Bmx tyrosine kinase transgene induces skin hyperplasia, inflammatory angiogenesis, and accelerated wound healing

Abstract

The Bmx gene, a member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in arterial endothelium and in certain hematopoietic and epithelial cells. Previous in vitro studies have implicated Bmx signaling in cell migration and survival and suggested that it contributes to the progression of prostate carcinomas. However, the function of Bmx in normal tissues in vivo is unknown. We show here that Bmx expression is induced in skin keratinocytes during wound healing. To analyze the role of Bmx in epidermal keratinocytes in vivo, we generated transgenic mice overexpressing Bmx in the skin. We show that Bmx overexpression accelerates keratinocyte proliferation and wound reepithelialization. Bmx expression also induces chronic inflammation and angiogenesis in the skin, and gene expression profiling suggests that this occurs via cytokine-mediated recruitment of inflammatory cells. Our studies provide the first data on Bmx function in vivo and form the basis of evaluation of its role in epithelial neoplasia.

Figure 1.

Bmx expression during wound healing analyzed in Bmx^LacZ/LacZ mice. In normal skin β-galactosidase staining was seen only in arterial endothelium (A, arrow). In the healing wound, the proliferating and migrating basal keratinocytes and the leading edge of the migrating epithelium stain strongly for β-galactosidase (here days 3 (B) and 4 (C), arrows). Very few cells are stained in the healed epidermis (D; here day 12). Counterstaining is with nuclear red.

Figure 2.

Comparison of K14-Bmx transgenic and control littermate mice. (A) Bmx expression (arrowhead) in TG mice analyzed by Western blotting of immunoprecipitates (IP) or total skin lysates (total). The asterisk indicates a nonspecific polypeptide. (B) Note the absent whiskers in TG mouse. H&E-stained cross sections from the ears of TG and WT mice (C and D). The arrowheads point to blood vessels in the cross sections. PCNA staining of the epidermis of back skin in TG (E) and WT mice (F). The arrowheads point to the proliferating basal cells. Sections of TG (G) and WT (H) ears stained for apoptotic cells (TUNEL; green, arrowheads) and cell nuclei (Hoechst; blue). Sections of tail skin of TG (I) and WT mice (J). Arrowhead shows the dermal papillae containing blood capillaries; arrow indicates the rete ridge-like changes in the skin; asterisk mark parakeratosis (retained cell nuclei in the horny layer of the epidermis). PCNA staining of tail sections of TG (K) and WT mice (L). Arrowheads indicate PCNA positive nuclei in the skin. (M) The percentage of BrdU-positive cells in MMC-E epithelial cells expressing mock vector (Mock), kinase-deficient Bmx (K445R) or Bmx (WT). Bars represent average counts from four separate infections and below are shown the relative protein amounts as detected by Western blotting. NS, not significant.

Figure 3.

Immunohistochemical analysis of the K14-Bmx mouse skin. TG and WT skin stained for keratin 5 (A, TG; B, WT), keratin 1 (C, TG; D, WT) and keratin 10 (E, TG; F, WT). Vertical bars next to A–F denote epidermal thickness. Staining for CD45 in TG (G) and WT (H) ear cross sections, positive cells stain red (arrowheads). Cell nuclei stained with Hoechst; blue. Quantification of CD45-positive cells +SD (I). The asterisk indicates statistical significance (p < 0.001, Student's unpaired t test). Staining of the skin for the granulocytic marker Ly6G (J, TG; K, WT) and the macrophage marker F4/80 (L, TG; M, WT). The drawn perpendicular lines in A–H and J–M indicate the basement membrane of the epidermis in the skin cross sections. Cell nuclei are stained with Hoechst; blue.

Figure 4.

Wound healing in the K14-Bmx and control littermate mice. Bars represent the percentage of the remaining wound area +SD on days 4, 5, and 6 after wounding. All wounds had healed on day 7. Student's unpaired t test was used for statistical analysis.

Figure 5.

Angiogenesis in the K14-Bmx mice. Whole-mount staining of TG and WT mouse ears for the vascular endothelial marker PECAM-1 (A–D) and for α-smooth muscle actin (SMA; E and F).

Figure 6.

Analysis of differential gene expression in TG and WT skin by RT-PCR. RT-PCR of WT and TG newborn skin total RNA for several of the genes that were up-regulated in the TG skin in Affymetrix analysis. See text for details.