섬유주세포에서 혈관내피성장인자가 YAP/TAZ 신호전달에 미치는 영향

Effects of Vascular Endothelial Growth Factor on YAP/TAZ Signaling in Trabecular Meshwork Cells

목적: 본 연구에서는 인간섬유주세포에서 VEGF가 Hippo 신호전달 관련 전사인자 yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) 신호전달에 영향을 미치는지 규명하고, 유도된 YAP/TAZ의 역할을 알아보고자 한다. 대상과 방법: 다양한 VEGF 농도를 인간섬유주세포에 처리하여 CCK-8 용액으로 세포독성을 확인하였다. TGFβ2 (5 ng/mL)와 VEGF (30 ng/mL) 처리 후 western blot과 RT-qPCR, 면역세포화학염색으로 YAP/TAZ 발현을 확인하였다. Fibronectin, collagen1, myocilin 발현은 western blot으로 확인하였다. Alexa Fluor 488-phalloidin으로 세포를 염색하여 액틴필라멘트를 관찰하였다. 결과: YAP과 TAZ의 발현은 TGFβ2와 VEGF의 24시간 처리 후 증가하였다. 모든 실험군에서 fibronectin과 collagen1이 증가한 반면myocilin은 TGFβ2와 TGFβ2+VEGF 처리군에서 증가하였다. 액틴필라멘트 염색으로 섬유주세포의 cross-linking을 확인하였다. 결론: VEGF가 YAP/TAZ 신호를 유도하여 섬유주세포의 섬유화를 증가시켰다. VEGF에 의한 기능적 변화가 확인된다면 섬유주세포의방수유출저항 기전으로 VEGF와 YAP/TAZ를 제시할 수 있을 것이다.

Purpose: To investigate the effects of vascular endothelial growth factor (VEGF) on yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding motif (TAZ), a Hippo pathway-related transcription factor, and the role of YAP/TAZ induced by trabecular meshwork stimulation. Methods: Human trabecular meshwork cells were cultured and treated with various VEGF concentrations to verify cell cytotoxicity using the CCK-8 solution. Transforming growth factor β-2 (TGFβ2; 5 ng/mL) and VEGF (30 ng/mL) were applied and YAP/TAZ expression was assessed by western blotting, reverse transcription quantitative polymerase chain reaction and immunocytochemistry. Fibronectin, collagen 1, and myocilin expression were also assessed by western blotting. The cells were stained using Alexa Fluor 488-phalloidin to observe F-actin changes. Results: YAP and TAZ expression increased following TGFβ2 and VEGF treatment for 24 hours. Fibronectin and collagen 1 increased significantly in all three treatment groups, while myocilin increased in the TGFβ2 and TGFβ2+VEGF groups. The F-actin staining showed increased cross-linking in the trabecular meshwork cells. Conclusions: VEGF induced YAP/TAZ signaling and increased trabecular meshwork cell fibrosis. Based on the functional changes caused by VEGF, it is suggested that VEGF and YAP/TAZ may increase aqueous humor outflow resistance in trabecular meshwork cells.