentiation markers, filaggrin (FLG) and loricrin (LOR), were induced only after 7 days of culture not by 3 days. When either MED1 (Figure 5a) or MED21 (Figure 5b) were silenced, both FLG and LOR levels were dramatically decreased, whereas silencing of MED10 and MED31 had less (FLG) or no (LOR) effect (Figure 5b). These results suggest that a new subunit MED21 and core subunit MED1 have a critical role in keratinocyte differentiation, especially in early and late differentiation, but less during midmarker formation.

Figure 5. The silencing of MED1 and MED21 results in defects in keratinocyte differentiation.

Figure 5

Figure 5

Keratinocytes were transfected with small interfering RNA (siRNA) in low calcium media for 2 days to block the expression of vitamin D receptor (VDR) and mediator subunits. Cells were then switched to high calcium (1.2 mm) to induce differentiation. Differentiation stages were monitored by measuring the mRNA levels of early markers, K1 and K10, middle markers, transglutaminase (TG) and involucrin (INV), and late markers, loricrin (LOR) and filaggrin (FLG). Differentiation progressed in time as was shown by the increase in these markers in the control group (a, b, closed circles). The effects on K1 and K10 in 3 days are shown in inserts. Data are shown as mean±SD of triplicate measurements (most SD are too small to visualize on the graph). The experiments were repeated twice to confirm their reproducibility.

Formation of the E-cadherin–β-catenin complex in the membrane is a critical step in calcium-induced differentiation (Xie and Bikle, 2007). To examine whether silencing VDR and the MED subunits affects this process, the translocation of E-cadherin and β-catenin to the membrane was examined. Keratinocytes were transfected with the different siRNAs in low calcium media (0.03 mm) then switched to high calcium (1.2 mm). Immunofluorescence evaluation showed that keratinocytes formed cell–cell contacts within 5 minutes, and E-cadherin and β-catenin colocalized to these regions, unlike in cells in low calcium (Figure 6a, sicontrol). When VDR, MED1, or MED21 were silenced, the translocation of both E-cadherin and β-catenin to the membrane decreased (Figure 6a, siVDR, siMED1, and siMED21). These results for E-cadherin were confirmed by western analysis. Total and plasma membrane lysates were prepared from keratinocytes and translocation of E-cadherin to the membrane was evaluated. Within 5 minutes of high-calcium treatment, E-cadherin levels increased in membrane fraction, although the total levels of E-cadherin did not change (Figure 6b sicontrol). When VDR, MED1, or MED21 were silenced, the E-cadherin translocation was significantly inhibited (Figure 6b siVDR, siMED1, and siMED21).

Figure 6. The function of MED1 and MED21 in calcium-induced E-cadherin translocation.

Figure 6

Keratinocytes were transfected with small interfering RNA (siRNA) to block expression of vitamin D receptor (VDR) or mediator subunits, and treated with calcium for 5 minutes to initiate keratinocyte adhesion. Translocation of E-cadherin (green) and β-catenin (red) to the membrane was visualized by immunofluorescence (green and red images were superimposed, so that sites of overlap are visualized as yellow) (a). Bars =20 μm. Calcium-induced E-cadherin translocation to the plasma membrane (b, western analysis, upper lanes) compared with that in total lysates (b, lower lanes). The mRNA expression of calcium-sensing receptor (CaR; c, left panel) and the calcium-induced translocation of CaR to the membrane (c, right panel) are also shown. CaR protein bands in sicontrol (lane 5, 6) were not run consecutively with one in siVDR and si vitamin D receptor interacting proteins (DRIP) (lane 1–4), but consistently appeared in this pattern.

The calcium-sensing receptor (CaR) has a critical role in mediating calcium-induced formation of the Ecadherin–β-catenin complex (Tu et al., 2008), and is regulated by 1,25(OH)2D3 (Ratnam et al., 1999). Therefore, we examined whether the loss of the ability of the keratinocyte to respond to calcium associated with the regulation through CaR. The mRNA levels of CaR decreased when VDR or MED1 is silenced (Figure 6c left panel). Translocation of CaR to the membrane was also affected. After high-calcium treatment, CaR levels increased in the controls (Figure 6c, western blot lanes 5, 6). When VDR or MED1 were silenced, both basal and calcium-induced translocation decreased (Figure 6c, western siVDR lanes 1, 2 and siMED1 lanes 3, 4). These results indicate that blocking MED21, MED1, or VDR reduces calcium-induced E-cadherin–β-catenin translocation to the membrane, and this reduction may be associated with decreased expression of CaR and/or its function in the membrane.

