A study on the role and mechanism of upregulated p62 protein in macrophage-derived foam cells

LIN Zhangjun; LI Qian; ZHANG Yuefan; RUI Yaocheng

doi:10.3969/j.issn.1006-0111.2019.05.004

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Article Navigation > Journal of Pharmaceutical Practice and Service > 2019 > 37(5): 400-405,426

LIN Zhangjun, LI Qian, ZHANG Yuefan, RUI Yaocheng. A study on the role and mechanism of upregulated p62 protein in macrophage-derived foam cells[J]. Journal of Pharmaceutical Practice and Service, 2019, 37(5): 400-405,426. doi: 10.3969/j.issn.1006-0111.2019.05.004

Citation:

LIN Zhangjun, LI Qian, ZHANG Yuefan, RUI Yaocheng. A study on the role and mechanism of upregulated p62 protein in macrophage-derived foam cells[J]. Journal of Pharmaceutical Practice and Service, 2019, 37(5): 400-405,426. doi: 10.3969/j.issn.1006-0111.2019.05.004

A study on the role and mechanism of upregulated p62 protein in macrophage-derived foam cells

doi: 10.3969/j.issn.1006-0111.2019.05.004

1.
Department of Pharmacology, School of Pharmacy, Naval Medical University, Shanghai 200433, China
2.
Department of Pharmacy, No. 901 Hospital of Joint Logistics Support Force of PLA, Hefei 230031, China

Received Date: 2019-06-23
Rev Recd Date: 2019-07-23

Abstract

Objective To investigate the effect of p62 protein on the lipid autophagy and the expression of inflammatory cytokines in macrophage-derived foam cells. Methods RAW264.7 cells were stimulated by oxidized low density lipoprotein to mimic the formation of macrophage-derived foam cells.The levels of p62 protein and mRNA in macrophage-derived foam cells were detected by Western blot and real-time qPCR.The protein levels of LC3,Plin2 and PEX2 were measured by Western blot in Ox-LDL treated Raw264.7 cells to determine the effects of p62 on autophagy in macrophage-derived foam cells.The TNFα and IL-6 mRNA levels were detected by real-time PCR. Results Ox-LDL up-regulated both autophagy levels and p62 protein levels in RAW264.7 cells.After interfering the expression of p62,the level of LC3-Ⅱ protein decreased,but the protein levels of lipid droplet-related protein Plin2 did not change significantly.The peroxisome-related protein PEX2 level increased.The expression of TNFα and IL-6 also increased.Further studies showed that Nrf2 interference significantly inhibited the up-regulation of p62 by Ox-LDL. Conclusion Ox-LDL mediates the upregulation of p62 through Nrf2 in macrophage-derived foam cells,and p62 may play a protective role in atherosclerosis.
- atherosclerosis,
- p62,
- autophagy,
- macrophage,
- inflammatory cytokines

