RhoA promotes epidermal stem cell proliferation via PKN1-cyclin D1 signaling

Abstract

Objective: Epidermal 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.

Methods: Human 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.

Results: RhoA 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.

Conclusion: 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.

Fig 1. Characterization of isolated and cultured cells.

(A) Cells behaved like clonogenic stem cells in vitro (×100). (B) Magnification of (A) (×200). (C) Magnification of (B) (×400). (D-F) The cells expressed integrin β1 and CK19 (immunofluorescence, ×1000).

Fig 2. Effect of RhoA on huESC proliferation.

(A) Transfection of RhoA^(+/+)(G14V), RhoA^(-/-)(T19N) or pGFP control plasmid (1.5 μg/ml each plasmid) into huESCs using Lipofectamine transfection reagent. (B) After transfection, cell proliferation was quantitated using a CCK-8 assay according to the manufacturer’s instructions. (C) DNA synthesis was quantified based on [³H]-thymidine uptake in disintegrations per minute (DPM). * P<0.05 versus control, **P<0.01 versus control. Data are shown as the mean ± SD of duplicate experiments on samples from three different donors (unpaired, two-tailed Student's t-test).

Fig 3. RhoA induces PKN1 activation and phosphorylation.

HuESCs were transfected for 48 h with RhoA^(+/+)(G14V), RhoA^(-/-)(T19N) or pGFP control plasmid (1.5 μg/ml each plasmid) using Lipofectamine transfection reagent. (A) Cell lysates were subjected to Western blot assays with anti-Thr⁷⁷⁴ phosphorylated (p-)PKN1, anti-total PKN1 and anti-GAPDH antibodies. (B) Thr⁷⁷⁴ p-PKN1/total-PKN1 was determined after densitometric analysis. *P<0.05 versus control, **P<0.01 versus control. The data are presented as the mean ± SD (error bars) of at least three separate experiments (unpaired, two-tailed Student's t-test).

Fig 4. RhoA regulates huESC proliferation via PKN1 signaling: evidence from siRNA-mediated PKN1 knock-down assays.

(A) A representative Western blot demonstrating the effects of two distinct PKN1 siRNAs. (B) Quantification of the Western blots in (A); **P<0.001 versus control siRNA. After Lipofectamine-mediated transfection of 1.5 μg/ml G14V plasmid, 1.5 μg/ml pGFP control plasmid, and 60 nM specific PKN1 siRNA into huESCs, a cell counting assay with CCK-8 (C) and a DNA synthesis assay with [³H]-thymidine incorporation (D) were used to assess cell proliferation at a 48-h timepoint. The data are presented as the mean ± SD of three independent experiments, each performed in duplicate (n = 3). The unpaired, two-tailed Student's t-test was used to assess significance: **P<0.001 versus control, ^##P<0.001 versus G14V + control siRNA.

Fig 5. RhoA regulates cyclin D1 expression through PKN1.

After Lipofectamine-mediated transfection of huESCs with 1.5 μg/ml G14V plasmid, 1.5 μg/ml pGFP control plasmid, and 60 nM specific PKN1 siRNA for 48 h, cyclin D1 protein (Western blot, A) and mRNA expression (real-time PCR, B) was detected. The data are presented as the mean ± SD of three independent experiments; an unpaired, two-tailed Student's t-test was used to assess significance: **P<0.01 versus control, ^#P<0.05 versus G14V + control siRNA, ^##P<0.001 versus G14V + control siRNA.

Fig 6. Immunohistochemical analysis of BrdU⁺IdU⁺-labeled cells.

After BrdU labeling of ESCs and IdU incorporation, immunofluorescence staining for BrdU and IdU was performed in mice treated with normal saline, U-46619, C3 transferase or U-46619+K-252a (A). 400× magnification. Double-positive cells are visible in the regenerated epidermis. Representative images of well-formed, BrdU and IdU-positive cells in the regenerated epidermis are shown at the same magnification (A). (B) Data analysis was performed using ImagePro Plus. Columns represent the mean double-positive cell count in the regenerated epidermis. The data are presented as the mean ± SD of three independent experiments; an unpaired, two-tailed Student's t-test were used to assess significance: *P<0.05 versus control group, **P<0.01 versus control group, ^##P<0.01 versus U-46619 group.