The Localization And Function Of Matrix Metalloproteinase-26 Under Endoplasmic Reticulum Stress

Matrix metalloproteinases?Matrix metalloproteinases,MMPs?are zinc ion-dependent endopeptidases that cause the destruction of inflammatory tissues.As the markers of chronic inflammatory diseases,MMPs degrade the matrix membrane and collagen,promote the metastasis of cancer cells.However,unexpected side effects have been reported when MMPs inhibitors were applied in clinical trials of cancers,revealing that MMPs possess the novel biological functions in inflammatory diseases and cancer progression.MMP-26?Matrix metalloproteinase-26,MMP-26,or Matrilysin-2?,the smallest member of the MMP family.Compared with other members of MMPs family,it has a high degree of structural similarity but perform distinct function in cells.MMP-26has a unique species and tissue expression specificity,unique cellular endoplasmic reticulum localization,and unique self-activation mechanisms and so on,which all indicate that MMP-26 may play different roles from other MMPs family members.Endoplasmic reticulum?ER?is one of the most important organelles in eukaryotic cells.It is an important site for protein synthesis and post-translational modification,lipid synthesis,calcium storage and maintenance of calcium homeostasis.ER stress is initiated when the ER dysfunction occurs between function and demand.Many physiological or pathological processes are accompanied by ER stress,and ER stress itself can also lead to or promote the development of diseases.In the previous work of our laboratory,we discovered that MMP-26 is specifically located in the ER of the cells.This is consistent with many previous reports about MMP-26,which should be secreted extracellularly,is mainly expressed in the cytoplasm and almost undetectable outside the cells.On the basis of these study,we questioned whether MMP-26 is still located in the ER under ER stress?Can MMP-26 participate in the ER stress process?Would the localization and function of MMP-26 under ER stress participate in the physiological and pathological process?These questions are the problems that this paper attempts to explain.In this study,MMP-26-GFP transfected cells were treated with ER stress inducers.Results revealed that MMP-26-GFP translocated to the Golgi apparatus under A23187?Calcium ionophore?,TG(Ca²⁺-ATPase inhibitor)and DTT?Dithiothreitol?stimulation,and was further secreted extracellularly.And the translocation under stress is related to the structure of MMP-26,regardless of proteolysis activity.We applied affinity chromatography to find the proteins interacting with MMP-26-GFP.The results showed the GRP78 was the one of the proteins that interacted with MMP-26-GFP.And the interaction between GRP78 and MMP-26-GFP was further verified by GST pull down assay.To identify the key motif for the interaction of MMP-26 and GRP78,we constructed MMP-26-GFP,MMP-26catE209A-GFP?MMP-26 catalytic domain inactive mutant?and MMP-26?80-125-GFP?MMP-26 deleted the 80-125 domain?transfected cells,and the results showed that the fragment of 80-125 amino acid is the key motif of MMP-26 and GRP78 interaction.We further analyzed the pattern of interaction between MMP-26and GRP78 using computer simulation.The ER location of MMP-26 was changed during the unfolded protein respond?UPR?.Whether or not MMP-26 affects the UPR during ER stress is also a concern of us.Professor Sang's research showed that the protein levels of GRP78 in MDA-MB-231 cells was increased when MMP-26 was knocked down by means of siRNA transfection.We examined the protein levels of GRP78 in GFP,MMP-26-GFP,shRNA-control and shRNA-MMP-26 transfected cells and the results showed that the protein levels of MMP-26-GFP transfected cells was lower than that in GFP expression cells,and the protein levels of GRP78 in shRNA-MMP-26transfected cells was higher than that in shRNA-control transfected cells.GRP78 is an important regulator of ER stress and perform anti-apoptosis function.In order to analyze whether MMP-26 affect the sensitivity to ER stress by regulating GRP78protein levels,the apoptosis rates of GFP,MMP-26-GFP,shRNA-control and shRNA-MMP-26 transfected cells was examined by MTT,flow cytometry and Hoechst 33342 staining under ER stress.The results showed that the apoptosis rates of MMP-26-GFP transfected cells under ER stress was significantly higher than that of GFP transfected cells,and the apoptosis rates of shRNA-MMP-26 cells was significantly less than that of shRNA-MMP-26 transfected cells.Transient transfection of HEK293T and HeLa cells with the MMP-26-GFP expression plasmids showed a decrease in cell survival with the increased of plasmid concentration,compared with the GFP transfected plasmids.These results indicated that MMP-26 increases the sensitivity of cells to ER stress.Further,we examined the protein levels of GRP78 and the activation of IRE1?,PERK and ATF-6 which are sensors to ER stress.The results showed that GRP78,IRE1?,PERK and ATF-6 were up-regulated or activated under a long-time or short-time ER stress.MMP-26downregulated the GRP78 protein levels and increased the phosphorylation of IRE1?.Although PERK and ATF-6 were also activated,there were no difference between several transfected cells.The above results indicated that MMP-26increases the sensitivity of cells to ER stress.To further verify whether the proteolysis activation of MMP-26 is an important factor that affected its regulation of ER stress sensitivity,we constructed MMP-26catE209A-GFP and MMP-26-wtE209A-GFP inactive mutants stably transfected cells and examined the sensitivity to ER stress over prolonged or short time ER stress.The results showed that the inactive mutants transfected cells did not affect the sensitivity to ER stress.GRP78 protein levels were significantly higher in MMP-26wtE209A-GFP transfected cells than that in MMP-26-GFP transfected cells,indicating that the protein levels of GRP78 was affected by MMP-26 is dependent on its activity.The effects of ER stress on UPR levels under a prolonged or short period of time were examined and the results showed that phosphorylation of IRE1?in MMP-26-wtE209A-GFP transfected cells was significantly less than that of MMP-26-GFP,and the GFP transfected cells did not differ significantly either.PERK and ATF-6 were activated under ER stress,but the activation levels did not receive the effect of the activity of MMP-26.The above results indicate that the activity of MMP-26 is important in regulating the sensitivity to the ER stress.One of our concerns is whether the ER location and function that involved in the UPR of MMP-26 plays a role in the pathophysiological process.Hormone is also one of inducers of ER stress.Estrogen and progesterone are important hormones during menstrual cycle.We examined the regulation of endogenous MMP-26 protein levels by estrogen and progesterone and the results showed that both estrogen and progesterone induced the increase of intracellular protein levels of MMP-26.We further examined the localization of MMP-26-GFP under progesterone treatment and found that MMP-26-GFP was translocated to the Golgi apparatus under the action of progesterone and estrogen.It showed that MMP-26 was located in the ER at homeostasis,progesterone and estrogen can induce the secretion of MMP-26 to the extracellular matrix with the concentration of progesterone increase in the menstrual cycle.Taken together,MMP-26 located in the ER and secreted under ER stress.MMP-26 interacted with GRP78,decrease the protein levels of GRP78 and increase the phosphorylation of IRE1?,finally increase the sensitivity of cells to ER stress.And the protein levels of MMP-26 under physiological conditions,extracellular secretion under environmental stress,and regulating the sensitivity to ER stress may be crucial for its involvement in the physiological and pathological process of human.