Programmed Cell Death 4 Modulates Lysosomal Function And Macrophage Anti-tumor Effect By Inhibiting TFEB Translation In An EIF4A-dependent Manner

ObjectiveTranscription factor EB(TFEB)is a key transcription factor regulating lysosomal biogenesis and lysosomal function,which can participate in the regulation of about 90%of lysosomal related genes and plays an important role in the overall coordination of the regulation of lysosome.TFEB related research has become a hotspot in the past decade,and targeted TFEB treatment of lysosomal storage disorders,lysosomal dysfunction related diseases and neurodegenerative diseases has been widely proven.However,the study of TFEB in tumors,tumor associated immune cells,and tumor immunotherapy is unclear.At present,studies on the regulation mechanism of TFEB mainly focus on its phosphorylation,dephosphorylation and subcellular localization,while studies on the transcription and translation of TFEB itself have not been reported.Programmed cell death 4(PDCD4)is a tumor suppressor gene and protein translation inhibitor that has been studied for a long time in our team.In most tumor cells,PDCD4 expression is down-regulated or absent.Replenishing or overexpressing PDCD4 can inhibit the proliferation,migration and invasion of tumor cells.In terms of inhibiting protein translation,PDCD4 can inhibit the initiation and extension of translation by eIF4A-dependent and eIF4A-independent methods,respectively,and finally inhibit protein expression.We have reported that PDCD4 knockout inhibited macrophage frothing and promoted lipid metabolism.PDCD4 inhibits the expression of autophagy-related gene ATG5.In mechanism,PDCD4 inhibits the translation of ATG5 in an eIF4A-dependent manner,and PDCD4 knockout can promote the expression of ATG5 and improve the level of autophagy.However,whether PDCD4 is involved in the regulation of lysosomal function remains to be explored experimentally.This study aims to determine whether PDCD4 is involved in the regulation of lysosomal biogenesis and lysosomal function through in vitro and in vivo experiments,and further explore the relevant regulatory mechanisms.To investigate whether PDCD4 regulates TFEB transcription or translation.It provides important theoretical basis for the understanding of TFEB regulation network.Our study provides new ideas for improving lysosomal function and treating lysosomal related diseases,neurodegenerative diseases and tumors.Methods1.PDCD4 regulates lysosomal biogenesis and lysosomal function through TFEB(1)Detection of lysosome morphology and number:Immunofluorescence staining of lysosome membrane proteins was used to detect the morphology,size and number of lysosomes in mouse macrophages and mouse embryonic fibroblasts after PDCD4 knockout or overexpression,respectively.(2)Lysosomal activity detection:Flow cytometry staining with a lysosomal specific activity probe was used to detect the regulation of lysosomal activity in mouse macrophages and mouse embryonic fibroblasts by PDCD4 knockout or overexpression.(3)Expression detection of lysosomal related proteins:The expression of lysosomal related membrane proteins and lysosomal enzymes in mouse macrophages and mouse embryonic fibroblasts after PDCD4 knockout or overexpression was detected by immunoblotting technique.(4)PDCD4 regulated lysosome through TFEB:TFEB expression was interfered in PDCD4 knockout mouse macrophages and mouse embryonic fibroblasts.Lysosomal morphology,size,number,activity and lysosomal related proteins were detected by immunofluorescence,flow cytometry and immunoblotting respectively.Real-time PCR was used to detect the expression of a range of TFEB regulated downstream genes after PDCD4 knockout and overexpression,as well as TFEB expression interference..2.The mechanism of PDCD4 regulating TFEB nuclear localization(1)TFEB activity and subcellular localization detection:Active TFEB can enter the nucleus to play its transcriptional regulation role.Cytoplasm and nuclear proteins were isolated from mouse macrophages and mouse embryonic fibroblasts with PDCD4 knocked out or overexpressed,and TFEB expression in cytoplasm and nucleus was detected by immunoblotting.The subcellular localization of TFEB was detected using immunofluorescence with specific staining.(2)Whether TFEB nuclear localization depends on mTORCl activity:EBSS starvation or mTORC1 specific inhibitors were used to inhibit mTORC1 activity in PDCD4 knockout or overexpressed mouse macrophages and mouse embryonic fibroblasts,respectively.The expression of TFEB in cytoplasm and nucleus was detected by immunoblotting and immunofluorescence.At the same time,mTORC1 activity was detected during the change of PDCD4 expression.Contrast this with the interference TFEB group.