Role of APE1/Ref-1 in hydrogen peroxide-induced apoptosis in human renal HK-2 cells

( Ha Yeon Kim ),( Jung Sun Park ),( Byeong Hwa Jeon ),( Hong Sang Choi ),( Chang Seong Kim ),( Seong Kwon Ma ),( Soo Wan Kim ),( Eun Hui Bae ) 대한신장학회 2024 Kidney Research and Clinical Practice Vol.43 No.2

Background: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multipotent protein that plays essential roles in cellular responses to oxidative stress. Methods: To examine the role of APE1/Ref-1 in ischemia-reperfusion (I/R) injuries and hydrogen peroxide (H₂O₂)-induced renal tubular apoptosis, we studied male C57BL6 mice and human proximal tubular epithelial (HK-2) cells treated with H₂O₂ at different concentrations. The colocalization of APE1/Ref-1 in the proximal tubule, distal tubule, thick ascending limb, and collecting duct was observed with confocal microscopy. The overexpression of APE1/Ref-1 with knockdown cell lines using an APE1/Ref-1-specific DNA or small interfering RNA (siRNA) was used for the apoptosis assay. The promotor activity of nuclear factor kappa B (NF-κB) was assessed and electrophoretic mobility shift assay was conducted. Results: APE1/Ref-1 was predominantly localized to the renal tubule nucleus. In renal I/R injuries, the levels of APE1/Ref-1 protein were increased compared with those in kidneys subjected to sham operations. The overexpression of APE1/Ref-1 in HK-2 cells enhanced the Bax/Bcl-2 ratio as a marker of apoptosis. Conversely, the suppression of APE1/Ref-1 expression by siRNA in 1-mM H₂O₂-treated HK-2 cells decreased the Bax/Bcl-2 ratio, the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and NF-κB. In HK-2 cells, the promoter activity of NF-κB increased following H₂O₂ exposure, and this effect was further enhanced by APE1/Ref-1 transfection. Conclusion: The inhibition of APE1/Ref-1 with siRNA attenuated H₂O₂-induced apoptosis through the modulation of mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 and the nuclear activation of NF-κB and proapoptotic factors.

Altered Secretory Activity of APE1/Ref-1 D148E Variants Identified in Human Patients With Bladder Cancer

이유란,임재성,신주현,최성아,주희경,전병화 대한배뇨장애요실금학회 2016 International Neurourology Journal Vol.20 No.S1

Purpose: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox modulation. Recently, serum and urinary APE1/Ref-1 levels were reported to be increased in patients with bladder cancer. Genetic variations of APE/Ref-1 are associated with the risk of cancer. However, the effect of APE1/Ref-1 variants on its secretory activity is yet unknown. Methods: APE1/Ref-1 variants were evaluated by DNA sequencing analysis of reverse transcription polymerase chain reaction products in coding DNA sequences (CDS) of APE1/Ref-1 in bladder tissue samples from patients with bladder cancer (n=10). Secretory activity of APE1/Ref-1 variants was evaluated with immunoblot and enzyme-linked immunosorbent assay of the culture medium supernatants. Results: Four different substitution mutants (D148E, I64V/D148E, W67R/D148E, and E86G/D148E) of APE1/Ref-1 were identified in bladder cancer specimens. However, deletion mutants of APE1/Ref-1 CDS were not found. The secretory activity of the APE1/Ref-1 variants (D148E, I64V/D148E, and E86G/D148E) was increased compared to that of wild type APE1/Ref- 1. Furthermore, the secretory activity in basal or hyperacetylated conditions was much higher than that in APE1/Ref-1 D148Etransfected HEK293 cells. Conclusions: Taken together, our data suggest that the increased secretory activity of D148E might contribute to increased serum levels of APE1/Ref-1 in patients with bladder cancer.

Cytoplasmic Localization and Redox Cysteine Residue of APE1/Ref-1 Are Associated with Its Anti-Inflammatory Activity in Cultured Endothelial Cells

박명수,전병화,김척승,주희경,이유란,강건,김수진,최성아,이상도,박진봉,김국성 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.5

Apurinic/apyrimidinic endonuclease1/redox factor-1 (APE1/ Ref-1) is a multifunctional protein involved in base excision DNA repair and transcriptional regulation of gene expression. APE1/Ref-1 is mainly localized in the nucleus, but cytoplasmic localization has also been reported. However, the functional role of cytoplasmic APE1/Ref-1 and its redox cysteine residue are still un-known. We investigated the role of cytoplasmic APE1/Ref-1 on tumor necrosis factor-alpha (TNF-alpha)-induced vascular cell adhesion molecule-1 (VCAM-1) expressions in endothelial cells. Endogenous APE1/Ref-1 was mainly observed in the nucleus, however, cytoplasmic APE1/Ref-1 was increased by TNF-alpha. Cytoplasmic APE1/ Ref-1 expression was not blunted by cycloheximide, a protein synthesis inhibitor, suggesting cytoplasmic trans-location of APE1/Ref-1. Transfection of an N-terminus deletion mutant APE1/Ref-1(29-318) inhibited TNF-alpha-induced VCAM-1 expression, indicating an anti-inflam- matory role for APE1/Ref-1 in the cytoplasm. In contrast, redox mutant of APE1/Ref-1 (C65A/C93A) transfection led to increased TNF-alpha-induced VCAM-1 expression. Our findings suggest cytoplasmic APE1/Ref-1 localization and redox cysteine residues of APE1/Ref-1 are associated with its anti-inflam-matory activity in endothelial cells.

