Abstract
ObjectiveEpidermal stem cells (ESCs) play a critical role in wound healing, but the mechanism underlying ESC proliferation is not well defined. Here, we explore the effects of RhoA on ESC proliferation and the possible underlying mechanism.MethodsHuman ESCs were enriched by rapid adhesion to collagen IV. RhoA(+/+)(G14V), RhoA(-/-)(T19N) and pGFP control plasmids were transfected into human ESCs. The effect of RhoA on cell proliferation was detected by cell proliferation and DNA synthesis assays. Induction of PKN1 activity by RhoA was determined by immunoblot analysis, and the effects of PKN1 on RhoA in terms of inducing cell proliferation and cyclin D1 expression were detected using specific siRNA targeting PKN1. The effects of U-46619 (a RhoA agonist) and C3 transferase (a RhoA antagonist) on ESC proliferation were observed in vivo.ResultsRhoA had a positive effect on ESC proliferation, and PKN1 activity was up-regulated by the active RhoA mutant (G14V) and suppressed by RhoA T19N. Moreover, the ability of RhoA to promote ESC proliferation and DNA synthesis was interrupted by PKN1 siRNA. Additionally, cyclin D1 protein and mRNA expression levels were up-regulated by RhoA G14V, and these effects were inhibited by siRNA-mediated knock-down of PKN1. RhoA also promoted ESC proliferation via PKN in vivo.ConclusionThis study shows that the effect of RhoA on ESC proliferation is mediated by activation of the PKN1-cyclin D1 pathway in vitro, suggesting that RhoA may serve as a new therapeutic target for wound healing.
Highlights
The skin is the largest organ of the body and the primary protective barrier against the environment [1]
RhoA had a positive effect on epidermal stem cells (ESCs) proliferation, and PKN1 activity was up-regulated by the active RhoA mutant (G14V) and suppressed by RhoA T19N
This study shows that the effect of RhoA on ESC proliferation is mediated by activation of the PKN1-cyclin D1 pathway in vitro, suggesting that RhoA may serve as a new therapeutic target for wound healing
Summary
The skin is the largest organ of the body and the primary protective barrier against the environment [1]. The epidermis, consisting of keratinocytes with variable degrees of differentiation, is constantly maintained by self-renewing epidermal stem cells (ESCs) [2], skin-specific adult stem cells with a strong proliferative capacity. ESCs differentiate into a variety of epidermal lineages to promote self-renewal and regeneration of the epidermis as well as wound healing [3]. Wound healing is a complex process mediated by various factors responsible for the regeneration and reorganization of damaged tissue into its normal architecture, and ESC differentiation, migration and proliferation are the basis of this process. The small GTPase RhoA, a member of the Rho GTPase family (RhoA, CDC42 and Rac), is activated by guanine nucleotide exchange factors (GEFs) [7]. PKN is involved in transforming growth factor (TGF)-beta1-induced expression of smooth muscle marker genes after RhoA activation [18]. PKNs have been implicated in signal transduction as effectors of Rho, Rac and PI3K (phosphoinositide 3-kinase) [19]
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