MicroRNA-21 is an important downstream component of BMP signalling in epidermal keratinocytes

Abstract

Bone morphogenetic proteins (BMPs) play essential roles in the control of skin development, postnatal tissue remodelling and tumorigenesis. To explore whether some of the effects of BMP signalling are mediated by microRNAs, we performed genome-wide microRNA (miRNA) screening in primary mouse keratinocytes after BMP4 treatment. Microarray analysis revealed substantial BMP4-dependent changes in the expression of distinct miRNAs, including miR-21. Real-time PCR confirmed that BMP4 dramatically inhibits miR-21 expression in the keratinocytes. Consistently, significantly increased levels of miR-21 were observed in transgenic mice overexpressing the BMP antagonist noggin under control of the K14 promoter (K14-noggin). By in situ hybridization, miR-21 expression was observed in the epidermis and hair follicle epithelium in normal mouse skin. In K14-noggin skin, miR-21 was prominently expressed in the epidermis, as well as in the peripheral portion of trichofolliculoma-like hair follicle-derived tumours that contain proliferating and poorly differentiated cells. By transfecting keratinocytes with a miR-21 mimic, we identified the existence of two groups of the BMP target genes, which are differentially regulated by miR-21. These included selected BMP-dependent tumour-suppressor genes (Pten, Pdcd4, Timp3 and Tpm1) negatively regulated by miR-21, as well as miR-21-independent Id1, Id2, Id3 and Msx2 that predominantly mediate the effects of BMPs on cell differentiation. In primary keratinocytes and HaCaT cells, miR-21 prevented the inhibitory effects of BMP4 on cell proliferation and migration. Thus, our study establishes a novel mechanism for the regulation of BMP-induced effects in the skin and suggests miRNAs are important modulators of the effects of growth factor signalling pathways on skin development and tumorigenesis.

Fig. 1.

miRNA expression profile of mouse primary keratinocytes treated with BMP4. (A,B) Microarray analysis. (A) A heat map depicting the miRNAs for which the expression significantly (P<0.01) changed after BMP4 treatment; red and green colours represent elevated and decreased expression of miRNAs, respectively. (B) Table of miRNAs showing ≥2-fold or higher changes in expression in the keratinocytes treated with BMP4 (levels of expression are shown in arbitrary units). (C,D) Detection of miR-21 by qRT-PCR. BMP4 treatment significantly (*P<0.01) reduced expression of the mature form of miR-21 in the primary mouse epidermal keratinocytes (PMK; C) and HaCaT cells (D).

Fig. 2.

Spatiotemporal expression of miR-21 in the skin. (A,B) Representative photomicrographs of in situ hybridization for miR-21. (A) miR-21 expression in the basal and suprabasal layers of the epidermis (arrowheads), in the HF outer and inner root sheaths (large and small arrows, respectively) and in the hair matrix (asterisk) in the skin of a 12-week-old wild-type mouse. (B) Prominent miR-21 expression in the epidermis (arrowheads) and in the periphery of the tumours (arrows) in K14-noggin skin. (C) Detection of miR-21 by qRT-PCR: there were significantly (*P<0.01) increased levels of miR-21 in the total skin of K14-noggin mice than in wild-type mice.

Fig. 3.

miR-21 modulates the effects of BMP4 on gene expression, cell proliferation and migration in keratinocytes. (A–E) qRT-PCR analysis of gene expression in the primary mouse keratinocytes relatively to Gapdh levels. (A) BMP4 treatment causes a significant increase in the expression of Id1, Id2, Id3 and Msx2 compared with untreated cells. (B) Upregulation in the expression of Pdcd4, Pten, Timp3 and Tpm1 transcripts by BMP4 treatment. (C) Expression of Id1, Id2, Id3 and Msx2 is not affected by transfection with miR-21 mimic (pro-miR-21). (D) Expression of Pdcd4, Pten, Timp3 and Tpm1 is decreased after treatment with miR-21 mimic. (E) miR-21 mimic prevents BMP4-induced expression of Pdcd4, Pten, Timp3 and Tpm1 in keratinocytes. (F) Expression of Pten, Pdcd4, Timp3 and Tpm1 in the skin of K14-noggin transgenic mice is decreased compared with wild-type mice. (G) Flow cytometry analysis of the cell cycle in primary keratinocytes: a significant decrease in number of proliferating cells caused by BMP-4 treatment was prevented by the miR-21 mimic. (H) Cell migration (scratch) assay. miR-21 mimic significantly increases HaCaT cell migration compared with the control, and interferes with the suppression of cell migration induced by BMP4; *P<0.01, **P<0.001.

Fig. 4.

Schematic illustrating an involvement of miR-21 in the modulation of BMP4-mediated effects on gene expression in keratinocytes. miR-21 modulates the BMP anti-tumour activity by negative regulation of the expression of BMP-dependent tumour-suppressor genes (Pten, Pdcd4, Timp3 and Tpm1) in keratinocytes. However, the effects of BMP on keratinocyte differentiation mediated through its targets Id1, Id2, Id3 and Msx2 are miR-21 independent.