Shigeyuki Kanazawa

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.

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.

Physiological ER Stress Mediates the Differentiation of Fibroblasts.

Recently, accumulating reports have suggested the importance of endoplasmic reticulum (ER) stress signaling in the differentiation of several tissues and cells, including myoblasts and osteoblasts. Secretory cells are easily subjected to ER stress during maturation of their secreted proteins. Skin fibroblasts produce and release several proteins, such as collagens, matrix metalloproteinases (MMPs), the tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAGs), and the production of these proteins is increased at wound sites. Differentiation of fibroblasts into myofibroblasts is one of the key factors for wound healing and that TGF-β can induce fibroblast differentiation into myofibroblasts, which express α-smooth muscle actin. Well-differentiated myofibroblasts show increased production of collagen and TGF-β, and bring about wound healing. In this study, we examined the effects of ER stress signaling on the differentiation of fibroblasts, which is required for wound healing, using constitutively ER stress-activated primary cultured fibroblasts. The cells expressed positive α-smooth muscle actin signals without TGF-β stimulation compared with control fibroblasts. Gel-contraction assays suggested that ER stress-treated primary fibroblasts caused stronger shrinkage of collagen gels than control cells. These results suggest that ER stress signaling could accelerate the differentiation of fibroblasts to myofibroblasts at injured sites. The present findings may provide important insights for developing therapies to improve wound healing.

Direct contact of fibroblasts with neuronal processes promotes differentiation to myofibroblasts and induces contraction of collagen matrix in vitro

Wound healing is often delayed in the patients whose sensory and autonomic innervation is impaired. We hypothesized that existence of neurites in the skin may promote wound healing by inducing differentiation of fibroblasts into myofibroblasts with consequent wound contraction. In the current study, we examined the effect of neurons on differentiation of fibroblasts and contraction of collagen matrix in vitro using a new co‐culture model. Neuronal cell line, PC12 cells, of which the neurite outgrowth can be controlled by adding nerve growth factor, was used. Rat dermal fibroblasts were co‐cultured with PC12 cells extending neurites or with PC12 cells lacking neurites. Then, differentiation of fibroblasts into myofibroblasts and contraction of the collagen matrix was evaluated. Finally, we examined whether direct or indirect contact with neurites of PC12 cells promoted the differentiation of fibroblasts. Our results showed that fibroblasts co‐cultured with PC12 extending neurites differentiated into myofibroblasts more effectively and contracted the collagen matrix stronger than those with PC12 lacking neurites. Direct contact of fibroblasts with neurites promoted more differentiation than indirect contact. In conclusion, direct contact of fibroblasts with neuronal processes is important for differentiation into myofibroblasts and induction of collagen gel contraction, leading to promotion of wound healing.

Molecular mechanism of kallikrein-related peptidase 8/neuropsin-induced hyperkeratosis in inflamed skin.

Background  Hyperkeratosis and acanthosis occur in inflamed skin. Proliferation and differentiation of keratinocytes are important processes during epidermal repair after inflammation. Neuropsin and its human homologue kallikrein‐related peptidase 8 (KLK8) have been reported to be involved in epidermal proliferation and differentiation, but the involved molecular mechanisms are obscure.

Hypertrophic scar contracture is mediated by the TRPC3 mechanical force transducer via NFkB activation