References

[1]	HANSSON G K.Inflammation,atherosclerosis,and coronary artery disease[J].N Engl J Med,2005,352(16):1685-1695.
[2]	LEVINE B,KROEMER G.Autophagy in the pathogenesis of disease[J].Cell,2008,132(1):27-42.
[3]	EVANS T D,SERGIN I,ZHANG X Y,et al.Target acquired:Selective autophagy in cardiometabolic disease[J].Sci Signal,2017,10(468):eaag2298.
[4]	OUIMET M,FRANKLIN V,MAK E,et al.Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase[J].Cell Metab,2011,13(6):655-667.
[5]	LIAO X H,SLUIMER J C,WANG Y,et al.Macrophage autophagy plays a protective role in advanced atherosclerosis[J].Cell Metab,2012,15(4):545-553.
[6]	LIPPAI M,LÖW P.The role of the selective adaptor p62 and ubiquitin-like proteins in autophagy[J].Biomed Res Int,2014,2014:832704.
[7]	MANLEY S,WILLIAMS J A,DING W X.Role of p62/SQSTM1 in liver physiology and pathogenesis[J].Exp Biol Med (Maywood),2013,238(5):525-538.
[8]	BITTO A,LERNER C A,NACARELLI T,et al.P62/SQSTM1 at the interface of aging,autophagy,and disease[J].Age (Dordr),2014,36(3):9626.
[9]	BURDELSKI C,REISWICH V,HUBE-MAGG C,et al.Cytoplasmic accumulation of sequestosome 1(p62) is a predictor of biochemical recurrence,rapid tumor cell proliferation,and genomic instability in prostate cancer[J].Clin Cancer Res,2015,21(15):3471-3479.
[10]	SERGIN I,BHATTACHARYA S,EMANUEL R,et al.Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis[J].Sci Signal,2016,9(409):ra2.
[11]	SERGIN I,EVANS T D,ZHANG X Y,et al.Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis[J].Nat Commun,2017,8:15750.
[12]	GROOTAERT M O J,ROTH L,SCHRIJVERS D M,et al.Defective autophagy in atherosclerosis:to die or to senesce?[J].Oxid Med Cell Longev,2018,2018:7687083.
[13]	JEONG S J,ZHANG X Y,RODRIGUEZ-VELEZ A,et al.P62/SQSTM1 and selective autophagy in cardiometabolic diseases[J].Antioxid Redox Signal,2019,31(6):458-471.
[14]	HU D,WU J,XU L F,et al.A method for the establishment of a cell line with stable expression of the GFP-LC3 reporter protein[J].Mol Med Rep,2012,6(4):783-786.
[15]	LIU N,WU C M,SUN L Z,et al.Sesamin enhances cholesterol efflux in RAW₂₆₄_.7 macrophages[J].Molecules,2014,19(6):7516-7527.
[16]	GUPTA N,GOSWAMI R,ALHARBI M O,et al.TRPV 4 is a regulator in P.gingivalis lipopolysaccharide-induced exacerbation of macrophage foam cell formation[J].Physiol Rep,2019,7(7):e14069.
[17]	BIERMANNS M,VON LAAR J,BROSIUS U,et al.The peroxisomal membrane targeting elements of human peroxin 2(PEX2)[J].Eur J Cell Biol,2003,82(4):155-162.
[18]	ISHⅡ T,ITOH K,TAKAHASHI S,et al.Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages[J].J Biol Chem,2000,275(21):16023-16029.
[19]	PARK Y M.CD36,a scavenger receptor implicated in atherosclerosis[J].Exp Mol Med,2014,46:e99.DOI: 10.1038/emm.2014.38.
[20]	CHOROMANSKA B,MYSLIWIEC P,CHOROMANSKA K,et al.The role of CD36 receptor in the pathogenesis of atherosclerosis[J].Adv Clin Exp Med,2017,26(4):717-722.
[21]	JEONG S J,LEE M N,OH G T.The role of macrophage lipophagy in reverse cholesterol transport[J].Endocrinol Metab (Seoul),2017,32(1):41-46.
[22]	SANDA G M,DELEANU M,TOMA L,et al.Oxidized LDL-exposed human macrophages display increased MMP-9 expression and secretion mediated by endoplasmic reticulum stress[J].J Cell Biochem,2017,118(4):661-669.
[23]	YAO S T,MIAO C,TIAN H,et al.Endoplasmic reticulum stress promotes macrophage-derived foam cell formation by up-regulating cluster of differentiation 36(CD36) expression[J].J Biol Chem,2014,289(7):4032-4042.
[24]	NING H F,LIU D,YU X C,et al.Oxidized low-density lipoprotein-induced p62/SQSTM1 accumulation in THP-1-derived macrophages promotes IL-18 secretion and cell death[J].Exp Ther Med,2017,14(6):5417-5423.
[25]	KIM J Y,OZATO K.The sequestosome 1/p62 attenuates cytokine gene expression in activated macrophages by inhibiting IFN regulatory factor 8 and TNF receptor-associated factor 6/NF-kappaB activity[J].J Immunol,2009,182(4):2131-2140.
[26]	KIRKIN V,LAMARK T,JOHANSEN T,et al.NBR1 cooperates with p62 in selective autophagy of ubiquitinated targets[J].Autophagy,2009,5(5):732-733.
[27]	NIAN Z Q,SUN Z Q,YU L X,et al.Fat-specific protein 27 undergoes ubiquitin-dependent degradation regulated by triacylglycerol synthesis and lipid droplet formation[J].J Biol Chem,2010,285(13):9604-9615.
[28]	FUJIMOTO T,OHSAKI Y.Proteasomal and autophagic pathways converge on lipid droplets[J].Autophagy,2006,2(4):299-301.
[29]	OUIMET M,MARCEL Y L.Regulation of lipid droplet cholesterol efflux from macrophage foam cells[J].Arterioscler Thromb Vasc Biol,2012,32(3):575-581.
[30]	TILL A,LAKHANI R,BURNETT S F,et al.Pexophagy:the selective degradation of peroxisomes[J].Int J Cell Biol,2012,2012:512721.
[31]	SARGENT G,VAN ZUTPHEN T,SHATSEVA T,et al.PEX2 is the E3 ubiquitin ligase required for pexophagy during starvation[J].J Cell Biol,2016,214(6):677-690.
[32]	JAIN A,LAMARK T,SJØTTEM E,et al.P62/SQSTM1 is a target gene for transcription factor NRF₂ and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription[J].J Biol Chem,2010,285(29):22576-22591.

