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A7125 - Proteasomal Degradation of Histone Protein Arginine Methyltransferases (PRMT6) Mediated by F-Box Protein FBXW17 Promote Cigarette Smoke Extract Induced Epithelial Cell Death
Author Block: X. He, T. Li, L. Chen, H. Zeng, Y. Chen; The Second Xiangya Hospital, Central South University, Changsha, China.
RATIONALE: Protein arginine N-methyltransferase 6 (PRMT6) is an essential nuclear enzyme which is involved in cell cycle regulation and apoptosis. In addition, PRMT6 may play a protective role in the pathogenesis of chronic obstructive pulmonary disease (COPD). But, very little has been known about the molecular regulation of PRMT6 expression. Ubiquitin-proteasome system (UPS) is abnormally activated in COPD, and is the major mechanism for protein degradation. This study is to investigate whether FBXW17, an ‘orphan’ member of the Skp1-Cullin-F-box family of E3 ubiquitin ligases, could mediate PRMT6 ubiquitin-proteasomal degradation and its role in epithelial cell death. METHODS: Plasmid delivering system mediated nucleofection was used to express PRMT6 or FBXW in Murine Lung Epithelial type II (MLE-12) cells, and siRNA was used to silence PRMT6. The expression of PRMT6 protein and mRNA was analyzed by Western blotting and quantitative real-time PCR (qRT-PCR). Apoptosis was tested using Annexin V (Av) and PropidiumIodide (PI) staining. Immuno-fluorescence assay was utilized to study PRMT6 nuclear relocation. Co-immunoprecipitation(co-IP) and confocal laser scanning were applied to show that the combination of FBXW17 and PRMT6. Cell proliferation was fully measured with MTT. RESULTS: The expression of PRMT6 protein was decreased in CSE treated or starvation condition, and its half-life was about 5 hours. Cell proliferation arrest and apoptosis was followed after CSE treatment. The knockdown of PRMT6 by siRNA dramatically induced MLE-12 cell death, as well as CSE treatment. Meanwhile, overexpression of PRMT6 in CSE treated cells significantly alleviated cell proliferation arrest and apoptosis. The expression of PRMT6 protein under the condition of proteasome inhibitor (MG132) treatment was gradually accumulated over time, while there was no significant change of PRMT6 protein in lysosome inhibitor(Leupeptin) group, which implying that PRMT6 was degraded in a proteasome manner. After scanning several F-box protein, we found FBXW17 overexpression significantly decreased PRMT6 protein expression in MLE cells, while not mRNA. Notably, in FBXW17 overexpressed cells, PRMT6 were significantly reduced in a dose-dependent and time-dependent manner, but not in randomly selected FBXW14 overexpressed cells, whether or not there is MG132 intervention. Co-IP and confocal laser scanning showed that FBXW17 and PRMT6 combined with each other and colocalized in nucleus. That is to say, PRMT6 protein was selectively degraded in a FBXW17 mediated proteasome manner and mainly located in nucleus. CONCLUSION: FBXW17 which specifically mediates proteasome degradation of PRMT6 could promote cell impairment such as proliferation arrest and apoptosis in response to CSE.