bFGF Regulates PI3-Kinase-Rac1-JNK Pathway and Promotes Fibroblast Migration in Wound Healing

Fibroblast proliferation and migration play important roles in wound healing. bFGF is known to promote both fibroblast proliferation and migration during the process of wound healing. However, the signal transduction of bFGF-induced fibroblast migration is still unclear, because bFGF can affect both proliferation and migration. Herein, we investigated the effect of bFGF on fibroblast migration regardless of its effect on fibroblast proliferation. We noticed involvement of the small GTPases of the Rho family, PI3-kinase, and JNK. bFGF activated RhoA, Rac1, PI3-kinase, and JNK in cultured fibroblasts. Inhibition of RhoA did not block bFGF-induced fibroblast migration, whereas inhibition of Rac1, PI3-kinase, or JNK blocked the fibroblast migration significantly. PI3-kinase-inhibited cells down-regulated the activities of Rac1 and JNK, and Rac1-inhibited cells down-regulated JNK activity, suggesting that PI3-kinase is upstream of Rac1 and that JNK is downstream of Rac1. Thus, we concluded that PI3-kinase, Rac1, and JNK were essential for bFGF-induced fibroblast migration, which is a novel pathway of bFGF-induced cell migration

A Low-Level Carbon Dioxide Laser Promotes Fibroblast Proliferation and Migration through Activation of Akt, ERK, and JNK.

Low-level laser therapy (LLLT) with various types of lasers promotes fibroblast proliferation and migration during the process of wound healing. Although LLLT with a carbon dioxide (CO2) laser was also reported to promote wound healing, the underlying mechanisms at the cellular level have not been previously described. Herein, we investigated the effect of LLLT with a CO2 laser on fibroblast proliferation and migration.Cultured human dermal fibroblasts were prepared. MTS and cell migration assays were performed with fibroblasts after LLLT with a CO2 laser at various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) to observe the effects of LLLT with a CO2 laser on the proliferation and migration of fibroblasts. The non-irradiated group served as the control. Moreover, western blot analysis was performed using fibroblasts after LLLT with a CO2 laser to analyze changes in the activities of Akt, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK), which are signaling molecules associated with cell proliferation and migration. Finally, the MTS assay, a cell migration assay, and western blot analysis were performed using fibroblasts treated with inhibitors of Akt, ERK, or JNK before LLLT with a CO2 laser.In MTS and cell migration assays, fibroblast proliferation and migration were promoted after LLLT with a CO2 laser at 1.0 J/cm2. Western blot analysis revealed that Akt, ERK, and JNK activities were promoted in fibroblasts after LLLT with a CO2 laser at 1.0 J/cm2. Moreover, inhibition of Akt, ERK, or JNK significantly blocked fibroblast proliferation and migration.These findings suggested that LLLT with a CO2 laser would accelerate wound healing by promoting the proliferation and migration of fibroblasts. Activation of Akt, ERK, and JNK was essential for CO2 laser-induced proliferation and migration of fibroblasts

L-arginine stimulates fibroblast proliferation through the GPRC6A-ERK1/2 and PI3K/Akt pathway.

L-arginine is considered a conditionally essential amino acid and has been shown to enhance wound healing. However, the molecular mechanisms through which arginine stimulates cutaneous wound repair remain unknown. Here, we evaluated the effects of arginine supplementation on fibroblast proliferation, which is a key process required for new tissue formation. We also sought to elucidate the signaling pathways involved in mediating the effects of arginine on fibroblasts by evaluation of extracellular signal-related kinase (ERK) 1/2 activation, which is important for cell growth, survival, and differentiation. Our data demonstrated that addition of 6 mM arginine significantly enhanced fibroblast proliferation, while arginine deprivation increased apoptosis, as observed by enhanced DNA fragmentation. In vitro kinase assays demonstrated that arginine supplementation activated ERK1/2, Akt, PKA and its downstream target, cAMP response element binding protein (CREB). Moreover, knockdown of GPRC6A using siRNA blocked fibroblast proliferation and decreased phosphorylation of ERK1/2, Akt and CREB. The present experiments demonstrated a critical role for the GPRC6A-ERK1/2 and PI3K/Akt signaling pathway in arginine-mediated fibroblast survival. Our findings provide novel mechanistic insights into the positive effects of arginine on wound healing

