C/EBPalpha and C/EBPbeta are required for Sebocyte differentiation and stratified squamous differentiation in adult mouse skin

Abstract

C/EBPalpha and C/EBPbeta are bZIP transcription factors that are highly expressed in the interfollicular epidermis and sebaceous glands of skin and yet germ line deletion of either family member alone has only mild or no effect on keratinocyte biology and their role in sebocyte biology has never been examined. To address possible functional redundancies and reveal functional roles of C/EBPalpha and C/EBPbeta in postnatal skin, mouse models were developed in which either family member could be acutely ablated alone or together in the epidermis and sebaceous glands of adult mice. Acute removal of either C/EBPalpha or C/EBPbeta alone in adult mouse skin revealed modest to no discernable changes in epidermis or sebaceous glands. In contrast, co-ablation of C/EBPalpha and C/EBPbeta in postnatal epidermis resulted in disruption of stratified squamous differentiation characterized by hyperproliferation of basal and suprabasal keratinocytes and a defective basal to spinous keratinocyte transition involving an expanded basal compartment and a diminished and delayed spinous compartment. Acute co-ablation of C/EBPalpha and C/EBPbeta in sebaceous glands resulted in severe morphological defects, and sebocyte differentiation was blocked as determined by lack of sebum production and reduced expression of stearoyl-CoA desaturase (SCD3) and melanocortin 5 receptor (MC5R), two markers of terminal sebocyte differentiation. Specialized sebocytes of Meibomian glands and preputial glands were also affected. Our results indicate that in adult mouse skin, C/EBPalpha and C/EBPbeta are critically involved in regulating sebocyte differentiation and epidermal homeostasis involving the basal to spinous keratinocyte transition and basal cell cycle withdrawal.

Figure 1. C/EBPα/β are co-expressed in skin and acute ablation C/EBPβ does not produce a major skin phenotype.

Co-IF staining of untreated mouse skin in: (A) interfollicular epidermis (IFE), and (B) the hair follicle outer root sheath (HF-ORS) and sebaceous gland (SG). (C) Immunoblot analysis of epidermal lysates from untreated and 4OHT-treated C/EBPβ^ff and IKOβ mice (n = 3 mice/genotype/treatment). (D) IHC staining for C/EBPβ in 4OHT-treated C/EBPβ^ff and 4OHT-treated IKOβ mouse skin. (E) H&E staining of 4OHT-treated C/EBPβ^ff and 4OHT-treated IKOβ mouse skin. (F) Quantification of nucleated cell layers in 4OHT-treated C/EBPβ^ff and IKOβ mice (n = 3 mice/genotype/treatment). (G) Quantification of basal BrdU positive cells in 4OHT-treated C/EBPβ^ff and IKOβ mice (n = 3 mice/genotype/treatment). (H) IHC staining for BrdU and markers of squamous differentiation. All scale bars represent 30 microns. *indicates significantly different from controls p<.05, Student's t test.

Figure 2. Acute ablation C/EBPα does not produce a major skin phenotype.

(A) Immunoblot analysis of epidermal lysates from untreated and 4OHT-treated C/EBPα^ff and IKOα mice (n = 3 mice/genotype/treatment). (B) IHC staining for C/EBPα in 4OHT-treated C/EBPα^ff and 4OHT-treated IKOα mouse skin. (C) H&E staining of 4OHT-treated C/EBPα^ff and 4OHT-treated IKOα mouse skin. (D) Quantification of nucleated cell layers and number of basal BrdU positive cells in 4OHT-treated C/EBPα^ff and IKOα mice (n = 3 mice/genotype/treatment). All scale bars represent 30 microns. *indicates significantly different from controls p<.05, Student's t test.

Figure 3. Acute co-ablation of C/EBPα/β results in epidermal morphological defects.

(A) Immunoblot analysis of epidermal lysates from untreated and 4OHT-treated α^ffβ^ff and IKOαβ mice (n = 3 mice/genotype/treatment). (B) Co-IF staining for C/EBPα and C/EBPβ in 4OHT-treated α^ffβ^ff and 4OHT-treated IKOαβ mouse skin. (C) Quantification of epidermal nucleated cell layers in 4OHT-treated mice. (D) H&E staining of 4OHT-treated α^ffβ^ff mice. (E) H&E staining of 4OHT-treated IKOαβ mice. (F) Characteristic H&E staining of lesion in 4OHT-treated IKOαβ mouse. (G) H&E staining of 4OHT-treated IKOαβ epidermis with (H) region enlarged displaying dysplasia. (I) H&E staining and TEM micrograph of keratohyalin granules in 4OHT-treated (I) α^ffβ^ff and 4OHT-treated (J) IKOαβ epidermis. (K) H&E staining of lesions from 4OHT-treated animals from different days after start of 4OHT treatment. Scale bars represent 30 microns unless otherwise notated. *indicates significantly different from controls p<.05, Student's t test.

Figure 4. Co-ablation of C/EBPα/β results in defects in epidermal squamous differentiation.

(A) IHC staining for markers of stratified squamous differentiation in 4OHT-treated controls and 4OHT-treated inducible double knockouts (IKOαβ). (B) Immunoblot analysis of epidermal lysates from animals treated with 4OHT for markers of stratified squamous differentiation (n = 3 mice/genotype). (C) Taqman® real time quantitative PCR (K10) and reverse transcriptase semi-quantitative PCR (all others) on mRNA from 4OHT-treated whole skin (n = 5 mice/genotype). Scale bars represent 30 microns.

Figure 5. Co-ablation of C/EBPα/β results in hyperproliferative basal and suprabasal keratinocytes.

(A) IHC staining for BrdU-positive keratinocytes in HF-ORS and IFE in 4OHT-treated α^ffβ^ff and 4OHT-treated IKOαβ epidermis. (B) Suprabasal BrdU-positive S-phase keratinocytes in 4OHT-treated IKOαβ mice. (C) Quantification of BrdU-positive basal and suprabasal IFE keratinocytes in 4OHT-treated α^ffβ^ff and 4OHT-treated IKOαβ mice (n = 3 mice/genotype, 3 strips per mouse; *p<0.05). Scale bars represent 30 microns.

Figure 6. Co-ablation of C/EBPα/β results in morphological and molecular defects in sebaceous, Meibomian and preputial glands.

(A) H&E staining of sebaceous glands/lobules of 4OHT-treated α^ffβ^ff and (B) 4OHT-treated IKOαβ mice. Co-IF staining in (C) Meibomian glands and (D) preputial glands in untreated controls. H&E staining of (E) Meibomian glands (circled) and (F) preputial glands (circled) of 4OHT-treated α^ffβ^ff and 4OHT-treated IKOαβ mice. (G) Oil Red O staining for lipids in 4OHT-treated α^ffβ^ff (arrows point to sebaceous glands) and 4OHT-treated IKOαβ mice (arrows point to sebaceous lobules). (H) FASN: Semi-quantitative reverse transcriptase PCR on whole skin mRNA from 4OHT-treated α^ffβ^ff and 4OHT-treated IKOαβ mice. SCD3 and MC5R: TaqMan® Real Time PCR. (n = 5 mice/treatment/genotype; *p<0.05). IHC staining for (I) keratin 14 and (J) FASN in 4OHT-treated α^ffβ^ff and 4OHT-treated IKOαβ mouse epidermis (AD-Adipose Tissue). Scale bars represent 30 microns.