Palmitoylation regulates epidermal homeostasis and hair follicle differentiation

Abstract

Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs). Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep) is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE) and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear beta-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis.

Figure 1. Identification and transgenic rescue of the dep mutation.

(A) Mapping the dep interval against the b-del complex. When dep is crossed with the b-IOZ deletion mutant (purple), offspring exhibit the hairloss phenotype. When crossed with the b-46UTHc deletion mutant (yellow), the hairloss phenotype disappears, indicating that the dep mutation lies within the genomic interval between the distal breakpoints of the 2 deletions. (B) Schematic of Zdhhc21 protein with dep C-terminal 3bp deletion resulting in the loss of a single highly conserved residue, phenylalanine (F) at position 233. The cysteine-rich domain containing a conserved DHHC motif is shown in blue on the cytoplasmic side. (C) The BAC clone RP23-76J17, which harbors the intact genomic sequences of Zdhhc21 and Cer1 successfully rescues the dep phenotype, shown at 6.5 weeks. (D) Transgenic dep mutant skin appears histologically normal and correct timing hair follicle differentiation is also restored. (E) Non-transgenic mutant littermate control.

Figure 2. dep mutation disrupts PAT activity and localization of Zdhhc21.

(A) [³H]Palmitate fluorography of individual Zdhhc21 (wild type, dep and C120S) HA-tagged constructs co-transfected with eNOS or Lck into HEK293 cells. Increased incorporation of [³H]palmitate into targets is observed with the wild type construct. Neither mutant shows palmitoylation activity above background. Immunoblots using anti-HA (Zdhhc21 constructs), anti-myc (eNOS) and anti-Lck control for loading. (B–I) Immunofluorescence of primary keratinocytes transfected with wild type (B–E) and dep (F–I) HA-tagged Zdhhc21 cDNAs. (B,F) Epidermal marker Keratin 14 (red) and anti-HA (green) antibody staining. While wild type protein shows discrete and compartmentalized localization, the mutant protein is diffuse. (C,G) cis-Golgi network marker GM130 (red) and anti-HA (green) antibody staining. (D,H) trans-Golgi marker Tgn138 (green) and anti-HA (red). (E,I) ER marker PDI (green) and anti-HA (red).

Figure 3. Zdhhc21 expression is restricted to differentiated post-mitotic lineages of hair follicles.

Confocal slices (1µm) of Zdhhc21 protein expression in wild type (A–E) and dep (F) P28 anagen follicles. (A) Ubiquitous Zdhhc21 (red) expression colocalizes with the outermost layer of AE15 (green) expression. (B) Punctate Zdhhc21 staining (red) colocalizes with the outermost layer of AE13 expression (green: weaker than inner cortex expression) and (C) Foxn1 expression (white arrowhead). (D) Single foci of Zdhhc21 staining (red: yellow arrowhead) per cell of the IRS cuticle is seen in the innermost Gata3-positive layer (green). (E) In wild type skin, punctae of Zdhhc21 (red) colocalize with GM130 (green), whilst diffuse staining is observed in dep mutants (F). Arrowheads mark same region in single channels.

Figure 4. Zdhhc21 is required for epidermal differentiation and patterning.

dep mutant skin displays pronounced defects postnatally associated with anagen stages of hair cycle including abnormal hair follicle differentiation, and interfollicular and sebaceous hyperplasia. Stages shown: anagen P28 (A–F), late embryonic morphogenesis E18.5 (G,J), early catagen P14 (H,K) and telogen P21 (I,L). Hematoxylin and eosin staining (A,D,G–L). Oil-Red-O staining of cryosections (B,E). Nile red wholemount staining of P28 tail skin(C,F). Insert in (A) shows histology of a “hairless” dorsal region at P28: note the more severe follicular phenotype. Filled arrowheads mark the interfollicular epidermis and open arrowheads point to examples of sebaceous glands.

Figure 5. Loss of Zdhhc21 disrupts balance between proliferation and differentiation in anagen interfollicular epidermis.

P28 wild type (A,A′,C,D,G,H,K,L) and dep (B,B′,E,F,I,J,M,N) skin. dep mutants show expansion of K5+ (red), p63+ (green) progenitor compartment (A–D). p63+ progenitors (green) are expanded into terminally differentiating K10+ (red) layers of dep IFE (D,F). Expression of transcriptional regulator of differentiation Gata3 in the basal and suprabasal keratinocytes is greatly reduced or absent in dep IFE with a parallel increase in proliferative basal phospho-ERK signals (green; Gata3, red; phospho-p42/44, G,H). (G–H″) single channels of region of interest in merge shown in (G,H).

Figure 6. Anagen signalling is delayed in dep follicles.

Wild type (A,C,E,G,I) and dep (B,D,F,H,J) P28 dorsal follicles. Several key signalling pathways required for postnatal hair development are not affected in dep mutants. Canonical BMP signalling (red; phospho-Smad-1/-5/-8, A,B), non-canonical BMP/TGF-β signalling (red; phospho-p38, C,D) and canonical TGF-β signalling (red: phospho-Smad-2/-3 E,F) are expressed in delayed dep follicles. Expression and nuclear localization of downstream canonical Wnt effectors β-catenin (green; β-catenin, G–J) and Lef-1 (red; Lef-1, I,J) are reduced in dep hair matrix and shaft (arrowed). Of the transcription factors regulating differentiation of anagen hair follicle lineages, expression of Wnt transcriptional target Foxn1 is reduced or absent in dep follicles (green; Foxn1 A,B), while Hoxc13 is expressed in all dep follicles (green; Hoxc13 C, D). Gata3 is expressed in all dep follicles although levels of expression may be slightly reduced. (green; Gata3 E,F). Along side merge panel are shown single channel of regions of interest to highlight staining.

Figure 7. Zdhhc21 palmitoylation of Fyn is required for localization in vitro and in vivo.

(A) [3H]Palmitate fluorography of subset of panel of all mammalian Zdhhc HA-tagged constructs co-transfected with Fyn::GFP into HEK293 cells. Increased incorporation of [3H]palmitate into targets is observed with Zdhhc15, −20 and −21. A myristoylation-deficient Fyn mutant is not palmitoylated following cotransfection with Zdhhc21 (G2A, final lane). (B–E) Immunofluorescence of HEK293 cotransfected with Fyn::GFP alone (B), Fyn::GFP and wild type HA-tagged Zdhhc21 cDNA (C), G2A mutant Fyn::GFP alone (D) and G2A mutant Fyn::GFP and HA-tagged Zdhhc21 (E). Zdhhc21 causes increased perinuclear accumulation of Fyn::GFP, a domain associated with localization of other palmitoylated targets including PSD-95 and GAP-43. This localization is not observed with G2A mutant Fyn::GFP (D). (F–I) Confocal images (0.5µm) of immunostaining of Fyn (red: F,G) and active SFKs (red: H–I) in P32 anagen dorsal hair follicles in wild type (F,H) and dep mutants (G,I). Arrowheads mark region of interest in IRS cuticle that are enlarged in single channel images (F′–I′) where Fyn localization to membranes between IRS cuticle cells is lost in dep mutants.