Altered Secretory Activity of APE1/Ref-1 D148E Variants Identified in Human Patients With Bladder Cancer

Lee, Yu Ran,Lim, Jae Sung,Shin, Ju Hyun,Choi, Sunga,Joo, Hee Kyoung,Jeon, Byeong Hwa Korean Continence Society 2016 International Neurourology Journal Vol.20 No.1

<P><B>Purpose:</B></P><P>Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox modulation. Recently, serum and urinary APE1/Ref-1 levels were reported to be increased in patients with bladder cancer. Genetic variations of APE/Ref-1 are associated with the risk of cancer. However, the effect of <I>APE1/Ref-1</I> variants on its secretory activity is yet unknown.</P><P><B>Methods:</B></P><P><I>APE1/Ref-1</I> variants were evaluated by DNA sequencing analysis of reverse transcription polymerase chain reaction products in coding DNA sequences (CDS) of APE1/Ref-1 in bladder tissue samples from patients with bladder cancer (n=10). Secretory activity of <I>APE1/Ref-1</I> variants was evaluated with immunoblot and enzyme-linked immunosorbent assay of the culture medium supernatants.</P><P><B>Results:</B></P><P>Four different substitution mutants (D148E, I64V/D148E, W67R/D148E, and E86G/D148E) of APE1/Ref-1 were identified in bladder cancer specimens. However, deletion mutants of APE1/Ref-1 CDS were not found. The secretory activity of the <I>APE1/Ref-1</I> variants (D148E, I64V/D148E, and E86G/D148E) was increased compared to that of wild type APE1/Ref-1. Furthermore, the secretory activity in basal or hyperacetylated conditions was much higher than that in <I>APE1/Ref-1</I> D148E-transfected HEK293 cells.</P><P><B>Conclusions:</B></P><P>Taken together, our data suggest that the increased secretory activity of D148E might contribute to increased serum levels of APE1/Ref-1 in patients with bladder cancer.</P>

APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions

Shweta Thakur,Bibekananda Sarkar,Ravi P Cholia,Nandini Gautam,Monisha Dhiman,Anil K Mantha 생화학분자생물학회 2014 Experimental and molecular medicine Vol.46 No.-

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1a, paired box gene 8, signal transducer activator of transcription 3and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1’s function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stressdependent responses. Thus, APE1/Ref-1 acts as a ‘hub-protein’ that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1’s versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed.

Ape1/Ref-1 Stimulates GDNF/GFRα1-mediated Downstream Signaling and Neuroblastoma Proliferation

강미영,김권영,윤영,강윤성,김홍범,윤차경,김동휘,김미화 대한약리학회 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.5

We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor α1 (GFRα1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFRα1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFRα1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFRα1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFRα1-specific RNA experiments demonstrated that the downregulation of GFRα1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLCγ-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFRα signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.

APE1/Ref-1 Inhibits Phosphate-Induced Calcification and Osteoblastic Phenotype Changes in Vascular Smooth Muscle Cells

Lee, Ki Mo,Lee, Eun Ok,Lee, Yu Ran,Joo, Hee Kyoung,Park, Myoung Soo,Kim, Cuk-Seong,Choi, Sunga,Jeong, Jin-Ok,Jeon, Byeong Hwa MDPI 2017 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.18 No.10

<P>Vascular calcification plays a role in the pathogenesis of atherosclerosis, diabetes, and chronic kidney disease; however, the role of apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) in inorganic phosphate (Pi)-induced vascular smooth muscle cell (VSMC) calcification remains unknown. In this study, we investigated the possible role of APE1/Ref-1 in Pi-induced VSMC calcification. We observed that Pi decreased endogenous APE1/Ref-1 expression and promoter activity in VSMCs, and that adenoviral overexpression of APE1/Ref-1 inhibited Pi-induced calcification in VSMCs and in an ex vivo organ culture of a rat aorta. However, a redox mutant of APE1/Ref-1(C65A/C93A) did not reduce Pi-induced calcification in VSMCs, suggesting APE1/Ref-1-mediated redox function against vascular calcification. Additionally, APE1/Ref-1 overexpression inhibited Pi-induced intracellular and mitochondrial reactive oxygen species production, and APE1/Ref-1 overexpression resulted in decreased Pi-induced lactate dehydrogenase activity, pro-apoptotic Bax levels, and increased anti-apoptotic Bcl-2 protein levels. Furthermore, APE1/Ref-1 inhibited Pi-induced osteoblastic differentiation associated with alkaline phosphatase activity and inhibited Pi-exposure-induced loss of the smooth muscle phenotype. Our findings provided valuable insights into the redox function of APE1/Ref-1 in preventing Pi-induced VSMC calcification by inhibiting oxidative stress and osteoblastic differentiation, resulting in prevention of altered osteoblastic phenotypes in VSMCs.</P>

