Anti-wrinkle effect of bone morphogenetic protein receptor 1a-extracellular domain (BMPR1a-ECD)

Abstract

Bone morphogenetic proteins (BMPs) have diverse and important roles in the proliferation and differentiation of adult stem cells in our tissues. Especially, BMPs are well known to be the main inducers of bone formation, by facilitating both proliferation and differentiation of bone stem cells. Interestingly, in skin stem cells, BMPs repress their proliferation but are indispensable for the proper differentiation into several lineages of skin cells. Here, we tested whether BMP antagonists have an effect on the prevention of wrinkle formation. For this study we used an in vivo wrinkle-induced mouse model. As a positive control, retinoic acid, one of the top anti-wrinkle effectors, showed a 44% improvement compared to the non-treated control. Surprisingly, bone morphogenetic protein receptor 1a extracellular domain (BMPR1a-ECD) exhibited an anti-wrinkle effect which was 6-fold greater than that of retinoic acid. Our results indicate that BMP antagonists will be good targets for skin or hair diseases.

Fig. 1.. Production and biological activity of BMP2, Noggin or BMPR1a-ECD protein. (A-C) 12% SDS-PAGE images. Lane 1 contains the protein ladder, lanes 2 and 3 indicate purified BMP2, Noggin or BMPR1a-ECD solution in the presence or absence of reducing agent, DTT. The active form of BMP2 or Noggin is a dimer. The migration of BMPR1a-ECD is not affected by the addition of DTT because it is a monomeric form. (D, E) Smad-1 luciferase reporter assay. C2C12 cells were transfected with Id1-Luc, Smad1 and beta-galactosidase plasmids using Fugene6. Luciferase activity was measured and normalized by beta-Gal value. Fold induction values are mean ± standard error from triplicated experiments, the χ axis shows the Noggin and BMP2 molar ratio in nM (D) or the BMPR1a-ECD and BMP2 molar ratio in nM (E), while the y axis shows the relative luciferase fold induction activity. *P ＜ 0.05, **P ＜ 0.01 and ***P ＜ 0.001 indicate significant differences as compared to the NT group. ^aP ＜ 0.05, ^bP ＜ 0.01 and ^cP ＜ 0.001 means significant different as compared to the 10 nM BMP2 (only) treated group.

Fig. 2.. Liposome size measurement in Lipo/BMP2, Lipo/Noggin or Lipo/BMPR1a-ECD sample. Hydrogenated lecithin comprised of more than 75% phosphocholine was mixed with BMP2, Noggin or BMPR1a-ECD protein solution to a final concentration of 1% lecithin and 0.001% of each test protein, in a total volume of 1 liter. These mixtures were homogenized at 800bar until the liposome size ranged from 100 to 200nm. In this figure, used here as an example, the liposome size in Lipo/BMPR1a-ECD samples was determined.

Fig. 3.. Dorsal skin photographs and wrinkle scores. Each 8 group is as follows. (A, B) Each sample was topically treated on the back of mice of each group as described in Materials and Methods. Group 1; UVB(-), group 2; UVB(+), group 3-8; UVB(+) with treatment of 15% ethanol (group 3), 0.01% retinoic acid (group 4) or 1% liposome (group 5), 1% liposome-encapsulated 0.001% BMP2 (group 6), 0.001% Noggin (group 7) or 0.001% BMPR1a-ECD (Group 8). ×2 magnified representative images are displayed in (B), and wrinkle scores (skin wrinkling grade: 0-10) in (C). *P ＜ 0.05, **P ＜ 0.01, ***P ＜ 0.001 vs. column. ANOVA.

Fig. 4.. BMPRIa-ECD prevents both abnormal epidermal thickness and loss of collagen triggered by UVB exposure in SKH-1 mice. SKH-1 hairless mice were irradiated with a total dose of 1.86 J/Cm² over the 11 week treatment period. Application of each sample is illustrated in the Materials and Methods section. Dorsal skin samples were obtained 24 h after the last sample application. Epidermal thickness was determined by H&E stained skin sections (A). The results are expressed as a mean ± SD of the thickness in μm (B). Bar size, 100 μm. *P ＜ 0.05. The same amount of total RNA was reverse-transcribed to generate its cDNA, and these cDNAs were used for the RT-PCR analysis of procollagen Iα (C) and MMP-1 (D) expression. The results are representative of 3 independent experiments and have been normalized based on GAPDH level. *P ＜ 0.05, **P ＜ 0.01, ***P ＜ 0.001 vs. controls. ANOVA.