RESUMO
Although the effects of low-intensity pulsed ultrasound (LIPUS) on diverse cell types have been fully studied, the functional role of LIPUS in keratinocytes remains poorly understood. This study aimed to investigate the effects of LIPUS on proliferation and migration of HaCaT cells as well as the regulatory mechanisms associated with signaling pathways. Human HaCaT cells were exposed or not to LIPUS, and cell proliferation and migration were measured by BrdU incorporation assay and Transwell assay, respectively. Expression of proteins associated with proliferation and migration was evaluated by western blot analysis. Expression of key kinases in the PI3K/AKT and JNK pathways was also evaluated by western blot analysis. Effects of LIPUS on the PI3K/AKT and JNK pathways, and whether LIPUS affected HaCaT cells via these two pathways were finally explored. When the parameter of LIPUS (number of cycles) was set at 300, cell viability was the highest after LIPUS stimulation. We then found that the percentage of BrdU positive cells was enhanced by LIPUS, along with up-regulation of cyclinD1, CDK6, CDK4, and VEGF. LIPUS promoted migration, as well as up-regulation of MMP-2 and MMP-9. Phosphorylation levels of key kinases in the PI3K/AKT and JNK pathways were increased by LIPUS. Inhibition of either PI3K/AKT pathway or JNK pathway attenuated effects of LIPUS on HaCaT cells, and co-inhibition of these two pathways showed augmented effects. LIPUS promoted proliferation and migration of HaCaT cells through activating the PI3K/AKT and JNK pathways.
Assuntos
Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Queratinócitos/efeitos da radiação , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Fosfatidilinositol 3-Quinases/efeitos da radiação , Proteínas Proto-Oncogênicas c-akt/efeitos da radiação , Ondas Ultrassônicas , Análise de Variância , Western Blotting , Bromodesoxiuridina , Linhagem Celular Transformada , Sobrevivência Celular/efeitos da radiação , Humanos , Queratinócitos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos da radiação , Regulação para CimaRESUMO
Although the effects of low-intensity pulsed ultrasound (LIPUS) on diverse cell types have been fully studied, the functional role of LIPUS in keratinocytes remains poorly understood. This study aimed to investigate the effects of LIPUS on proliferation and migration of HaCaT cells as well as the regulatory mechanisms associated with signaling pathways. Human HaCaT cells were exposed or not to LIPUS, and cell proliferation and migration were measured by BrdU incorporation assay and Transwell assay, respectively. Expression of proteins associated with proliferation and migration was evaluated by western blot analysis. Expression of key kinases in the PI3K/AKT and JNK pathways was also evaluated by western blot analysis. Effects of LIPUS on the PI3K/AKT and JNK pathways, and whether LIPUS affected HaCaT cells via these two pathways were finally explored. When the parameter of LIPUS (number of cycles) was set at 300, cell viability was the highest after LIPUS stimulation. We then found that the percentage of BrdU positive cells was enhanced by LIPUS, along with up-regulation of cyclinD1, CDK6, CDK4, and VEGF. LIPUS promoted migration, as well as up-regulation of MMP-2 and MMP-9. Phosphorylation levels of key kinases in the PI3K/AKT and JNK pathways were increased by LIPUS. Inhibition of either PI3K/AKT pathway or JNK pathway attenuated effects of LIPUS on HaCaT cells, and co-inhibition of these two pathways showed augmented effects. LIPUS promoted proliferation and migration of HaCaT cells through activating the PI3K/AKT and JNK pathways.
Assuntos
Queratinócitos/efeitos da radiação , Movimento Celular/efeitos da radiação , Fosfatidilinositol 3-Quinases/efeitos da radiação , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Proliferação de Células/efeitos da radiação , Ondas Ultrassônicas , Bromodesoxiuridina , Linhagem Celular Transformada , Transdução de Sinais/efeitos da radiação , Queratinócitos/metabolismo , Regulação para Cima , Sobrevivência Celular/efeitos da radiação , Western Blotting , Reprodutibilidade dos Testes , Análise de Variância , Fosfatidilinositol 3-Quinases/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismoRESUMO
Skin is the largest organ in our body and strategically placed to provide a metabolically active biological barrier against a range of noxious stressors. A lot of inflammatory cytokines, which are increased after ultraviolet (UV) irradiation produced by keratinocytes or other immunocytes, are closely related to pigmentary changes, including interleukin-18 (IL-18) and interferon-γ (IFN-γ). In this study, the effect of cross-talk between IL-18 and IFN-γ on melanogenesis was investigated. Treatment with IL-18 resulted in a dose-dependent increase of melanogenesis, while IFN-γ made an opposite effect. This influence of IL-18 and IFN-γ was mediated by regulations of microphthalmia-associated transcription factor (MITF) and its downstream enzymatic cascade expressions. Furthermore, IFN-γ inhibited basal and IL-18-induced melanogenesis. IFN-γ increased signal transducer and activator of transcription-1 (STAT-1) phosphorylation to play its position in regulating melanin pigmentation, and its inhibitory effect could be prevented by Janus Kinase 1 (JAK 1) inhibitor. IFN-γ could inhibit melanogenesis by decreasing melanocyte dendrite formation. In addition, IFN-γ inhibited the expressions of Rab Pases to suppress the mature and transport of melanosomes. IL-18 could rapidly induce Akt and PTEN phosphorylation and p65 expression in B16F10 cells. When treatment with IL-18 and IFN-γ together, the phosphorylation level of Protein Kinase B (Akt) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) and expression of p65 NF-κB were inhibited, compared with treated with IL-18 only. Our studies indicated that IFN-γ could directly induce B16F10 cells apoptosis in vitro. Furthermore, we demonstrated that IFN-γ markedly up-regulated IL-18 binding protein (BP) production in normal human foreskin-derived epidermal keratinocytes in dose-dependent manner. UVB irradiation induced protease-activated receptor-2 (PAR-2) expression in NHEK, IFN-γ could inhibit this enhancement in a dose-dependent manner. These data suggest that IFN-γ plays a role in regulating inflammation- or UV-induced pigmentary changes, in direct/indirect manner.