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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A study on the role and mechanism of upregulated p62 protein in macrophage-derived foam cells

1. Department of Pharmacology, School of Pharmacy, Naval Medical University, Shanghai 200433, China

2. Department of Pharmacy, No. 901 Hospital of Joint Logistics Support Force of PLA, Hefei 230031, China

Received Date: 2019-06-23

Rev Recd Date: 2019-07-23

Key words:

Abstract: Objective To investigate the effect of p62 protein on the lipid autophagy and the expression of inflammatory cytokines in macrophage-derived foam cells. Methods RAW264.7 cells were stimulated by oxidized low density lipoprotein to mimic the formation of macrophage-derived foam cells.The levels of p62 protein and mRNA in macrophage-derived foam cells were detected by Western blot and real-time qPCR.The protein levels of LC3,Plin2 and PEX2 were measured by Western blot in Ox-LDL treated Raw264.7 cells to determine the effects of p62 on autophagy in macrophage-derived foam cells.The TNFα and IL-6 mRNA levels were detected by real-time PCR. Results Ox-LDL up-regulated both autophagy levels and p62 protein levels in RAW264.7 cells.After interfering the expression of p62,the level of LC3-Ⅱ protein decreased,but the protein levels of lipid droplet-related protein Plin2 did not change significantly.The peroxisome-related protein PEX2 level increased.The expression of TNFα and IL-6 also increased.Further studies showed that Nrf2 interference significantly inhibited the up-regulation of p62 by Ox-LDL. Conclusion Ox-LDL mediates the upregulation of p62 through Nrf2 in macrophage-derived foam cells,and p62 may play a protective role in atherosclerosis.

