rhPGRN promotes cell proliferation by regulating autophagy and endoplasmic reticulum stress through MAPK/PI3K pathway |
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| Authors: | LUO Rui QIN Qi-zhong LIU Min YIN Dan-yang GUO Feng-jin |
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| Institution: | Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing 400016, China |
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| Abstract: | AIM To extract and purify recombinant human progranulin (rhPGRN) and to examine its effect on the proliferation, autophagy and endoplasmic reticulum stress (ERS) in human chondrocyte C28I2 and mouse macrophage RAW264.7. METHODS The histidine-tagged protein was specifically affinity-purified using Ni-NTA Sefinose resin, and the concentration and purity of the target protein were verified by Coomassie blue staining, BCA method and Western blot. The effects of rhPGRN on the proliferation, autophagy and ERS in C28I2 and RAW264.7 cells were detected by cell counting, Western blot and RT-qPCR. RESULTS The highly purified biologically active human recombinant protein rhPGRN was successfully extracted from the cell line with stable PGRN transfection. rhPGRN promoted the proliferation of the C28I2 cells and RAW264.7 cells, up-regulated the mRNA expression of cell cycle-related molecules (PCNA, cyclin B1 and cyclin D1) and the protein expression of Ki67, and increased the phosphorylation levels of proliferation-related signaling molecules ERK and Akt. Treatment with ERK pathway inhibitor U0126 inhibited rhPGRN-promoted proliferation, autophagy and ERS in the cells. The rhPGRN-induced autophagy of the cells was also inhibited by PI3K/Akt pathway inhibitor 3-methyladenine. The rhPGRN-promoted protein expression of Ki67 was down-regulated by autophagy inhibitor bafilomycin A1 and ERS inhibitor 4-phenylbutyric acid. CONCLUSION These results not only established a method for stable extraction of biologically active high-concentration high-purity recombinant protein rhPGRN, but also confirmed that the biological effect of rhPGRN on promoting cell proliferation was achieved through regulating autophagy and ERS via MAPK and PI3K/Akt pathway. |
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| Keywords: | Recombinant human progranulin Eukaryotic expression Cell proliferation Autophagy Endoplasmic reticulum stress |
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