Keratinocyte specific MED1 knockout mice
We generated conditional MED1 null mice using a Cre-lox system to specifically delete MED1 expression in keratinocytes. Floxed MED1 mice, in which two lox P sites were inserted into the introns upstream and downstream of exons 8, 9 and 10 of the MED1 gene (C57/BL6 background) (Jia et al., 2004
) were bred with transgenic mice expressing Cre recombinase under the control of the Keratin 14 (K14) promoter (C57/BL6 background, Jackson Lab) (). Homozygous floxed mice with the Cre transgene (KO) were compared to control littermates that had floxed MED1 alleles but no Cre (CON). The recombination was detected in the whole skin as well as in epidermis and dermis containing the hair follicles (). However, excision was not detected in other tissues except small amounts in lung probably in bronchial epithelia expressing K14 (), confirming tissue specific excision. A marked reduction in MED1 mRNA was seen in the epidermis of null mice (). Deletion of MED1 protein was shown by western analysis. A 200 kDa MED1 protein band was absent in keratinocytes separated from the null skin ( KO). As MED1 is a nuclear protein, it was detected in the nuclear fraction but not in the cytoplasm (). A different coactivator, SRC3, also involved in transcriptional regulation, was equally detected in both control and null keratinocytes. SRC3 is localized in both the nucleus and cytoplasm. The coactivator p300, VDR and SRC3 were also equally detected (). These results were confirmed by immuno-histochemistry. As shown in , MED1 was absent in keratinocytes in the null skin. In contrast, it was detected in keratinocytes of both IFE and HF the control skin at P17. These results demonstrate that both transcript and protein of MED1 were deleted from keratinocytes.
Generation of conditional MED1 null mice
Disruption of the MED1 gene resulted in abnormalities in hair differentiation and cycling
The MED1 floxed mice with or without Cre transgene were born in the expected Mendelian ratios. The growth of the null mice was slightly retarded (10–30% smaller than control). The MED1 null mice did not show abnormalities in the appearance of the skin and hair at least through P12 (anagen). However, changes in their hair coats were first recognized at P17, at which point hair loss was visually observed in the skin of head and neck (). The hair density decreased in ventral skin (), and in dorsal skin as shown by hairs remaining on the depilation tapes after hairs were plucked (). Hair loss was relatively mild, but changes in hair coats were sufficient to distinguish them before PCR genotyping. Hair loss increased gradually with age (). As these animals were generated in the C57/BL6 background, skin pigmentation and epidermal thickness were correlated to hair follicle cycling and used as a tool to stage their hair cycle (Slominski and Paus, 1993
). Histological examination showed that null skin was normal in neonates (P3) (data not shown) to first anagen (P12) ( P12). The control skin underwent catagen beginning at the head and moving caudally. Even by P17, the lower back showed late anagen morphology ( P17 CON). In contrast, most of the null skin was pale pink macroscopically, and the hair follicles were in an advanced stage of the regression ( P17 KO). The IFE was hyperplastic ( P17 KO). Telogen at P21 was similar in KO and controls (data not shown).
Deletion of MED1 resulted in hair loss
Deletion of MED1 caused rapid regression of HFs in the morphogenic hair cycle
Proliferation and hair follicle differentiation were assessed at P17 (). The control skin was in late anagen at P17 but HFs in KO were regressing (, enlarged images are shown in B). Hair bulbs containing PCNA positive cells were already lost ( PCNA staining in brown), and hair shafts were thinner () in the KO. Hair differentiation was assessed by hair keratin Ha1/KRT31 (Ha1). The Ha1 was highly expressed in the inner root sheath (IRS) in the control skin ( Ha1staining in green), but absent in the null skin (). Hair differentiation was also evaluated by measuring the mRNA levels of hair keratins during synchronous hair follicle cycling (P3 to P21) by isolating RNA from whole skin. Ha1 expression increased from P3 to P12 and remained high to P17, then sharply decreased by P21 ( open bar) paralleling the activation and regression during hair cycling in the controls. However, MED1 null skin showed lower Ha1 levels during all the stages, most significantly at P17 ( closed bar). Expression of other hair keratins, Ha2 and Krt2-16, were also decreased significantly at P17 ().
