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APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex.
Shimomura Y
,
Agalliu D
,
Vonica A
,
Luria V
,
Wajid M
,
Baumer A
,
Belli S
,
Petukhova L
,
Schinzel A
,
Brivanlou AH
,
Barres BA
,
Christiano AM
.
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Hereditary hypotrichosis simplex is a rare autosomal dominant form of hair loss characterized by hair follicle miniaturization. Using genetic linkage analysis, we mapped a new locus for the disease to chromosome 18p11.22, and identified a mutation (Leu9Arg) in the adenomatosis polyposis down-regulated 1 (APCDD1) gene in three families. We show that APCDD1 is a membrane-bound glycoprotein that is abundantly expressed in human hair follicles, and can interact in vitro with WNT3A and LRP5-two essential components of Wnt signalling. Functional studies show that APCDD1 inhibits Wnt signalling in a cell-autonomous manner and functions upstream of beta-catenin. Moreover, APCDD1 represses activation of Wnt reporters and target genes, and inhibits the biological effects of Wnt signalling during both the generation of neurons from progenitors in the developing chick nervous system, and axis specification in Xenopus laevis embryos. The mutation Leu9Arg is located in the signal peptide of APCDD1, and perturbs its translational processing from the endoplasmic reticulum to the plasma membrane. APCDD1(L9R) probably functions in a dominant-negative manner to inhibit the stability and membrane localization of the wild-type protein. These findings describe a novel inhibitor of the Wnt signalling pathway with an essential role in human hair growth. As APCDD1 is expressed in a broad repertoire of cell types, our findings indicate that APCDD1 may regulate a diversity of biological processes controlled by Wnt signalling.
Figure 1: The HHS phenotype maps on chromosome 18p11.2 at a point mutation in the APCDD1 gene. a–d, Clinical appearance of HHS patients (a, b) and their hair shafts (c, d). Scale bar, 100 μm. e, Haplotype analysis of a Pakistani family, HHS1. The linked haplotype is indicated in red. The critical recombination events are indicated by black arrowheads. kb, kilobases. f, In situ hybridization with an APCDD1 antisense mRNA probe in human hair follicles. APCDD1 is present in the dermal papilla (DP), matrix (Mx), hair shaft cortex (HSCx), and cuticle (HSCu) of the hair follicle. g–j, Immunofluorescence in human HFs with a mouse polyclonal anti-APCDD1 antibody (Abnova). The expression of APCDD1 protein in the HSCx (white box in g) overlaps with that of E- and P-cadherin (pan-cadherin) (h–j). Counterstaining with DAPI is shown in blue (g, j). Scale bars, 100 μm (f, g) and 20 μm (h). k, Schematic of APCDD1 protein and the position of the Leu9Arg mutation. l, A western blot with the mouse polyclonal anti-APCDD1 antibody (Abnova) from cell lysates of human scalp skin shows two fragments around 58 and 130 kDa in size. A similar pattern was observed with the haemagglutinin (HA)-tagged wild-type APCDD1 overexpressed in HEK293T cells.
Figure 2: Wild-type, but not Leu9Arg mutant APCDD1, inhibits canonical Wnt signalling.a, Co-immunoprecipitation assays show that the extracellular domain of APCDD1 (APCDD1δTM) strongly interacts with the extracellular domain of LRP5 (LRP5-EC) and WNT3A. The extracellular domain of a non-Wnt related single transmembrane receptor CD40 (CD40-EC) was used as a negative control. IP, immunoprecipitation; WB, western blot. b, TOP/FOP-flash reporter assays in HEK293T cells. TOP-flash, TCF-β-catenin reporter (active); FOP-flash, scrambled consensus reporter (inactive). c, Effect of APCDD1 overexpression on transcriptional activity of the Wnt-specific sia reporter gene induced by wnt8 or β-catenin in Xenopus. APCDD1 (1 ng RNA) inhibited wnt8- (50 pg RNA), but not β-catenin-(1 ng RNA) induced transcription. The number above the column indicates fold repression by APCDD1. d, The Leu9Arg mutant APCDD1 has a dominant-negative effect on wild-type APCDD1 in Xenopus. Activity of the sia reporter gene induced by wnt8 RNA (50 pg) was inhibited by co-injection of wild-type APCDD1 RNA (1 ng), but not by co-expression of wild-type and the Leu9Arg mutant. e, Western blot of APCDD1 levels in Xenopus embryos. APCDD1(L9R) affects wild-type protein levels in Xenopus embryos. Coexpressed β-Galactosidase (β-Gal) is used as a loading control. f, Western blot analysis of lysates from HEK293T cells transfected with wild-type, APCDD1(L9R) or APCDD1(L9V). g, The expression level of HA-tagged wild-type APCDD1 (WT–HA) is decreased by co-expression with APCDD1(L9R). β-actin was used as a normalization control (f, g). h–j, Immunofluorescence in transfected HEK293T cells with an APCCD1 antibody (green). The cell membrane was labelled with rhodamine–phalloidin (red). Scale bar, 20 μm. k, Quantification of subcellular localization of APCDD1 isoforms. Error bars represent mean ± s.e.m. (n = 20 cells per group). Wild type versus Leu9Arg, P < 8 × 10-7; wild type versus wild type + Leu9Arg, P < 3 × 10-6; Leu9Arg versus wild type + Leu9Arg, P = 0.99. The reported P-values are adjusted with the Bonferroni correction.
FIGURE 4. APCDD1 inhibits the Wnt pathway in Xenopus embryos. a, b, Dorsal (do) overexpression of human APCDD1 (hAPCDD1) reduces axial and anterior structures (b). Scale bar, 4 mm (a). c, d, Ventral (ve) overexpression of hAPCDD1 produces a secondary axis (arrow in c) in cell autonomous fashion (GFP tracer under ultraviolet (UV) light, arrow in d). e–g, Phenotype of Xapcdd1 protein depletion and its rescue. Dorsal depletion by MO produced a ventralized phenotype (e), which was rescued by Xapcdd1 RNA (f) and by dominant-negative wnt8 RNA (DN-wnt8) (g). h, APCDD1 is required in the signal-receiving cells. wnt8 RNA and siamois reporter gene were injected in adjacent cells. hAPCDD1 inhibited signalling only when co-injected with the reporter. +, Coexpression in the same cell; /, expression in different cells. i, Potential mechanism of action for wild-type and mutant APCDD1. Top, wild-type APCDD1 (L) is processed in the ER and localized at the cell membrane, where it may inhibit Wnt signalling by interacting with Wnt and LRP proteins. Bottom, when wild-type and APCDD1(L9R) (R) are co-expressed, wild-type APCDD1 is retained and degraded in the ER with the mutant, thus activating the Wnt signalling pathway.
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