Discussion

In this study, we characterized the DRIP–Mediator complex involved in VDR function in primary keratinocytes. Newly described subunits of this complex were identified that were not previously found in the mediator complexes of immorta­lized keratinocytes SCC12B2 or Namalwa B cells (Rachez et al., 1999; Oda et al., 2003). Normal keratinocytes may therefore use a functionally distinct mediator assembly for VDR regulation. The differences in function may be inferred when these subunits are assigned to the module structures proposed in yeast mediator (Blazek et al., 2005; Chadick and Asturias, 2005). In this scheme, the middle module, which is proposed to bind directly to RNA Pol II to activate or repress transcription, is represented by the core subunit MED1. The MED1 is essential for binding to VDR and activation of transcription. Our data show that MED1 has a critical role in VDR regulation of keratinocyte proliferation and differentia­tion. Two other core components found in the yeast middle module, DRIP34/MED7 and DRIP36/MED4, were not detected in our mediator complex. However, MED10, MED21, and MED31, which were found in the keratinocyte mediator complex, could potentially substitute for these two subunits in the middle module. The subunit MED21, when silenced, promoted keratinocyte proliferation and the expression of cyclin D1. It also blocked calcium-induced differentiation and calcium-induced membrane localization of the E-cadherin–β-catenin complex. These results are comparable with those seen with the silencing of MED1. However, MED21 silencing did not block 1,25(OH)2D3 induction of 24OHase unlike the effect of MED1 silencing. Neither MED1 nor MED21 silencing blocked 1,25(OH)2D3 induction of CAMP or PADI1, consistent with the role of the mediator complex in the early, but not the terminal events in VDR regulated keratinocyte differentiation. In contrast reducing the expression of MED10 inhibited proliferation and pro­moted both 24OHase and CAMP induction by 1,25(OH)2D3. The repressor function of MED10 in transcription was also previously reported in yeast (Tabtiang and Herskowitz, 1998). However, the effects of MED10 silencing on calcium induction of the differentiation markers were comparable, if not as extensive as those seen, with MED21 and MED1 silencing. Silencing MED31 had comparable effects with MED10 on calcium induced differentiation markers, but its impact on keratinocyte proliferation and 1,25(OH)2D3 induction of 24OHase and CAMP was negligible. These results indicate that the mediator complex in keratinocytes contains functionally distinct subunits to facilitate and/or repress VDR function.

Our results further indicate that MED21/MED1 are involved in 1,25(OH)2D3/VDR regulation of Wnt signaling. MED21/MED1 in association with VDR may compete with LEF/TCF for β-catenin and, hence, limit the induction of Wnt target genes involved with proliferation such as cyclin D1. However, other Wnt target genes involved with terminal differentiation such as PADI1, which are activated by 1,25(OH)2D3/VDR, were not involved in proliferation. Thus, VDR can either repress or activate Wnt target gene expression depending on the gene, and for these genes mediator subunits may be differentially involved in their repression and activation.

Part of the mechanism by which VDR and mediator subunits regulate Wnt signaling could also be attributed to the ability of 1,25(OH)2D3/VDR to regulate the distribution of β-catenin within the cell by facilitating calcium-induced formation of the E-cadherin–β-catenin complex in the membrane. Formation of this complex is required for calcium-induced differentiation of the keratinocyte (Xie and Bikle, 2007). Silencing MED1 and MED21, as well as VDR prevents the calcium-induced formation of this complex in the membrane. Recently we have shown that the CaR is required for the formation of the E-cadherin–β-catenin complex. The CaR forms a complex with the scaffolding protein filamin A (paper submitted) and activates Rho signaling (Tu et al., 2008), which would promote E-cadherin translocation, accompanied by cytoskeleton rearrangement. It also stimulates Fyn/Src kinases, which then phosphorylate β-, γ-,and p120-catenin, promoting their binding to E-cadherin at the cell membrane (Tu et al., 2008). In earlier studies we had shown that CaR in keratinocytes was induced by 1,25(OH)2D3 (Ratnam et al., 1999). In this study we show that silencing MED1, as well as VDR suppresses the expression of CaR and its translocation to the membrane after calcium stimulation. This suggests a mechanism, outlined above, by which VDR/Mediator is required for E-cadherin–β-catenin complex formation, so is necessary for the differentiation process.