(3)Whether TFEB nuclear localization depends on ERK2 activity:Immunoblotting was used to detect ERK2 activity in PDCD4 knockout mouse macrophages and mouse embryonic fibroblasts.3.The molecular mechanism of PDCD4 regulating TFEB expression(1)Detection the influence of PDCD4 on TFEB mRNA expression:In a variety of cell lines,knockout,interference,or gradient overexpression of PDCD4 were performed.Real-time PCR was used to detect the influence of PDCD4 expression changes on TFEB mRNA expression.(2)Detection of PDCD4 regulation on TFEB protein levels:In a variety of cell lines,knockout,interference,or gradient overexpression of PDCD4 were used to detect the effect of changes in PDCD4 expression on TFEB protein levels using immunoblotting.(3)To detect the regulation of PDCD4 on TFEB degradation and stability:PDCD4 knockout system was used to detect the effect of PDCD4 on TFEB degradation and stability after MG 132 blocked the protein degradation pathway and CHX blocked the protein synthesis pathway through western blotting.The effect of PDCD4 knockout on TFEB ubiquitin modification was detected by western blotting.(4)To detect whether PDCD4 regulation of TFEB translation depends on eIF4A:eIF4A is interfered or overexpressed in mouse macrophages and mouse embryonic fibroblasts that are PDCD4 knocked out or overexpressed,respectively;immunoblot detection of PDCD4 regulated TFEB depends on eIF4A or not.(5)To clarify the molecular mechanism and structural domain of PDCD4 regulating TFEB translation:RNA-protein immunoprecipitation technology was used to detect whether PDCD4 bound TFEB mRNA.Reporter gene technology was used to detect the three 5 'UTR of TFEB regulated by PDCD4.According to the two ways of PDCD4 inhibition of protein translation,PDCD4 functional domain truncated constitutive particles were constructed,and PDCD4 knockout mouse embryonic fibroblasts were used to explore the molecular mechanism and structural domain of PDCD4 regulation of TFEB translation.4.PDCD4 regulates macrophage polarization and antitumor effects through TFEB(1)Detection the influence of PDCD4 on macrophage polarization:In vitro experiments,wild type and PDCD4 knockout mouse macrophages were used,LPS and IL-4 were used to induce macrophage polarization.Real-time PCR,immunoblot and flow cytometry were used to detect the expression of relevant molecular markers of macrophage M1 and M2 polarization,so as to explore the effect of PDCD4 on macrophage polarization.(2)To detect the regulation of PDCD4 on tumor associated macrophage polarization:Tumor culture medium and tumor-macrophage co-culture were used to detect the effect of PDCD4 on the polarization of macrophages in the tumor microenvironment through real-time PCR,immunoblotting and flow cytometry.(3)To detect the anti-tumor effect of PDCD4 on macrophage:The tumor-macrophage co-culture system was used in vitro to detect the proliferation ability of tumor cells.The effects of PDCD4 knockout macrophages on tumor growth and quality were measured using two in vivo mouse subcutaneous tumor models.Results1.PDCD4 negatively regulates lysosomal function through TFEB(1)PDCD4 negatively regulates lysosome quantity:Immunofluorescence was used to stain the lysosome marker protein LAMP1 to detect the effects of PDCD4 knockout or overexpression on mouse macrophages and mouse embryonic fibroblasts,respectively.The results showed that PDCD4 had no effect on the morphology and size of lysosomes,but significantly changed the number of lysosomes.(2)PDCD4 negatively regulates lysosomal activity:Flow cytometry staining with Lysotracker,a lysosomal specific activity probe,was used to detect the lysosomal activity of mouse macrophages and mouse embryonic fibroblasts with PDCD4 knockout or overexpression.The results showed that PDCD4 knockout significantly increased lysosomal activity,while overexpression of PDCD4 inhibited lysosomal activity.(3)PDCD4 inhibits the expression of lysosomal related proteins:Immunoblotting technique was used to detect the expression of lysosomal related membrane proteins and lysosomal enzymes in mouse macrophages and mouse embryonic fibroblasts after PDCD4 knockout or overexpression.The results showed that PDCD4 knockout significantly increased the expression of lysosomal associated membrane protein 1(LAMP1)and cathepsin B and D(CTSB/CTSD).Overexpression of PDCD4 inhibited lysosomal related protein expression.