Previously we demonstrated that Ahnak mediates transforming growth factor-β (TGFβ)-induced epithelial-mesenchymal transition (EMT) during tumor metastasis. It is well-known that circulating tumor cells (CTCs) invade the vasculature of adjacent target tissues before working to adapt to the host environments. Currently, the molecular mechanism by which infiltrated tumor cells interact with host cells to survive within target tissue environments is far from clear. Here, we show that Ahnak regulates tumor metastasis through PCSK9 expression. To validate the molecular function of Ahnak in metastasis, B16F10 melanoma cells were injected into WT and Ahnak knockout (Ahnak−/−) mice. Ahnak−/− mice were more resistant to the pulmonary metastasis of B16F10 cells compared to wild-type (WT) mice. To investigate the host function of Ahnak in recipient organs against metastasis of melanoma cells, transcriptomic analyses of primary pulmonary endothelial cells from WT or Ahnak−/− mice in the absence or presence of TGFβ stimulation were performed. We found PCSK9, along with several other candidate genes, was involved in the invasion of melanoma cells into lung tissues. PCSK9 expression in the pulmonary artery was higher in WT mice than Ahnak−/− mice. To evaluate the host function of PCSK9 in lung tissues during the metastasis of melanoma cells, we established lung epithelial cell-specific tamoxifen-induced PCSK9 conditional KO mice (Scgb1a1-Cre/PCSK9fl/fl). The pulmonary metastasis of B16F10 cells in Scgb1a1-Cre/PCSK9fl/fl mice was significantly suppressed, indicating that PCSK9 plays an important role in the metastasis of melanoma cells. Taken together, our data demonstrate that Ahnak regulates metastatic colonization through the regulation of PCSK9 expression.
Abstract We hypothesized that NADPH oxidase 4 (Nox4) is involved in the formation of neointimal atherosclerotic plaques through the migration of smooth muscle cells (SMCs) in response to flagellin. Here, we demonstrate that TLR5-mediated Nox4 activation regulates the migration of SMCs, leading to neointimal plaque formation in atherosclerosis. To investigate the molecular mechanism by which the TLR5-Nox4 cascade mediates SMC migration, we analyzed the signaling cascade in primary vascular SMCs (VSMCs) from wild-type (WT) or Nox4 KO mice. Stimulation of VSMCs from Nox4 KO mice with flagellin failed to induce H 2 O 2 production and Rac activation compared with stimulation of VSMCs from WT mice. Moreover, the migration of Nox4-deficient VSMCs was attenuated in response to flagellin in transwell migration and wound healing assays. Finally, we performed partial carotid artery ligation in ApoE KO and Nox4ApoE DKO mice fed a high-fat diet (HFD) with or without recombinant FliC (rFliC) injection. Injection of rFliC into ApoE KO mice fed a HFD resulted in significantly increased SMC migration into the intimal layer, whereas SMC accumulation was not detected in Nox4ApoE DKO mice. We conclude that activation of the TLR5-Nox4 cascade plays an important role in the formation of neointimal atherosclerotic plaques.
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