039 Constitutive TGFβ1 Overexpression in Mouse Keratinocytes Delays Cutaneous Wound Healing

Abstract

TGFβ1 is a multipotent secreted cytokine that plays a pivotal role in wound healing. Previously, both positive and negative effects of TGFβ1 on cutaneous wound healing have been reported. The mechanisms underlying these effects of TGFβ1 remain to be elucidated. In the present study, we used a mouse model to examine the effects of cutaneous wound healing, wherein the latent form of TGFβ1 is constitutively expressed in basal keratinocytes of the mouse epidermis, targeted by the keratin 5 promoter (K5.TGFβ1). In wild-type mice with 6-mm full thickness excisional cutaneous wounds, TGFβ1 protein levels were elevated immediately after wounding, and reached a peak level on day 3 postwounding, which was 3–5-fold higher than that in the nonwounded skin, and sharply declined afterward. This elevated expression pattern suggests that only transient TGFβ1 overexpression is required for normal wound healing. In contrast, K5.TGFβ1 mice with prolonged TGFβ1 transgene expression at the level equivalent to the above peak level in wild-type mice exhibited a significant delay in healing of 6-mm full thickness excisional wounds when compared to wild-type mice. Histological analysis of wounds revealed delayed reepithelialization, increased inflammation and prolonged granular tissue accumulation in K5.TGFβ1wt mice. Immunohistochemistry analysis revealed excessive leukocyte infiltration in transgenic wounds when compared to wild-type wounds. Understandably, the accumulation of leukocytes was also accompanied by increased expression of inflammatory cytokines, chemokines, angiogenic factors and fibrotic factors as determined by qRT-PCR analysis. Although the above factors override the growth inhibitory effect of TGFβ1 on epidermal proliferation away from the wound edge, proliferation at the leading edge of the migrating keratinocytes was reduced in transgenic skin compared to the nontransgenic skin, evident by PCNA staining. Keratinocyte proliferation was also reduced in vitro when assessed by H3-thymidine incorporation. To assess the direct effect of TGFβ1 on keratinocyte migration, we performed an in vitro migration assay and found that K5.TGFβ1 keratinocytes had delayed migration when compared with wild-type cells. Our study suggests that prolonged TGFβ1 expression in keratinocytes delays cutaneous wound healing by excessive inflammation, coupled to a direct inhibitory effect on keratinocyte proliferation and migration.