Previous studies showed that VDR binds to the DRIP Mediator complex in the presence of 1,25(OH)2D3 (Oda et al., 2003). However, our current results suggest that MED1 may be involved in 1,25(OH)2D3-independent repression by VDR of the cyclin D1 and Gli 1 genes. Such 1,25(OH)2 D3-independent actions of VDR have previously been reported in keratinocyte culture systems, in which VDR forms transcriptional regulatory complexes with retinoid X receptor (RXR) in the absence of 1,25(OH)2D3 (Ellison et al., 2007) and stimulates the expression of various differentiation markers (Hawker et al., 2007). Keratinocyte specific modulatory proteins, signaling pathways or alternative ligands may potentially be involved in the function of VDR-mediator in the absence 1,25(OH)2D3.

Our previous studies (Oda et al., 2003) did not exclude the presence of an alternative ligands in keratinocytes because the mediator complex was isolated from dialyzed nuclear extracts from which small molecule ligands were removed, then 1,25(OH)2D3 was externally added to GST-VDR affinity beads. Recently, a potential alternative ligand, 20-hydroxyvitamin D3, was reported to activate VDR and induce keratinocyte differentiation (Zbytek et al., 2008). This potential ligand may be produced from vitamin D3 using cytochrome P450scc, which is present in keratinocytes (Tuckey et al., 2008). It is also possible that MED1 alters the expression of genes like cyclin D1 and Gli 1 through other transcription factors than VDR, factors that would not require 1,25(OH)2D3 as ligand.

Our results show that both MED1 and MED21 have critical roles for both in proliferation and some aspects of differentia­tion. These results do not entirely support our initial model (Oda et al., 2003), wherein mediator was isolated from proliferating keratinocytes, whereas other coactivator SRCs were found from differentiated keratinocytes. In addition, our preliminary results using GST-VDR affinity beads showed DRIP complex was found in partially differentiated keratinocytes too (data not shown). We now modify our previous model by proposing that mediator regulate cell proliferation, that both mediator and SRCs are used during the earlier and mid stages of differentiation, but that terminal differentiation processes including barrier formation (Oda et al., 2009) and innate immunity (Schauber et al., 2008) are dependent on SRCs, but not MED1. However, LOR and FLG, so called “late” differentiation markers, require both coactivators perhaps because these genes are initially induced in less differentiated keratinocytes than the genes involved in permeability barrier or innate immunity.

MED1 is known to bind to other nuclear receptors, such as PPAR, which have also been found to have a role in keratinocyte differentiation (Hanley et al., 1998, 2000; Schmuth et al., 2004). Therefore, the effect of MED1 may not be specific for VDR. However, phenotypes resulting from MED1 silencing are similar to those caused by the loss of VDR, suggesting that MED1 and MED21 work primarily through VDR to regulate proliferation and differentiation in keratinocytes.

In this study, we characterized the mediator complex from primary keratinocytes. The newly described subunit MED21 and core subunit MED1 were found to facilitate VDR function and to regulate keratinocyte proliferation and differentiation. In examining genes that are regulated by β-catenin and VDR, we found that MED21/MED1 facilitates some, but not all actions of VDR in their transcription. These studies further increase our understand­ing of the biological function of the transcriptional coactivator complex mediator in normal skin and may further increase our understanding of the aberrant VDR and/or Wnt signaling in hyperproliferative skin tumors, such as trichofolliculomas (hair follicle tumors), basal, and squamous cell carcinomas.

Materials and Methods

Cell culture

Normal epidermal keratinocytes were isolated from neonatal-human foreskin and grown in serum-free keratinocyte growth medium (Cascade Biologics, Portland OR) as previously described (Gibson et al., 1996). Second passage keratinocytes were cultured with keratinocyte growth medium containing 0.03mm calcium to maintain them in a proliferating stage. To induce keratinocyte adhesion, cells were treated with the same medium containing high calcium (1.2 mm) for 5 minutes. To induce differentiation, cells were switched to high-calcium medium and maintained for 3–7 days. The study was conducted according to Declaration of Helsinki Principles.