Eyebrow Reconstruction Using a Composite Skin Graft from Sideburns

Summary: Wide resection of malignant skin tumors in the upper orbital region often results in soft-tissue defects involving the eyebrow. We used composite skin grafts from the area around the sideburns for 1-stage reconstruction of skin and eyebrow defects. The results were aesthetically satisfying because the hair and shape of these regions were similar to those of the original eyebrow, and donor-site closure was easy with inconspicuous scar. The survival of full-thickness skin graft area of composite grafts from sideburn facilitates revascularization of thicker hair follicles in the graft and allows safe, natural eyebrow reconstruction

Effect of inhibition of Akt, ERK, or JNK on LLLT-induced activation of signaling molecules in HDFs.

<p>(A) Akt activity in HDFs treated with or without the inhibitor of Akt was analyzed by immunoblotting at 15 min after LLLT (1.0 J/cm²). Densitometric measurement of p-Akt was normalized to the amount of total Akt. (B) ERK activity in HDFs treated with or without the inhibitor of ERK was analyzed by immunoblotting at 15 min after LLLT (1.0 J/cm²). Densitometric measurement of p-ERK was normalized to the amount of total ERK. (C) JNK activity in HDFs treated with or without the inhibitor of JNK was analyzed by immunoblotting at 15 min after LLLT (1.0 J/cm²). Densitometric measurement of p-JNK was normalized to the amount of total JNK. The results are presented as fold change compared with the non-inhibited group. Results are expressed as the mean ± SD of three independent experiments. ***p<0.001, ****p<0.0001 (Tukey-Kramer test).</p

Effects of LLLT with a CO₂ laser on HDF migration.

<p>A cell migration assay of HDFs treated with LLLT under several irradiation powers (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm²). The non-irradiated group served as the control. (A) Migration rates (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm²) are expressed as migration distance/time (μm/h). The non-irradiated group served as the control. Results are expressed as the mean ± SD of three independent experiments. *p<0.05, ***p<0.001 compared with the non-irradiated group (Tukey-Kramer test). (B) Images show the wounded cell monolayers at 0, 12, and 24 h after wounding with non-irradiated or irradiated (1.0 J/cm²) HDFs. The line indicates the wound edge at the start of the experiment (0 h). Bar = 300 nm. The migration of irradiated HDFs was promoted compared with the control.</p

The CO₂ laser machine equipped with a homogenizer.

<p>(A) The CO₂ laser system was equipped with a homogenizer (arrow) attached to the end of the articulated arm in order to equalize the profile of the laser beam. Power densities were generated in a round homogeneous spot with a diameter of 35 mm. (B) The homogenizer was composed of (1) the beam shaper, (2) the aspherical lens (a diameter of 25.4 mm) and (3) the plano-convex lens (a diameter of 50.8 mm). The distance from the beam shaper to the aspherical lens was 60 mm, the aspherical lens to the plano-convex lens was 125 mm, and the plano-convex lens to the samples was 70 mm.</p

Effect of inhibition of Akt, ERK, or JNK on LLLT-induced HDF proliferation.

<p>(A) HDF proliferation was measured by the MTS assay after pretreatment with the inhibitor of Akt and subsequent LLLT irradiation (1.0 J/cm²). (B) HDF proliferation assay after pretreatment with the inhibitor of ERK and subsequent LLLT irradiation (1.0 J/cm²). (C) HDF proliferation assay after pretreatment with the inhibitor of JNK and subsequent LLLT irradiation (1.0 J/cm²). Results are expressed as the mean ± SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Tukey-Kramer test).</p