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. 2006 Nov 28;103(48):18166-71.
doi: 10.1073/pnas.0608899103. Epub 2006 Nov 17.

Bone morphogenetic protein signaling regulates the size of hair follicles and modulates the expression of cell cycle-associated genes

Andrey A Sharov  1 , Tatyana Y Sharova, Andrei N Mardaryev, Alice Tommasi di Vignano, Ruzanna Atoyan, Lorin Weiner, Shi Yang, Janice L Brissette, G Paolo Dotto, Vladimir A Botchkarev
Affiliations

Bone morphogenetic protein signaling regulates the size of hair follicles and modulates the expression of cell cycle-associated genes

Andrey A Sharov et al. Proc Natl Acad Sci U S A. .

Abstract

Bone morphogenetic protein (BMP) signaling is involved in the regulation of a large variety of developmental programs, including those controlling organ sizes. Here, we show that transgenic (TG) mice overexpressing the BMP antagonist noggin (promoter, K5) are characterized by a marked increase in size of anagen hair follicles (HFs) and by the replacement of zig-zag and auchen hairs by awl-like hairs, compared with the age-matched WT controls. Markedly enlarged anagen HFs of TG mice show increased proliferation in the matrix and an increased number of hair cortex and medulla cells compared with WT HFs. Microarray and real-time PCR analyses of the laser-captured hair matrix cells show a strong decrease in expression of Cdk inhibitor p27(Kip1) and increased expression of selected cyclins in TG vs. WT mice. Similar to TG mice, p27(Kip1) knockout mice also show an increased size of anagen HFs associated with increased cell proliferation in the hair bulb. Primary epidermal keratinocytes (KC) from TG mice exhibit significantly increased proliferation and decreased p27(Kip1) expression, compared with WT KC. Alternatively, activation of BMP signaling in HaCaT KC induces growth arrest, stimulates p27(Kip1) expression, and positively regulates p27(Kip1) promoter activity, thus further supporting a role of p27(Kip1) in mediating the effects of BMP signaling on HF size. These data suggest that BMP signaling plays an important role in regulating cell proliferation and controls the size of anagen HFs by modulating the expression of cell-cycle-associated genes in hair matrix KC.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Increase of HF size in K5-Noggin mice is accompanied by increase of cell number in the hair matrix, cortex, and medulla. At distinct time points of postnatal development (postnatal day 6.5, 10–12 weeks, 6 months), hairs and dorsal skin of WT and TG mice were harvested. Skin cryosections were processed for histoenzymatic visualization of alkaline phosphatase (A–F), and histomorphometric analyses of the HFs and plucked hairs were performed (G–L). Statistical analyses were performed by using Student's t test, ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001. (A–F) Progressive increase of the hair bulb size (arrows) in TG vs. WT mice. (G) Lack of small HFs and significant (P < 0.001) increase in proportion of intermediate HFs in TG mice versus WT mice. (H and I) Replacement of zig-zag and auchene hairs in TG mice by awl-like hairs. (J, K, and L) Awl-like hairs of TG mice show significantly increased diameter (P < 0.01) and number of medulla cells (P < 0.05), compared with zig-zag hairs of WT mice. G, guard; SC, subcutis; Zz, zig-zag.
Fig. 2.
Fig. 2.
Molecular analyses of anagen HFs from WT and TG mice suggest differences in expression of cell-cycle-associated genes. Skin of 12-week-old WT and TG mice was synchronized at anagen VI stage of the hair cycle by depilation. Skin was harvested, and cryosections were processed for laser-capture microdissection, RNA isolation and amplification, microarray and real-time PCR analyses (A–G), and for immunohistochemistry (H–J). (A and B) Anagen VI HF before and after laser-capture microdissection. (C) RT-PCR of the hair matrix- and FP-specific genes in RNA samples obtained from the corresponding areas of anagen VI HFs of WT and TG mice. (D–G) Real-time PCR of genes expressed in the hair matrix or FP that show differences between intermediate HFs of TG mice and intermediate or small HFs in WT mice. (H) pSmad1/5 in the hair matrix (large arrows), hair shaft (small arrows), and outer root sheath (arrowheads) in small and intermediate HFs of WT and TG mice. (I) Ectopic appearance of BrdU+ (green fluorescence, arrowhead) and Ki-67+ cells (red fluorescence, arrowhead) in differentiating cells of the hair shaft in TG mice. (J) Decrease in p27Kip1 expression in hair matrix of intermediate HFs in WT and TG mice (arrows).
Fig. 3.
Fig. 3.
Increased size and cell proliferation in anagen HFs of p27Kip1 knockout mice. Dorsal skin of 12-week-old WT and p27Kip1 knockout (−/−) mice was harvested 12 days after depilation. Skin cryosections were processed for either histoenzymatic visualization of alkaline phosphatase (A and B) or immunofluorescent stainings with anti-Ki-67 antiserum (C and D). Histomorphometric analyses of the HFs and plucked hairs were performed (E and F). Statistical analysis was performed by using Student's t test, ∗, P < 0.05. (A and B) Anagen VI HFs in p27Kip1 −/− mice (arrows) and WT mice. (C and D) Proliferating cells in the matrix (arrows) and ectopic appearance of Ki-67 in differentiating cells above the FP (arrowhead) in the HF of p27Kip1 −/− mice. (E) Decrease of small HFs and increase of intermediate HFs in p27Kip1 −/− mice. (F) Increase in number of medulla cells in awl hairs of p27Kip1 −/− mice.
Fig. 4.
Fig. 4.
Effects of BMP signaling on KC growth arrest, p27Kip1 expression and promoter activity. Primary mouse KC were obtained from newborn WT and TG mice. Human HaCaT KC were incubated for 24 h with rhBMP4, and a combination of BMP4 and noggin or diluent control and were harvested 6–24 h after beginning the experiment. Flow cytometry of cell cycle (A and B), real-time PCR (C and D), Western blot (E), and promoter assay (F) were performed. (A) A significant increase in number of cells in S phase and decrease of G0/G1 cells in TG vs. WT mice. (B) Significant increase in number of cells in G0/G1 phase and decrease in number of proliferating cells after BMP-4 treatment, compared with control. (C) Real-time PCR of the Cdk2, cyclin M3, p21Cip1, and p27Kip1 transcripts in primary KC of WT and TG mice. (D) Increase in p27Kip1 transcripts in HaCaT cells 6–24 h after BMP-4 treatment and inhibition of these effects by noggin. (E) Increase in p27Kip1 protein expression, Smad1/5 phosphorylation and decrease of pRb protein in HaCaT cells after BMP-4 treatment. (F) Effects of cotransfection with vectors containing constitutively active BMPR-IA (Alk3QD), BMPR-IB (Alk6QD), pCMVmSmad1, and/or pCMVSmad5 on transcription of the p27PF-Luc reporter plasmid containing human p27 promoter region and pGVB2L-Luc (negative control). (G) Scheme illustrating mechanisms of the effects of BMP signaling on proliferation and differentiation of hair matrix KC.
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