Angiotensin II facilitates neointimal formation by increasing vascular smooth muscle cell migration: Involvement of APE/Ref-1-mediated overexpression of sphingosine-1-phosphate receptor 1

Lee, Dong-Youb,Won, Kyung-Jong,Lee, Kang Pa,Jung, Seung Hyo,Baek, Suji,Chung, Hyun Woo,Choi, Wahn Soo,Lee, Hwan Myung,Lee, Byeong Han,Jeon, Byeong Hwa,Kim, Bokyung Elsevier 2018 Toxicology and applied pharmacology Vol.347 No.-

<P><B>Abstract</B></P> <P>Angiotensin II (Ang II) is implicated in the development of cardiovascular disorders including hypertension and atherosclerosis. However, the role of Ang II in the interaction between apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) and sphingosine-1-phosphate (S1P) signals in relation to vascular disorders remains to be clarified. This study aimed to determine whether APE/Ref-1 plays a role in epigenetic regulation of the S1P receptor (S1PR) in response to Ang II in vascular smooth muscle cell (VSMC) migration and vascular neointima formation. Ang II augmented the expression of S1PR1 in aortic smooth muscle cells of Sprague Dawley rats (RASMCs), which was attenuated by Ang II receptor (AT) 1 inhibitors, antioxidants, and APE/Ref-1 knockdown with small interference RNA. Ang II stimulation produced H<SUB>2</SUB>O<SUB>2</SUB>, and exogenous H<SUB>2</SUB>O<SUB>2</SUB> elevated S1PR1 expression in RASMCs. Moreover, Ang II caused translocation of cytoplasmic APE/Ref-1 into the nucleus in RASMCs. H3 histone acetylation and APE/Ref-1 binding at the S1PR1 promoter were increased in RASMCs treated with Ang II. In addition, Ang II induced migration in RASMCs, which was suppressed by AT1 and S1PR1 inhibitors. The expression of S1PR1, and colocalization of APE/Ref-1 and acetylated histone H3 in vascular neointima, were greater in Ang II-infused rats compared with a control group. These findings demonstrate that Ang II stimulates the epigenetic regulation of S1PR1 expression via H<SUB>2</SUB>O<SUB>2</SUB>-mediated APE/Ref-1 translocation, which may consequently be involved in Ang II-induced VSMC migration and vascular neointima formation. Therefore, APE/Ref-1-mediated overexpression of S1PR1 may be implicated in the vascular dysfunction evoked by Ang II.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Ang II increased S1PR1 expression and H<SUB>2</SUB>O<SUB>2</SUB> generation in VSMCs. </LI> <LI> H<SUB>2</SUB>O<SUB>2</SUB> elevated S1PR1 expression in VSMCs. </LI> <LI> Ang II epigenetically enhanced S1PR1 expression via APE/Ref-1 translocation by H<SUB>2</SUB>O<SUB>2</SUB>. </LI> <LI> These events may be linked to Ang II-increased VSMC migration and vascular neointima. </LI> </UL> </P>

Ape1/Ref-1 Stimulates GDNF/GFR ${\alpha}$ 1-mediated Downstream Signaling and Neuroblastoma Proliferation

Kang, Mi-Young,Kim, Kweon-Young,Yoon, Young,Kang, Yoon-Sung,Kim, Hong-Beum,Youn, Cha-Kyung,Kim, Dong-Hui,Kim, Mi-Hwa The Korean Society of Pharmacology 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.5

We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor ${\alpha}$ 1 (GFR ${\alpha}$ 1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFR ${\alpha}$ 1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFR ${\alpha}$ 1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFR ${\alpha}$ 1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFR ${\alpha}$ 1-specific RNA experiments demonstrated that the downregulation of GFR ${\alpha}$ 1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLC ${\gamma}$-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFR ${\alpha}$ signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.

Ape1/Ref-1 Stimulates GDNF/GFRՁ1-mediated Downstream Signaling and Neuroblastoma Proliferation

Mi-Young Kang,Kweon Young Kim,Young Yoon,Yoonsung Kang,Hong Beum Kim,Cha Kyung Youn,Dong-Hui Kim,Mi-Hwa Kim 대한생리학회-대한약리학회 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.5

We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor Ձ1 (GFRՁ1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFRՁ1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFRՁ1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFRՁ1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFRՁ1-specific RNA experiments demonstrated that the downregulation of GFRՁ1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLCՃ-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFRՁ signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.