Coactivator MED1 Ablation in Keratinocytes Results in Hair Cycling Defects and Epidermal Alternations

The transcriptional coactivator complex Mediator facilitates transcription of nuclear hormone receptors and other transcription factors. We have previously isolated the Mediator complex from primary keratinocytes as the vitamin D receptor interacting protein complex. We identified a role for Mediator in keratinocyte proliferation and differentiation in cultured keratinocytes. Here, we investigated the in vivo role of Mediator by generating conditional null mice, where a critical subunit of the Mediator complex, MED1, is deleted from their keratinocytes. The MED1 ablation resulted in aberrant hair differentiation and cycling leading to hair loss. During the first hair follicle cycle, MED1 deletion resulted in a rapid regression of the hair follicles. Hair differentiation was reduced, and β-catenin/vitamin D receptor (VDR) regulated gene expression was dramatically decreased. In the subsequent adult hair cycle, MED1 ablation activated the initiation of hair follicle cycling. Shh signaling was increased, but terminal differentiation was not sufficient. Deletion of MED1 also caused hyper-proliferation of interfollicular epidermal keratinocytes, and increased the expression of epidermal differentiation markers. These results indicate that MED1 plays a critical role in regulating hair/epidermal proliferation and differentiation.

2 (0.25% trypsin, 37 °C, 1h). Nuclear extracts and cytoplasmic fractions were prepared by using NE-PER Nuclear Protein Extraction Kit (Pierce). MED1 protein was detected by western analysis as described (Oda et al., 2003;Tu et al., 2008). Briefly, 20 µg of nuclear extracts or cytoplasmic fractions were electrophoresed through SDS-PAGE and electroblotted onto a PVDF membrane.
After blocking, the blots were incubated with antibodies raised against MED1 (TRAP220 C-19 Santa Cruz Biotechnology)(1:1000). Subsequently, the blot was incubated with secondary antibody conjugated with HRP (Amersham), and bound antibody was visualized using a chemiluminescence system (SuperSignal ULTRA) (Pierce) by exposing X-ray film.

Histological analysis and K1/FLG immunostaining;
The lower portion of the dorsal skin was excised from the MED1 null and their littermate control mice, fixed with 4% paraformaldehyde overnight. The skin was embedded in paraffin, and cut into 5µm thick sections. H&E staining was performed as described previously (Bikle et al., 2006). Morphology of HF and IFE was evaluated in approximately 1cm lengths of dorsal skin of KO and CON (n=3) at different ages of mice, and reproducibility was confirmed by using at least two litters of mice for these analyses.
The K1 and FLG were detected by immuno-staining. After deparaffinization, antigen retrieval was performed using unmasking Solution (Vector Laboratories #H-3300), and the section was blocked by Block solution (sc-2051, Santa Cruz Biotechnology), and quenched with 3% H 2 O 2 in methanol. The sections were then incubated with primary antibodies against K1 (1:1000) and FLG (1:500), and signals were detected by using InnunoCruz rabbit™ LSAB Staining System (sc-2051, Santa Cruz Biotechnology). They are counterstained by hematoxylin. composite images of three colors, Ha1 green, K14 red, DAPI bue, were prepared by multidimentional analysis by using Axioskop software.

Measurement of mRNA expression by RT-QPCR
Total RNAs were isolated from whole skin excised from lower dorsal part of the null mice and from equivalent skin from their littermate control mice using STAT 60 (Invitrogen) and cleaned up with RNeasy Mini RNA purification kit (74134, Qiagen Valencia CA). cDNA synthesis was performed using TaqMan Reverse Transcription Reagents kit using 50µM random hexamer provided (Applied Biosystems, N808-0127, Foster City, CA). Real-time quantitative PCR was performed using the synthesized cDNA 4 as a template in a 20µl reaction mixture using Power SYBR Green (Applied Biosystems, 1102115) with the 7300 Real Time PCR system (Applied Biosystems). Relative mRNA levels compared to the control gens GAPDH was determined by the ΔΔCt method. Primers for QPCR analysis were designed using Primer Express software (Applied Biosystem) to span the exon and intron boundaries, or were derived from Primer bank. Primer sets were verified by drawing dissociation curves. Primer sequences for QPCR are available in supplement Table SII. These experiments were repeated in more than two litters of knockout mice (n=3 or more in each litter) to confirm reproducibility.

Statistical analysis Values are expressed as the average ± standard deviation (SD). Unpaired
Student t test was performed for determination of p-values using Excel or Graphpad Prism software (Graphpad Software). In case the p-values were smaller than 0.05 or 0.01, differences were considered to be statistically significant.
(C) Epidermal thickness was measured as length of green bars, which spanned the epidermis by drawing top (red) and bottom (yellow) of the epidermis by using Bioquant. Average thickness was calculated by accumulating green bars in 0.3-1.5 cm length of the skin in each mouse.