Purification of VDR binding proteins from keratinocytes

VDR binding proteins were purified using GST-VDR affinity beads as described previously (Oda et al., 2003). Briefly, nuclear extracts were prepared from proliferating keratinocytes as described and incubated with GST-VDR (LBD) affinity beads in the presence of 1 × 10−6 m 1,25(OH)2D3. Bound proteins were eluted with 0.2% sarkosyl, separated by SDS-PAGE, and visualized by silver staining (Oda et al., 2003).

Protein identification by MS

Protein bands were excised from the SDS gel. Minced gel pieces were reduced using dithiothreitol (DTT). Free cysteines were alkylated using iodoacetamide, and the proteins were digested using trypsin (12.5 ng μl−1 in 25 mm ammonium bicarbonate) at 37C overnight. Peptides were extracted with 50% acetonitrile, 2% formic acid. Extracted peptides were vacuum centrifuged to dryness, resuspended in 0.1% formic acid and analyzed by LC-MSMS using a QSTAR Pulsar (AB Sciex, Foster City, CA). A Pepmap column (75 mm internal diameter × 150 mm, Dionex, Sunnyvale, CA) was used for HPLC separation, using a gradient of 0–40% acetonitrile or 0.1% formic acid over 30 minutes. When a peptide was observed above minimum threshold intensity in the mass spectrum, it was automatically selected for fragmentation by collision-induced dissociation. Fragmentation spectra were analyzed against the Uniprot protein database (21 April 2006) using Protein Prospector (Chalkley et al., 2005) (http://prospector.ucsf.edu). Only proteins identified by two or more different peptides were reported. Bait protein, several bacterial proteins, and keratin peptides were also identified in addition to the proteins reported in Table 1.

siRNA silencing and measurement of mRNA expression

A pool of four siRNA duplexes with UU overhangs and a 5′ phosphate on the antisense strand specific for human MED1 (SMART pool PLUS M-004267-00 5′-GGTCTGATTTGGTTAAGAA-3′, 5′-GTTCAGAGATCA TAGGAAT-3′, 5′-TAAATAGGGTTCAGAGATC-3′, 5′-CAAGCTGGGT GAATTAGAA-3′), human VDR (siGENOME duplexes MQ-003448-00-0005, 5′-TGAAGAAGCTGAACTTGCA-3′, 5′-GCAACCAAGACTA CAAGTA-3′, 5′-TCAATGCTATGACCTGTGA-3′, 5′-CCATTGAGGT CATCATGTT-3′), human MED10L-014834-01-0005, human MED21 (SURB7) L-016221-00-0005, and human MED31L-027282-00-0005 were designed and synthesized by Dharmacon Research (Lafayette, CO) using SMART selection and SMART pooling technologies. As a control a nonspecific SMARTpool (D-0012206-13-05) were used. Keratinocytes (12-well plate) were transfected with 20 nM siRNA oligonucleotides using 2 μl siLentFect (Bio-Rad, Hercules, CA) in keratinocyte growth media containing 0.03 mm or 1.2 mm calcium. The cells were transfected twice (24 hours apart) to maximize the silencing effect. The cells were then treated with either EtOH or 1 × 10−8−1 × 10−7 m 1,25(OH)2D3 overnight. To maximize their effects on 24OHase, cells were treated with a lower concentration of 1,25(OH)2D3 (1 × 10−9m), which causes less induction of 24OHase but increases sensitivity to inhibition of 1,25(OH)2D3 induced transactivation.

QPCR analysis

Total RNA was isolated from keratinocytes using RNeasy RNA purification kit (Qiagen, Valencia, CA), and cDNA was prepared using reverse transcription kit (Applied Biosystems, Foster City, CA). The mRNA expression of human 24OHase, CAMP, Cyclin D1, Gli 1, K15, PADI1, K1, K10, transglutaminase, involucrin, LOR, FLG was measured by QPCR using SYBR green master mix (Applied Biosystems) in real-time PCR instruments 7900 HT or 7300 (Applied Biosystems). Relative expression of these genes compared with mitochondrial ribosomal protein L19 was calculated. Primers for QPCR analysis were designed using Primer Express (Applied Biosystem) to span the exon and intron boundaries. Primer sets were verified by drawing dissociation curves. Primer sequences for QPCR are available in supplements. These experiments were repeated using two or three different batches of primary keratino­cytes to confirm reproducibility.

