Fig 1.
CsA treatment down-regulates SFRP1 in the DP of human HFs ex vivo.
(A) Microarray analysis of CsA-treated (6 hours) human HFs (n = 3 male patient samples). (B–E) Characterisation of SFRP1 mRNA (in situ hybridisation; B–D) and protein (immunofluorescence; E) expression in the human HF bulb. (F) Schematic of SFRP1 in the human HF bulb. (G–J) SFRP1 protein analysis after CsA treatment (48 hours) in the human HF bulb (n = 10 HFs Control, 11 HFs CsA; from 2 male patient samples). (K–N) In situ hybridisation of SFRP1 mRNA with CsA treatment (48 hours). (O) qRT-PCR analysis of SFRP1 after CsA treatment (48 hours) (n = 3 male patient samples). (H and J) Two-tailed unpaired t test, (I) Mann-Whitney test, and (O) one-sample t test. Data are expressed as mean ± SEM. Scale bars = 50 μm. Underlying data can be found in S1 Data. ANP32E, acidic nuclear phosphoprotein 32 family member E; APOBEC3A, apolipoprotein B mRNA editing enzyme catalytic subunit 3A; BDP1, B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB; CCND2, cyclin D2; CsA, Cyclosporine A; CLCN5, chloride voltage-gated channel 5; CXCL8, C-X-C motif chemokine ligand 8; DAB2, DAB2, clathrin adaptor protein; DP, dermal papilla; EEA1, early endosome antigen 1; FDCSP, follicular dendritic cell secreted protein; FLG2, filaggrin family member 2; HF, hair follicle; HM, hair matrix; KISS1R, KISS1 receptor; MALAT1, metastasis associated lung adenocarcinoma transcript 1; NLRP2, NLR family pyrin domain containing 2; Pre-Cx, pre-cortex; qRT-PCR, quantitative real-time PCR; PPIB, peptidylprolyl isomerase B; PTX3, pentraxin 3; RTCA, RNA 3′-terminal phosphate cyclase; SFRP1, secreted frizzled related protein 1; SLFN11, schlafen family member 11; SPP1, secreted phosphoprotein 1; TET2, tet methylcytosine dioxygenase 2.
Fig 2.
SFRP1 modulates canonical β-catenin activity at the ligand level in the human HF bulb ex vivo.
(A–F) The human HF bulb expresses the core components of the canonical β-catenin pathway. Immunofluorescence of β-catenin (A), in situ hybridisation of AXIN2 (B), LEF1 (C), and WLS (D). (G–O) qRT-PCR analysis of SFRP1 and the direct β-catenin target genes AXIN2 and LEF1 in the human HF with IWP-2 (G, J, M), rhSFRP1 (H, K, N), and WAY-316606 (I, L, O) treatment (n = 4 male patient samples). (P–T) Analysis of AXIN2 mRNA by in situ hybridisation after WAY-316606 treatment (n = 15 HFs control, 14 HFs WAY-316606; from 3 male patient samples). (G–O) One-sample t test, (Q and S) two-tailed unpaired t test (R and T), and Mann-Whitney test. Data are expressed as mean ± SEM. Scale bars = 50 μm. Underlying data can be found in S1 Data. AXIN2, axis inhibition protein 2; DP, dermal papilla; HF, hair follicle; IWP-2, inhibitor of Wnt production-2; LEF1, lymphoid enhancer binding factor 1; n.s, not significant; PPIB, peptidylprolyl isomerase B; Pre-Cx, pre-cortex, qRT-PCR, quantitative real-time PCR; rhSFRP1, recombinant human SFRP1; SFRP1, secreted frizzled related protein 1; WAY, WAY-316606; WLS, Wntless.
Fig 3.
Inhibiting SFRP1 activity with WAY-316606 enhances human hair growth, increases K85 protein expression, and inhibits spontaneous catagen ex vivo.
(A) Hair shaft elongation of human HFs ex vivo with WAY-316606 treatment (n = 31 HFs Control, 30 HFs WAY-316606; from 3 male patient samples). (B) K85 protein quantification using immunofluorescence after 48 hours of treatment with WAY-316606 (n = 16 HFs control, 14 HFs WAY-316606; from 3 male patient samples). (C) Macroscopic quantification of hair cycle stage on day 6 with WAY-316606 treatment. (D–H) Validation of HF cycle stage with Ki-67/TUNEL analysis (n = 21 HFs control, 20 HFs WAY-316606; from 3 male patient samples) and Masson's-Fontana (n = 17 HFs control, 20 HFs WAY-316606; from 3 male patient samples). (I) Working hypothesis. Data are expressed as mean ± SEM. (A, B, E, G, and H) Two-tailed unpaired t test and (F) Mann-Whitney test. Scale bars, A = 1 mm; B, D, and H = 50 μm. Underlying data can be found in S1 Data. AL, Auber’s line; Axin2, axis inhibition protein 2; CsA, Cyclosporine A; DC, differentiating cell; DP, dermal papilla; DPC, dermal papilla cell; HF, hair follicle; K85, Keratin 85; LEF1, lymphoid enhancer binding factor 1; SFRP1, secreted frizzled related protein 1; TAC, transient amplifying cell; WAY, WAY-316606.