Ablation of MED1 decreased hair differentiation at P17
Deletion of MED1 increased epidermal differentiation markers
The expression of the early epidermal differentiation marker keratin 1 (K1), and late differentiation marker filaggrin (FLG) was evaluated at P17. The protein levels of both K1 ( upper panels) and FLG (lower panels) increased in the null skin. The mRNA expression of K1 and FLG was highest at P3 and markedly decreased at P17 in the control skin ( open bar). In contrast, their expressions were higher in KO compared to control at P3, P17 () and 10 weeks ().
Epidermal differentiation and proliferation increased upon the deletion of MED1
Ablation of MED1 increased keratinocyte proliferation
Keratinocyte proliferation in IFE was evaluated by PCNA staining and epidermal thickness. There is not a significant difference between null and control mice in the skin of neonates to P12 (data not shown). However, the null skin showed hyperplasia of IFE at P17 (), P25 () and at 10 weeks (). These differences were quantified and shown to be significant at P17, P25 and 10 weeks (, Supplemental Table SIII
, Fig. S1
MED1 ablation resulted in abnormal HF anagen activation in the adult hair follicle cycle
The deletion of MED1 activated HF progression during the adult hair cycle
At P25, the control skin was mostly in telogen ( CON). In contrast, large areas of the null skin were now in anagen characterized by thicker epidermis ( A KO) and numerous PCNA positive cells in enlarged hair bulbs and the outer root sheath (ORS) (5D KO). However, hair shafts were thinner in KO ( left two panels). At 6 weeks, control skin remained in prolonged telogen, and by 9–10 weeks, the entire back of control mice was pale pink (when shaved) indicating telogen (). In contrast, large areas of null skin were pigmented with epidermis thickening, indicating anagen stage VI (). At 6 month, the null skin was still in anagen even though hair loss progressed ( left two panels) shown by pigmented anagen skin after hairs were shaved (1C right two panels). These changes were consistent within a litter (at least 3 mice per group) and between litters (2 or more at each time point).
The MED1 ablation resulted in abnormal hair follicle differentiation
Hair differentiation was assessed by Ha1 expression. The control skin did not express Ha1 in the telogen HF at 10 week ( I CON). In contrast, Ha1 was expressed in the IRS of the null skin ( I KO). However, Ha1 levels at 10 weeks (10W KO) when compared to those in normal anagen (P12 CON) indicated a reduction in Ha1 expression in the null HF at 10 weeks (). These results suggest that MED1 ablation activates HF progression, but terminal differentiation is defective.
MED1 deletion did not affect calcium metabolism
We analyzed serum levels of calcium because of concern for hypocalcemia seen in VDR null mice, which may secondarily affect skin physiology. Serum levels of calcium were normal in MED1 null mice fed standard mouse chow (Supplemental Table SI
Differential regulation of signaling pathways involved in HF progression and differentiation
RNA was isolated from whole skin of null and littermate control mice. The mRNA levels of genes known to be involved in HF progression and differentiation that we examined are listed in . Their expression was measured at i) P12 when the null skin was in normal anagen; ii) P17 when the null HF was rapidly regressing; iii) 10 weeks when the HF was abnormally activated. Fold changes of null skin (KO) compared to control skin (CON) were calculated (). At P12, the expression of most genes was not significantly changed ( P12 shown as black). At P17, the expression of hair differentiation genes such as PADI1, PADI3, S100A3, Tubb3, Dlx3 and hair keratins was dramatically decreased ( P17 green). In contrast, the exression of all the genes examined increased at 10 weeks. Most significant increases were in Hh signaling ( 10W, light red). At 10 weeks, Shh, PADI1, PADI3, S100A3, and hair keratins were undetectable in CON, but increased in the KO ( 10W, yellow (+) indicating that the mRNA levels were undetectable in CON but increased in KO preventing a calculation of the ratio). The mRNA levels of these genes in KO at 10 week (10W KO) were then compared to those in normal anagen (P12 CON) (). The expression of Shh, Patch1, and Gli1 was higher in the 10 week anagen of the KO compared to the P12 anagen of CON (). However, hair keratins were lower in the 10wk KO anagen than in the P12 CON anagen suggesting incomplete hair differentiation (). The BMP components, Lef1, PADI1, PADI3 were also lower. These results indicate that deletion of MED1 accelerates HF regression at P17, with a reduction in the expression of hair keratins and VDR/β-catenin target genes, whereas at 10 weeks, HF progression is activated and Hh pathway components are elevated but hair differentiation is incomplete.
Signaling pathways impacted by the deletion of MED1