(4)PDCD4 negatively regulates lysosome through TFEB:?The TFEB expression is interfered in mouse macrophages and mouse embryonic fibroblasts that are PDCD4 knocked out or overexpressed respectively,and the number,activity and related protein expression of lysosomes are detected by immunofluorescence,flow cytometry and immunoblot.The results showed that interfering TFEB can reverse the increase in lysosome number,activity and related protein expression caused by PDCD4 knockout,suggesting that PDCD4 regulates lysosome through TFEB.?Real-time PCR was used to detect the expression of a series of downstream genes regulated by TFEB.The results showed that PDCD4 negatively regulated the expression of TFEB downstream genes.In the PDCD4 knockout system,interfering TFEB can reverse the increased expression caused by PDCD4 knockout.2.PDCD4 inhibits TFEB nuclear localization independent of mTOR and ERK activity(1)PDCD4 inhibits TFEB nuclear localization:?Cytoplasm and nuclear proteins were isolated from PDCD4 knockout mouse macrophages and mouse embryonic fibroblasts,and TFEB expression in cytoplasm and nucleus was detected by immunoblot.The results showed that PDCD4 inhibited the expression of TFEB in the nucleus and the knockout of PDCD4 significantly increased the expression of TFEB in the nucleus.?The subcellular localization of TFEB was detected by immunofluorescence.The results showed that PDCD4 knockout promoted TFEB nuclear localization,while PDCD4 overexpression inhibited its nuclear localization.(2)TFEB nuclear localization independent of mTORCl activity:In mouse macrophages and mouse embryonic fibroblasts with PDCD4 knockout or overexpression,EBSS starvation or mTORC1 specific inhibitors were used to inhibit mTORC1 activity respectively,and TFEB expression in cytoplasm and nucleus was detected.At the same time,mTORC1 activity was detected during the change of PDCD4 expression.Contrast this with the interference TFEB group.The western blotting results showed that mTORC1 inhibition did not change the negative regulation of PDCD4 on TFEB nuclear translocation.Knockout or overexpression of PDCD4 did not affect mTORC1 activity.The above results indicated that PDCD4 regulation of TFEB nuclear localization does not depend on mTORC1 activity.(3)TFEB nuclear localization independent of ERK2 activity:PDCD4 was knocked out in mouse macrophages and mouse embryonic fibroblasts,and ERK2 activity was detected by immunoblot.The results showed that PDCD4 expression did not affect ERK2 activity,suggesting that PDCD4 regulation of TFEB nuclear localization was not dependent on ERK2 activity.3.PDCD4 regulates TFEB at the global level in eIF4A-dependent manner through its MA3 domain(1)PDCD4 does not affect TFEB mRNA expression:In a variety of cell lines,knockdown,interference,or gradient overexpression of PDCD4 is used to detect the effect of changes in PDCD4 on TFEB mRNA expression by real-time PCR.The results showed that changing the expression of PDCD4 had no significant effect on the expression of TFEB mRNA.(2)PDCD4 inhibits TFEB protein expression:In a variety of cell lines,knockout,interference,or gradient overexpression of PDCD4 was used to detect the effect of changes in PDCD4 expression on TFEB protein levels by immunoblotting.The results showed that knockout or interference of PDCD4 significantly increased TFEB protein expression,while overexpression of PDCD4 significantly inhibited TFEB expression in a gradient-dependent manner.(3)PDCD4 does not affect the degradation and stability of TFEB:?The effect of PDCD4 on TFEB was detected by immunoblotting to block protein synthesis or protein degradation.The results showed that PDCD4 did not affect the degradation pathway and protein stability of TFEB.?The effect of PDCD4 knockout on TFEB ubiquitin modification was detected by immunoblotting.The results showed that PDCD4 did not affect the ubiquitination of TFEB.(4)PDCD4 inhibition of TFEB translation is dependent on eIF4A:eIF4A is interfered or overexpressed in mouse macrophages and mouse embryonic fibroblasts that are PDCD4 knocked out or overexpressed,respectively.Immunoblotting is used to detect whether PDCD4 regulation of TFEB is dependent on eIF4A.The results showed that interfering with eIF4A in the PDCD4 knockout system can inhibit TFEB expression,while simultaneously overexpressing eIF4A in the PDCD4 overexpressed system can enhance TFEB expression,indicating that PDCD4 inhibition of TFEB translation is an eIF4A-dependent mode.(5)PDCD4 inhibits TFEB translation by its MA3 domain:?Using RNA-protein immunoprecipitation,PDCD4 was found to bind with TFEB mRNA.?It was found that PDCD4 regulates the three 5'UTRs of TFEB and mainly acts on the second type of 5'UTR by reporter gene technique.?According to the two ways of PDCD4 inhibition of protein translation,PDCD4 functional domain truncated plasmids were constructed,and the full-length and each truncated plasmids were overexpressed in PDCD4 knockout mouse embryonic fibroblasts.The results showed that PDCD4 inhibited TFEB translation through its MA3 domain,and demonstrated that PDCD4 inhibited TFEB nuclear translocation by inhibiting its overall protein expression levels.4.PDCD4 regulates macrophage polarization and antitumor effects through TFEB(1)PDCD4 knockout promotes macrophage M1 polarization:LPS and IL-4 were used to induce macrophage polarization in vitro,and relevant molecular markers of M1 and M2 polarization were detected through real-time PCR,immunoblot and flow cytometry.The results showed that PDCD4 knockout could significantly promote the polarization of macrophages toward M1 and inhibit the polarization of macrophages toward M2.