Proliferation

Keratinocyte proliferation was evaluated by measurement of cell numbers using Cell Proliferation kit II (XTT; Roche Applied Science, Indianapolis, IN) according to the manufacturer's instruction. Proliferation was also assessed by the measurement of DNA synthesis. Keratinocytes were labeled with BrdU for 6 hours. The incorporated BrdU was detected by a BrdU kit (Zymed, Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Digital images of three representative fields per experimental condition were acquired and quantified using a computer-assisted program (BIOQUANT, Nashville, TN). Percentages of BrdU-positive cells per total cells were calculated. To normalize the data in different experiment, the percentage of BrdU-positive cells to that in the sicontrol group was further calculated. Student's t-test was used for statistic analysis.

Immunofluorescence

The localization of E-cadherin and β-catenin was examined as described (Tu et al., 2008). Briefly, keratinocytes cultured on coverslips were transfected with siRNA in low calcium medium (0.03 mm) for 3 days. Cells were treated with high calcium (1.2mm) medium for 5 minutes, then fixed and permeabilized. After blocking, cells were incubated with 10μg ml−1 of antibodies against E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA) and β-catenin (Santa Cruz) and subsequently incubated with fluorescein- or Texas Red-conjugated secondary antibody (20μg ml−1, Molecular Probes, Invitrogen).

Cell lysate preparation

Total cell lysates and membrane proteins were prepared from keratinocytes as described (Tu et al., 2008). Briefly, keratinocytes were lysed in Nonidet P-40 lysis buffer (0.5%; total lysates). Membrane lysates and nuclear extracts were prepared using the Mem-PER Eukaryotic Membrane Protein Extraction reagent Kit (Pierce Biotechnology, Rockford, IL) and Nuclear Extraction Kit (Pierce Biotechnology) according to the manufacturer's protocols.

Protein analysis by western blot

The protein expression of VDR, MED1, and E-cadherin was detected by western analysis as described (Oda et al., 2003; Tu et al., 2008). Briefly, 20μg of total RIPA lysates (for VDR) or nuclear extracts (for MED1) or total NP-40 lysates (total protein for E-cadherin) or membrane lysates (for E-cadherin) were electrophoresed through SDS-PAGE and electroblotted onto a PVDF membrane. After blocking, the blots were incubated with antibodies raised against VDR (Santa Cruz D-6; 1:200), or MED1 (Santa Cruz TRAP220 C-19; 1:1000) or E-cadherin (Santa Cruz H-108; 1:1000) or CaR (ADD; 1:500). Subsequently, the blot was incubated with secondary antibody conjugated with HRP (GE Healthcare/Amersham, Piscataway, NJ), and bound antibody was visualized using a chemiluminescence system (SuperSignal ULTRA, Pierce Biotechnology).

The Committee for Human Research (IRB) of the University of California, San Francisco and the Veterans Affairs Medical Center approved all described studies with the human keratinocytes.

Supplementary Material

Supplemental information

Acknowledgments

We thank Dr Chialing Tu for CaR studies. We also thank Ms Sally Pennypacker for assistance with the keratinocyte cultures. We also thank Dr Matt Petit for paper preparation. This work was supported by NIH grants R01 AR050023 and P01 AR39448 (DDB), by the American Institute of Cancer Research (DDB), and by NCRR grants RR01614 and RR012961 (ALB).

Abbreviations

CAMP

cathelicidin

DRIP

vitamin D receptor interacting protein

FLG

filaggrin

Gli 1

glioma-associated oncogene homolog

LOR

loricrin

MS

mass spectrometry

24OHase

24 hydroxylase

1,25(OH)2D3 or 1,25D3

1,25-dihydroxyvitamin D3

PADI

peptidyl arginine deiminase

RNA Pol

RNA polymerase

SCC

squamous carcinoma cells

SRC

steroid receptor coactivator

VDR

vitamin D receptor

Footnotes

Conflict of Interest: The authors state no conflict of interest.

Supplementary Material: Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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