Dataset Information

MAGEH1 interacts with GADD45G and induces renal tubular cell apoptosis

ABSTRACT:

Background

Melanoma-associated antigen H1 (MAGEH1) is a protein that belongs to melanoma-associated antigen (MAGE) superfamily. Growth arrest and DNA damage 45G (GADD45G) is a member of the DNA damage-inducible gene family which responds to environmental stresses. We have previously shown that GADD45G is a protein that promotes apoptosis of renal tubular cells in response to a nephrotoxic injury. In this study, we show evidence that MAGEH1 interacts with GADD45G and is involved in the induction of nephrotoxin-induced apoptosis of renal tubular cells.

Methods

Primary human renal tubular epithelial (HRE) cells and human kidney 2 (HK-2) cells were used in this study. To produce stable cell lines in which MAGEH1 expression was silenced, HRE cells were transduced with a lentiviral vector encoding a single guide RNA construct targeting the MAGEH1 gene. To knockdown GADD45G expression in HRE cells, a vector containing short hairpin RNA (shRNA) was used. We used short interfering RNAs (siRNA) to achieve transient silencing of genes in HK-2 cells. Recombinant adenoviruses were synthesized to overexpress MAGEH1 and GADD45G proteins. Human protein microarray was used to identify proteins that binds to GADD45G. Co-immunoprecipitation assays were then performed to confirm microarray results. Cell death was induced by cyclosporine A (CsA). Real-time quantitative PCR assay was used to evaluate gene expression levels. The degree of apoptosis and necrosis of cultured cells was evaluated by flow cytometry. Expression levels of caspases were examined using western blot analysis.

Results

We found that GADD45G bound to one protein spotted in the protein microarray, which was subsequently identified as MAGEH1. We confirmed the interaction between GADD45G and MAGEH1 protein using the co-immunoprecipitation assay. MAGEH1 gene expression was not altered by CsA-induced cytotoxic injury, whereas GADD45G gene expression was increased significantly upon CsA treatment. MAGEH1 expression was significantly downregulated in GADD45G knockdown HRE stable cells suggesting that MAGEH1 expression may be dependent on GADD45G expression. CsA-induced apoptosis was significantly reduced in MAGEH1 knockdown HRE stable cells which led to an increased survival of these cells. Similar results were observed in GADD45G knockdown HRE stable cells. Accordingly, CsA-induced apoptosis was significantly decreased in MAGEH1 siRNA and GADD45G siRNA transfected HK-2 cells. CsA-induced activation of caspase-7 and caspase-9 was inhibited in MAGEH1 knockdown HRE stable cells, and similarly in GADD45G knockdown HRE stable cells.

Conclusions

To the best of our knowledge, this is the first study to show that MAGEH1 interacts with GADD45G and that MAGEH1 is involved in caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.

SUBMITTER: Shin G

PROVIDER: S-EPMC8598065 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundThe morbidity of nephrolithiasis is 2-3 times higher in males than in females, suggesting that androgen plays a key role in nephrolithiasis. The death of renal tubular epithelial cells (TECs) is an important pathophysiological process contributing to the development of nephrolithiasis. Therefore, the aim of this study is to investigate whether androgen directly induces TECs apoptosis and necrosis and its underlying mechanisms in kidney stone formation.Materials and methodsWe compared serum testosterone level between male and female healthy volunteers and kidney stone patients. The in vivo nephrolithiasis model was established using glyoxylic acid, and calcium deposits were detected by van Kossa staining. In the in vitro study using mouse TECs (TCMK-1 cells) and human TECs (HK-2 cells), apoptosis, necrosis, and the expression of BH3-only protein Bcl-2-like 19 kDa-interacting protein 3 (BNIP3) were examined incubated with different doses of testosterone using flow cytometry. Levels of apoptosis-related proteins transfected with the BNIP3 siRNA were examined by western blotting. The mitochondrial potential (ΔΨm) was detected by JC-1 staining and flow cytometry. We monitored BNIP3 expression in the testosterone-induced TECs injury model after treatment with hypoxia inducible factor 1α (HIF-1α) and/or hypoxia inducible factor 2α (HIF-2α) inhibitors to determine the upstream protein regulating BNIP3 expression. Additionally, ChIP and luciferase assays were performed to confirm the interaction between HIF-1α and BNIP3.ResultsBoth male and female patients have significantly higher testosterones compared with healthy volunteers. More calcium deposits in the medulla were detected in male mice compared to female and castrated male mice. Testosterone induced TECs apoptosis and necrosis and increased BNIP3 expression in a dose-dependent manner. Testosterone also increased Bax expression, decreased Bcl-2 expression and induced a loss of ΔΨm. This effect was reversed by BNIP3 knockdown. HIF-1α inhibition significantly decreased BNIP3 expression and protected TECs from testosterone-induced apoptosis and necrosis. HIF-2α inhibition, however, did not influence BNIP3 expression or TECs apoptosis or necrosis. Finally, HIF-1α interacted with the BNIP3 promoter region.ConclusionBased on these results, testosterone induced renal TECs death by activating the HIF-1α/BNIP3 pathway.

Project description:BackgroundGrowth 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.MethodsPrimary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GADD45γ expression was silenced, HRE cells were transduced with a plasmid encoding GADD45γ-specific shRNA. The recombinant adenovirus containing the GADD45γ gene was synthesized to overexpress GADD45γ 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.ResultsExpression of the GADD45γ 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 GADD45γ, and significantly augmented by the overexpression of GADD45γ. The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or CsA was reduced by silencing of GADD45γ, and was augmented by the overexpression of GADD45γ, indicating that caspase activation is dependent on the expression of GADD45γ. 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.ConclusionsTo our knowledge, this is the first study to show that GADD45γ is required for the caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.