(2)PDCD4 knockout inhibits tumor associated macrophages M2 polarization:Tumor culture medium and tumor-macrophage co-culture were used to detect the effect of PDCD4 on macrophage polarization in tumor microenvironment through real-time PCR,immunoblotting and flow cytometry.The results showed that PDCD4 knockout could inhibit the M2 polarization of tumor associated macrophages.(3)PDCD4 knockout can improve the anti-tumor effect of macrophages:?Tumor-macrophage co-culture system was used to detect the proliferation ability of tumor cells.The results showed that PDCD4 knockout macrophages could inhibit tumor cell proliferation,and TFEB interference could reverse its inhibitory of tumor cells.?The effect of PDCD4 knockout macrophages on tumor growth and quality was detected by two mouse subcutaneous tumor model.The results showed that PDCD4 knockout macrophages could significantly inhibit tumor growth and tumor quality,and the interference of TFEB expression in PDCD4 knockout macrophages could reverse its inhibitory ability on tumor growth and quality and reduce its anti-tumor effect.It suggested that PDCD4 knockout could improve the anti-tumor effect of macrophages by upregulating TFEB expression.Conclusions1.PDCD4 can negatively regulate lysosomal biogenesis and lysosomal function.2.PDCD4 negatively regulates TFEB nuclear localization independent of mTORC1 and ERK2 activities.3.PDCD4 inhibits TFEB expression and nuclear localization by inhibiting TFEB translation at the global level,and realizes negative regulation of lysosomal biogenesis and lysosomal function.4.PDCD4 inhibits TFEB translation in an eIF4A-dependent manner through its MA3 domain.PDCD4 can regulate the three 5'UTRs of TFEB but mainly works on the second 5'UTR.5.PDCD4 knockout can inhibit the macrophage M2 polarization,and improve the anti-tumor effect of macrophage by increasing the expression of TFEB.Originality and Significance1.PDCD4 is an important tumor suppressant gene and protein translation inhibitor.Our experiment identified a new target gene of PDCD4,TFEB,which proved that PDCD4 can inhibit TFEB translation in an eIF4A-dependent manner through its MA3 domain.For the first time,PDCD4 was found to negatively regulate lysosomal biogenesis and function by inhibiting TFEB translation,providing a new theoretical basis for clarifying the role of PDCD4 in regulating lysosome.2.Combined with the previously published mechanism of PDCD4 inhibiting autophagy,our experiment fully elaborated the regulatory mechanism of PDCD4 in lysosome and autophagy,providing an important theoretical basis for regulating cell energy perception,maintaining cell metabolic homeostasis,and understanding cell autophagy-lysosome pathways.3.TFEB is an important transcription factor in regulating lysosome.Currently,the regulation mechanism of TFEB is mainly focused on its phosphorylation modification.Our study firstly clarifies the regulation mechanism of TFEB at the protein translation level,providing an important theoretical basis for fully understanding the entire regulation network of TFEB.It provides a good theoretical support for targeted therapy of lysosomal storage disorders,neurodegenerative diseases,tumors and other lysosomal related diseases.4.PDCD4 molecule is a tumor suppressor gene,and overexpression of PDCD4 in tumor cells can inhibit the lysosomal function,thus inhibiting their proliferation and migration.However,in tumor associated immune cells,it is necessary to knock out or reduce PDCD4 expression to improve cell lysosomal function and enhance the biological function of immune cells.This suggests that PDCD4 has opposite effects in tumor cells and tumor associated immune cells,and our study provides very important theoretical guidance for targeted PDCD4 therapy for tumors.Limitations1.TFEB belongs to the MIT-TFE family,and our study focuses on the regulatory mechanism and physiological effects of PDCD4 on TFEB.However,whether PDCD4 can be involved in regulating other members of the MIT-TFE family is unclear and further experimental studies are needed.2.TFEB can regulate a large number of lysosomal related diseases,and our study focuses on the role and significance of TFEB in tumor associated macrophages.However,the PDCD4 regulation of TFEB in other lysosomal related diseases,such as neurodegenerative and metabolic diseases,remains to be further investigated experimentally.