IADR Abstract Archives
Elucidation of the Function of Bone Morphogenetic Protein-7 in the Oral Epithelium
Objectives: Bone-morphogenetic protein-7 (BMP-7) is a member of TGF-β superfamily with pleiotropic functions. Besides its main function on osteogenesis, its biological effects on various cell types have been reported. BMP-7 exerts its functions through binding to the cognate receptors BMPR type I and type II. Although the expression of these receptors in oral epithelial cells have been reported, the effects of BMP-7 have never been examined. In the present report, we attempted to verify the biological function of BMP-7 on oral epithelial cells.
Methods: Oral squamous cell carcinoma-derived cell lines, Ca9-22, HSC3 and KB, were maintained with 10% FCS-RPMI 1640 medium supplemeted with . The expression of BMPR type I and type II was examined by reverse-transcriptase polymerase chain reaction (RT-PCR). For detection of phosphorylation status of BMP-7, Ca9-22 was stimulated with various concentration of rhBMP-7 for 30, 60, 180 amd 360 min. After stimulation, the cell lysates were harvested and subjected to Western blotting. For the comprehensive analysis of gene expression profiles after BMP-7 stimulation, the cells were stimulated with 100 mg/ml of rhBMP-7 for 1 h. Total RNA was purified from the cells and subjected to microarray analysis. The detection of IL-17F mRNA was performed with its specific primers with real-time PCR. The 5’ untranslated region of IL-17F was cloned with PCR, sucloned to pGL4-basic vector and used for luciferase assay. The production of IL-17F protein was examined by ELISA.
Results: Both BMPRs type I and type II expression was detected in OSCCs. Stimulation of OSCCs with rhBMP-7 clearly induced the phosphorylation of Smad 1, 5, 8. The most prominent phosphorylation was observed after 1 h of rhBMP-7 stimulation at a concentration of 100 μg/ml. Micorarray analysis revealed that stimulation of OSCC with rhBMP-7 induced the expression of various genes. Among them, we focused on IL-17F. Real-time PCR confirmed the augmented expression of IL-17F in OSCC. The induced production of IL-17F was confirmed by ELISA in protein level.
Conclusions: In the present study, we confirmed that OSCC can respond to BMP-7 and produce IL-17F. As IL-17F plays important roles for the protection of mucosal surface from opportunistic infection, BMP-7 might contribute to the barrier function of oral mucosa.
Methods: Oral squamous cell carcinoma-derived cell lines, Ca9-22, HSC3 and KB, were maintained with 10% FCS-RPMI 1640 medium supplemeted with . The expression of BMPR type I and type II was examined by reverse-transcriptase polymerase chain reaction (RT-PCR). For detection of phosphorylation status of BMP-7, Ca9-22 was stimulated with various concentration of rhBMP-7 for 30, 60, 180 amd 360 min. After stimulation, the cell lysates were harvested and subjected to Western blotting. For the comprehensive analysis of gene expression profiles after BMP-7 stimulation, the cells were stimulated with 100 mg/ml of rhBMP-7 for 1 h. Total RNA was purified from the cells and subjected to microarray analysis. The detection of IL-17F mRNA was performed with its specific primers with real-time PCR. The 5’ untranslated region of IL-17F was cloned with PCR, sucloned to pGL4-basic vector and used for luciferase assay. The production of IL-17F protein was examined by ELISA.
Results: Both BMPRs type I and type II expression was detected in OSCCs. Stimulation of OSCCs with rhBMP-7 clearly induced the phosphorylation of Smad 1, 5, 8. The most prominent phosphorylation was observed after 1 h of rhBMP-7 stimulation at a concentration of 100 μg/ml. Micorarray analysis revealed that stimulation of OSCC with rhBMP-7 induced the expression of various genes. Among them, we focused on IL-17F. Real-time PCR confirmed the augmented expression of IL-17F in OSCC. The induced production of IL-17F was confirmed by ELISA in protein level.
Conclusions: In the present study, we confirmed that OSCC can respond to BMP-7 and produce IL-17F. As IL-17F plays important roles for the protection of mucosal surface from opportunistic infection, BMP-7 might contribute to the barrier function of oral mucosa.