ABSTRACT
OBJECTIVE: It has been reported that tumor-associated lymphangiogenesis plays an important role in lymph node metastasis and contributes to the poor survival of lung adenocarcinoma (LUAD) patients. As yet, however, the molecular mechanism underlying LUAD-associated lymphangiogenesis has remained elusive. METHODS: Immunohistochemistry (IHC) was used to determine the expression of integrin subunit alpha 6 (ITGA6) and the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) in clinicopathologically characterized LUAD specimens. The effect of ITGA6 overexpression on lymphangiogenesis and lymphatic metastasis was examined by tube formation, scratch wound-healing, and cell migration assays in vitro and a popliteal lymph node metastasis model in vivo. Mechanistically, overexpression of ITGA6 and activation of NF-κB signaling were examined by real-time PCR, ubiquitination and dual-luciferase reporter assays. Finally, high ITGA6 expression in LUAD tissue samples was related to copy number variation (CNV) using the TCGA database. RESULTS: We found that ITGA6 overexpression correlated with microlymphatic vessel density in LUAD specimens (p < 0.01). Importantly, by using a popliteal lymph node metastasis model, we found that ITGA6 upregulation significantly enhanced lymphangiogenesis and lymphatic metastasis in vivo (p < 0.05). In addition, we found that ITGA6 overexpression enhanced the capability of A549 and H1299 LUAD cells to induce tube formation and migration in human lymphatic endothelial cells (HLECs). Mechanistically, we found that ITGA6 sustained NF-κB activity via binding and promoting K63 polyubiquitination of TNF receptor-associated factor 2 (TRAF2). Finally, CNV analysis revealed ITGA6 amplification of 27.5% in the LUAD tissue samples in the TCGA database. CONCLUSIONS: Taken together, our results uncover a plausible role for ITGA6 in mediating lymphangiogenesis and lymphatic metastasis and may provide a basis for targeting ITGA6 to treat LUAD lymphatic metastasis.
Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/metabolism , Cell Line, Tumor , DNA Copy Number Variations , Endothelial Cells/metabolism , Humans , Integrin alpha6/metabolism , Integrin alpha6/pharmacology , Lung Neoplasms/pathology , Lymphangiogenesis , Lymphatic Metastasis , NF-kappa B/metabolism , Signal Transduction , Vascular Endothelial Growth Factor C/genetics , Vascular Endothelial Growth Factor C/metabolism , Vascular Endothelial Growth Factor C/pharmacologyABSTRACT
OBJECTIVES: Although integrins have been shown to be associated with proliferation and differentiation in some stem cells, the regulatory effect of integrin α6 (ITGα6) on the human dental pulp stem cells (hDPSCs) has not been reported. Here, we detected the roles of ITGα6 in hDPSCs. MATERIALS AND METHODS: Attached to Cytodex 3 microcarriers, hDPSCs grown under stimulated microgravity (SMG) or conventional culture conditions were measured the proliferation and different gene expression. Further, ITGα6 was silenced in hDPSCs, and its effect on proliferation, differentiation, and cytoskeletal organization was analyzed. RESULTS: SMG conditions increased the number of Ki67-positive hDPSCs and progression into S phase of cell cycle. WB analysis showed the expression of ITGα6 was upregulated in hDPSCs under SMG conditions. Knockdown of ITGα6 decreased the expression of stemness markers, CD105 and STRO-1 in hDPSCs, but promoted the osteogenic and odontogenic differentiation by increased ALP expression and Alizarin Red nodules. Moreover, RNA-seq demonstrated that RHO/ROCK signaling pathway upregulated silencing ITGα6-hDPSCs. Treatment with Y-27632 inhibited the effect of ITGα6 depletion on hDPSCs stemness, rearranged the cytoskeleton, promoted the pluripotency, proliferation ability, and inhibited the differentiation. CONCLUSION: ITGα6 promotes hDPSCs stemness via inhibiting RHO/ROCK and restoring cytoskeleton.
Subject(s)
Dental Pulp , Stem Cells , Cell Differentiation/genetics , Cell Proliferation/genetics , Cells, Cultured , Humans , Integrin alpha6/genetics , Integrin alpha6/metabolism , Integrin alpha6/pharmacologyABSTRACT
BACKGROUND: Idiopathic pulmonary fibrosis is a devastating disorder for which there is no effective treatment. Transforming growth factor (TGF)-beta plays a critical role in provoking fibrosis. Interleukin (IL)-10 is a potent immunosuppressive cytokine but its effect on the fibrosing process is unclear. A study was undertaken to examine whether IL-10 affects the production and activation of TGF-beta and thus can attenuate the fibrosis. METHODS: Mice were given an intratracheal injection of bleomycin. On day 1 or 14, IL-10 gene was delivered by rapid intravenous injection of Ringer's solution containing plasmid. Two weeks after the plasmid injection the mice were examined for fibrosis. The effect of IL-10 on TGF-beta production by alveolar macrophages was assessed. RESULTS: Even when delivered during the fibrosing phase, IL-10 gene significantly suppressed the pathological findings, hydroxyproline content, and production of both active and total forms of TGF-beta1 in the lung. Immunohistochemical analyses showed that alveolar macrophages were one of the major sources of TGF-beta1 and IL-10 diminished the intensity of the staining. IL-10 also suppressed the expression of alphaV beta6 integrin, a molecule that plays an important role in TGF-beta activation, on lung epithelial cells. Alveolar macrophages from bleomycin injected mice produced TGF-beta1 spontaneously ex vivo, which was significantly suppressed by treatment of the mice in vivo or by treatment of the explanted macrophages ex vivo with IL-10. CONCLUSION: IL-10 suppresses the production and activation of TGF-beta in the lung and thus attenuates pulmonary fibrosis, even when delivered in the chronic phase.
