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Growth arrest and DNA damage 45? is required for caspase-dependent renal tubular cell apoptosis.

ABSTRACT: BACKGROUND:Growth Arrest and DNA Damage 45? (GADD45?) is a member of the DNA damage-inducible gene family which responds to environmental stresses. Apoptosis is a critical mode of renal tubular cell death in nephrotoxin-induced acute kidney injury. In this study, we investigated the role of GADD45? in renal tubular cell apoptosis induced by nephrotoxic drugs. METHODS:Primary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GADD45? expression was silenced, HRE cells were transduced with a plasmid encoding GADD45?-specific shRNA. The recombinant adenovirus containing the GADD45? gene was synthesized to overexpress GADD45? protein. Cell death was induced by cisplatin and cyclosporine A (CsA). To prevent apoptotic cell death, pan-caspase inhibitor ZVAD-FMK was used. To prevent non-apoptotic cell death, necrostatin-1 and ferrostatin-1 were used. The degree of apoptosis and necrosis of cultured cells were evaluated by flow cytometry. RESULTS:Expression of the GADD45? gene was significantly upregulated in response to treatment with CsA and cisplatin. Apoptosis and necrosis induced by these drugs were significantly reduced by silencing of GADD45?, and significantly augmented by the overexpression of GADD45?. The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or CsA was reduced by silencing of GADD45?, and was augmented by the overexpression of GADD45?, indicating that caspase activation is dependent on the expression of GADD45?. ZVAD-FMK significantly inhibited apoptosis induced by cisplatin or CsA, indicating a role of caspases in mediating apoptotic cell death. ZVAD-FMK was effective to prevent necrosis as well, indicating that the observed necrosis was a secondary event following apoptosis at least in part. CONCLUSIONS:To our knowledge, this is the first study to show that GADD45? is required for the caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.

SUBMITTER: Shin GT

PROVIDER: S-EPMC6386268 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Growth arrest and DNA damage 45γ is required for caspase-dependent renal tubular cell apoptosis.

Shin Gyu-Tae GT Lee Hwa Joung HJ Park Ji Eun JE

PloS one 20190222 2

<h4>Background</h4>Growth Arrest and DNA Damage 45γ (GADD45γ) is a member of the DNA damage-inducible gene family which responds to environmental stresses. Apoptosis is a critical mode of renal tubular cell death in nephrotoxin-induced acute kidney injury. In this study, we investigated the role of GADD45γ in renal tubular cell apoptosis induced by nephrotoxic drugs.<h4>Methods</h4>Primary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GA ...[more]

PMID: 30794682

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Project description:BackgroundAcute kidney injury (AKI) is a common clinically critical illness with serious consequences for the patients. Shenshuaikang enema (SE) is a Chinese herbal compound that is used to treat AKI in clinical practice. However, its mechanism of action remains unclear.AimThe aim of this study was to investigate the therapeutic effect of SE and explore the molecular mechanisms using network pharmacology and in vitro experiments.Materials and methodsThe herb-component-target network was constructed based on network pharmacology. The predicted targets and pathways were validated using in vitro experiments. A renal tubular epithelial cell line (HK-2 cells) was exposed to hypoxia and reoxygenation (H/R) using air-tight conditions for five hours and treated with different concentrations of SE (25%, 50%, and 75%) to assess cell viability and apoptosis and determine the optimal experimental dose. Subsequently, H/R-injured HK-2 cells were pretreated with the optimal SE dose and then randomly divided into three groups, the SE, SE-SP600125 (inhibitor of JNK), and SE-NAC (antioxidant) groups. The cell vitality, apoptosis, and death were evaluated using the cell counting kit 8 (CCK8) and carboxyfluorescein succinimidyl ester/propidium iodide (CFSF/PI) staining. The apoptosis-related protein JNK and Caspase-3 were assessed by Western blot. Expression of JNK and Caspase-3 genes was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR).Results123 active components and 226 targets were identified from four herbs that composed the herb-compound-target network based on transcriptomics and network pharmacology analyses. The KEGG pathway analyses revealed that the mitochondrial apoptosis pathway was involved in the therapeutic AKI effects of SE. Cell vitality of H/R-induced HK-2 cells was obviously increased when treating them with SE, and the apoptosis was significantly inhibited, especially in the SE (50%) group at 4 and 12 h after modeling. Pretreatment with antioxidant NAC obviously prevented cell death compared to the SE (50%) group, while no obvious reduction of apoptosis was observed in the SP600125 group. JNK expression level was significantly increased in the SE (50%) group compared to the SP600125 (P < 0.01) and the NAC group (P < 0.05). Caspase-3 was downregulated in the SE (50%) group compared to the SP600125 (P < 0.01) and NAC group (P < 0.05). Caspase-3 activation in the SP600125 group was higher than that in the NAC group (P < 0.05). Moreover, the oxidative damage-dependent JNK/Caspase-3 pathway was identified in the H/R-injured HK-2 cells by inhibiting the JNK activation and oxidative damage.ConclusionsOur findings suggested that the H/R-triggered apoptosis in HK-2 cells was abrogated by SE by upregulating the oxidative damage-dependent JNK to trigger suppression of Caspase-3.

Project description:Growth arrest DNA damage-inducible gene 45 beta (GADD45beta) has been known to regulate cell growth, apoptotic cell death, and cellular response to DNA damage. Down-regulation of GADD45beta has been verified to be specific in hepatocellular cancer (HCC) and consistent with the p53 mutant, and degree of malignancy of HCC. This observation was further confirmed by eight HCC cell lines and paired human normal and HCC tumor tissues by Northern blot and immunohistochemistry. To better understand the transcription regulation, we cloned and characterized the active promoter region of GADD45beta in luciferase-expressing vector. Using the luciferase assay, three nuclear factor-kappaB binding sites, one E2F-1 binding site, and one putative inhibition region were identified in the proximal promoter of GADD45beta from -865/+6. Of interest, no marked putative binding sites could be identified in the inhibition region between -520/-470, which corresponds to CpG-rich region. The demethylating agent 5-Aza-dC was used and demonstrated restoration of the GADD45beta expression in HepG2 in a dose-dependent manner. The methylation status in the promoter was further examined in one normal liver cell, eight HCC cell lines, eight HCC tissues, and five corresponding nonneoplastic liver tissues. Methylation-specific polymerase chain reaction and sequencing of the sodium bisulfite-treated DNA from HCC cell lines and HCC samples revealed a high percentage of hypermethylation of the CpG islands. Comparatively, the five nonneoplastic correspondent liver tissues demonstrated very low levels of methylation. To further understand the functional role of GADD45beta under-expression in HCC the GADD45beta cDNA constructed plasmid was transfected into HepG2 (p53 WT) and Hep3B (p53 null) cells. The transforming growth factor-beta was assayed by enzyme-linked immunosorbent assay, which revealed a decrease to 40% in transfectant of HepG2, but no significant change in Hep3B transfectant. Whereas, Hep3B co-transfected with p53 and GADD45beta demonstrated significantly reduced transforming growth factor-beta. The colony formation was further examined and revealed a decrease in HepG2-GADD45beta transfectant and Hep3B-p53/GADD45beta co-transfectant. These findings suggested that methylation might play a crucial role in the epigenetic regulation of GADD45beta in hepatocyte transformation that may be directed by p53 status. Thus, our results provided a deeper understanding of the molecular mechanism of GADD45beta down-regulation in HCC.

Dataset Information

Growth arrest and DNA damage 45? is required for caspase-dependent renal tubular cell apoptosis.

Publications

Growth arrest and DNA damage 45γ is required for caspase-dependent renal tubular cell apoptosis.

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