Wound healing process is a complex and highly orchestrated process that ultimately results in the formation of scar tissue. Hypertrophic scar contracture is considered to be a pathologic and exaggerated wound healing response that is known to be triggered by repetitive mechanical forces. We now show that Transient Receptor Potential (TRP) C3 regulates the expression of fibronectin, a key regulatory molecule involved in the wound healing process, in response to mechanical strain via the NFkB pathway. TRPC3 is highly expressed in human hypertrophic scar tissue and mechanical stimuli are known to upregulate TRPC3 expression in human skin fibroblasts in vitro. TRPC3 overexpressing fibroblasts subjected to repetitive stretching forces showed robust expression levels of fibronectin. Furthermore, mechanical stretching of TRPC3 overexpressing fibroblasts induced the activation of nuclear factor-kappa B (NFκB), a regulator fibronectin expression, which was able to be attenuated by pharmacologic blockade of either TRPC3 or NFκB. Finally, transplantation of TRPC3 overexpressing fibroblasts into mice promoted wound contraction and increased fibronectin levels in vivo. These observations demonstrate that mechanical stretching drives fibronectin expression via the TRPC3-NFkB axis, leading to intractable wound contracture. This model explains how mechanical strain on cutaneous wounds might contribute to pathologic scarring.

Objective assessment of facial skin aging and the associated environmental factors in Japanese monozygotic twins

Twin studies, especially those involving monozygotic (MZ) twins, facilitate the analysis of factors affecting skin aging while controlling for age, gender, and genetic susceptibility. The purpose of this study was to objectively assess various features of facial skin and analyze the effects of environmental factors on these features in MZ twins. At the Osaka Twin Research Center, 67 pairs of MZ twins underwent medical interviews and photographic assessments, using the VISIA® Complexion Analysis System. First, the average scores of the right and left cheek skin spots, wrinkles, pores, texture, and erythema were calculated; the differences between the scores were then compared in each pair of twins. Next, using the results of medical interviews and VISIA data, we investigated the effects of environmental factors on skin aging. The data were analyzed using Pearsons correlation coefficient test and the Wilcoxon signed‐rank test. The intrapair differences in facial texture scores significantly increased as the age of the twins increased (P = 0.03). Among the twin pairs who provided answers to the questions regarding history differences in medical interviews, the twins who smoked or did not use skin protection showed significantly higher facial texture or wrinkle scores compared with the twins not exposed to cigarettes or protectants (P = 0.04 and 0.03, respectively). The study demonstrated that skin aging among Japanese MZ twins, especially in terms of facial texture, was significantly influenced by environmental factors. In addition, smoking and skin protectant use were important environmental factors influencing skin aging.

Anti-inflammatory effect of electroacupuncture in the C3H/HeJ mouse model of alopecia areata

Accumulating experimental evidence has indicated that electroacupuncture (EA) stimulation may enhance immune function in several animal models of inflammatory diseases.1 ,2 However, there are few clinical data on EA stimulation for autoimmune diseases and the mechanisms underlying the therapeutic effect of EA stimulation for autoimmune diseases remain unclear. Mast cells are the central players in allergic inflammation, and it has recently been reported that mast cells are involved in autoimmune diseases and chronic inflammation.3 ,4 Significant increases in mast cell degranulation were observed in these autoimmune diseases. Furthermore, severe mast cell degranulation and the accumulation of inflammatory cells around the anagen (growth phase) hair follicles were observed in autoimmune diseases such as the mouse model for alopecia areata (AA).5 This self-attack of the hair follicle cells by inflammatory cells changes the hair matrix cell phase to the telogen phase and results in hair loss.6 A …

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

Background 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. Materials and Methods 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. Results 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. Conclusions 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.

Reconstruction of through-and-through oromandibular defects by the double-skin paddle fibula osteocutaneous flap: Can the skin paddle always be divided?

Abstract Reconstruction of the through-and-through defects of the oral cavity, involving oral mucosa, bone, and external skin is a major challenge. A single fibula osteocutaneous flap providing two skin islands is an option for such composite reconstruction. The number, location, and size of skin perforators were studied in the distal two thirds of the lower legs in 22 cases of fibula osteocutaneous flap mandibular reconstruction, and whether the skin paddle of the fibula flap could always be divided completely based on two distal perforators was examined. In this study, only 50% of the flaps had two or more distal perforators; thus, it was concluded that the skin paddle of the fibula osteocutaneous flap could not always be divided based on two distal skin perforators.