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[1]	HANSSON G K.Inflammation,atherosclerosis,and coronary artery disease[J].N Engl J Med,2005,352(16):1685-1695.
[2]	LEVINE B,KROEMER G.Autophagy in the pathogenesis of disease[J].Cell,2008,132(1):27-42.
[3]	EVANS T D,SERGIN I,ZHANG X Y,et al.Target acquired:Selective autophagy in cardiometabolic disease[J].Sci Signal,2017,10(468):eaag2298.
[4]	OUIMET M,FRANKLIN V,MAK E,et al.Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase[J].Cell Metab,2011,13(6):655-667.
[5]	LIAO X H,SLUIMER J C,WANG Y,et al.Macrophage autophagy plays a protective role in advanced atherosclerosis[J].Cell Metab,2012,15(4):545-553.
[6]	LIPPAI M,LÖW P.The role of the selective adaptor p62 and ubiquitin-like proteins in autophagy[J].Biomed Res Int,2014,2014:832704.
[7]	MANLEY S,WILLIAMS J A,DING W X.Role of p62/SQSTM1 in liver physiology and pathogenesis[J].Exp Biol Med (Maywood),2013,238(5):525-538.
[8]	BITTO A,LERNER C A,NACARELLI T,et al.P62/SQSTM1 at the interface of aging,autophagy,and disease[J].Age (Dordr),2014,36(3):9626.
[9]	BURDELSKI C,REISWICH V,HUBE-MAGG C,et al.Cytoplasmic accumulation of sequestosome 1(p62) is a predictor of biochemical recurrence,rapid tumor cell proliferation,and genomic instability in prostate cancer[J].Clin Cancer Res,2015,21(15):3471-3479.
[10]	SERGIN I,BHATTACHARYA S,EMANUEL R,et al.Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis[J].Sci Signal,2016,9(409):ra2.
[11]	SERGIN I,EVANS T D,ZHANG X Y,et al.Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis[J].Nat Commun,2017,8:15750.
[12]	GROOTAERT M O J,ROTH L,SCHRIJVERS D M,et al.Defective autophagy in atherosclerosis:to die or to senesce?[J].Oxid Med Cell Longev,2018,2018:7687083.
[13]	JEONG S J,ZHANG X Y,RODRIGUEZ-VELEZ A,et al.P62/SQSTM1 and selective autophagy in cardiometabolic diseases[J].Antioxid Redox Signal,2019,31(6):458-471.
[14]	HU D,WU J,XU L F,et al.A method for the establishment of a cell line with stable expression of the GFP-LC3 reporter protein[J].Mol Med Rep,2012,6(4):783-786.
[15]	LIU N,WU C M,SUN L Z,et al.Sesamin enhances cholesterol efflux in RAW₂₆₄_.7 macrophages[J].Molecules,2014,19(6):7516-7527.
[16]	GUPTA N,GOSWAMI R,ALHARBI M O,et al.TRPV 4 is a regulator in P.gingivalis lipopolysaccharide-induced exacerbation of macrophage foam cell formation[J].Physiol Rep,2019,7(7):e14069.
[17]	BIERMANNS M,VON LAAR J,BROSIUS U,et al.The peroxisomal membrane targeting elements of human peroxin 2(PEX2)[J].Eur J Cell Biol,2003,82(4):155-162.
[18]	ISHⅡ T,ITOH K,TAKAHASHI S,et al.Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages[J].J Biol Chem,2000,275(21):16023-16029.
[19]	PARK Y M.CD36,a scavenger receptor implicated in atherosclerosis[J].Exp Mol Med,2014,46:e99.DOI:10.1038/emm.2014.38.
[20]	CHOROMANSKA B,MYSLIWIEC P,CHOROMANSKA K,et al.The role of CD36 receptor in the pathogenesis of atherosclerosis[J].Adv Clin Exp Med,2017,26(4):717-722.
[21]	JEONG S J,LEE M N,OH G T.The role of macrophage lipophagy in reverse cholesterol transport[J].Endocrinol Metab (Seoul),2017,32(1):41-46.
[22]	SANDA G M,DELEANU M,TOMA L,et al.Oxidized LDL-exposed human macrophages display increased MMP-9 expression and secretion mediated by endoplasmic reticulum stress[J].J Cell Biochem,2017,118(4):661-669.
[23]	YAO S T,MIAO C,TIAN H,et al.Endoplasmic reticulum stress promotes macrophage-derived foam cell formation by up-regulating cluster of differentiation 36(CD36) expression[J].J Biol Chem,2014,289(7):4032-4042.
[24]	NING H F,LIU D,YU X C,et al.Oxidized low-density lipoprotein-induced p62/SQSTM1 accumulation in THP-1-derived macrophages promotes IL-18 secretion and cell death[J].Exp Ther Med,2017,14(6):5417-5423.
[25]	KIM J Y,OZATO K.The sequestosome 1/p62 attenuates cytokine gene expression in activated macrophages by inhibiting IFN regulatory factor 8 and TNF receptor-associated factor 6/NF-kappaB activity[J].J Immunol,2009,182(4):2131-2140.
[26]	KIRKIN V,LAMARK T,JOHANSEN T,et al.NBR1 cooperates with p62 in selective autophagy of ubiquitinated targets[J].Autophagy,2009,5(5):732-733.
[27]	NIAN Z Q,SUN Z Q,YU L X,et al.Fat-specific protein 27 undergoes ubiquitin-dependent degradation regulated by triacylglycerol synthesis and lipid droplet formation[J].J Biol Chem,2010,285(13):9604-9615.
[28]	FUJIMOTO T,OHSAKI Y.Proteasomal and autophagic pathways converge on lipid droplets[J].Autophagy,2006,2(4):299-301.
[29]	OUIMET M,MARCEL Y L.Regulation of lipid droplet cholesterol efflux from macrophage foam cells[J].Arterioscler Thromb Vasc Biol,2012,32(3):575-581.
[30]	TILL A,LAKHANI R,BURNETT S F,et al.Pexophagy:the selective degradation of peroxisomes[J].Int J Cell Biol,2012,2012:512721.
[31]	SARGENT G,VAN ZUTPHEN T,SHATSEVA T,et al.PEX2 is the E3 ubiquitin ligase required for pexophagy during starvation[J].J Cell Biol,2016,214(6):677-690.
[32]	JAIN A,LAMARK T,SJØTTEM E,et al.P62/SQSTM1 is a target gene for transcription factor NRF₂ and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription[J].J Biol Chem,2010,285(29):22576-22591.

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A study on the role and mechanism of upregulated p62 protein in macrophage-derived foam cells

doi: 10.3969/j.issn.1006-0111.2019.05.004

Abstract

References

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通讯作者: 陈斌, bchen63@163.com

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