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.

Genome-wide expression analysis in a Fabry disease human podocyte cell line

“…This work has identified several genes, such as EBP, MAGEH1, PLEKHO1, SMG1 , and TIA1 , that are reported to be involved in apoptosis through various mechanisms. MAGEH1, a member of the MAGE gene family, is involved in causing caspase-dependent apoptosis of renal tubular cells through the use of nephrotoxic drugs [ 72 ]. Studies have shown that PLEKHO1, a member of the Pleckstrin homology domain-containing family, is involved in regulating apoptosis.…”

Identification of apoptosis-related key genes and the associated regulation mechanism in thoracic aortic aneurysm

“…GADD45G plays an essential regulatory role in DNA repair, cell cycle regulation, aging and genotoxic stress responses, and other cellular functions [ 24 ]. A large number of studies have shown that GADD45G is involved in the regulation of a variety of cell signaling pathways in tumor cells, and the significant reduction of their expression is closely related to tumor formation and progression [ 25 – 27 ]. The evaluation of the diagnostic potential of CASP6, AKT3, JUN, and DFFA measuring their AUC values revealed that they could discriminate TAAs and healthy samples with excellent specificity and sensitivity both in training and validation sets.…”

Overexpression of GADD45g inhibits proliferation and invasion of acute myeloid leukemia cell lines with FLT3-ITD+ by downregulating the JNK pathway

“…GADD45g gene, localized on chromosome 9q22.1-q22.2, is an evolutionarily conserved small molecule encoding an 18-kDa protein that plays a critical role in cell cycle regulation, apoptosis, and DNA damage repair 5,6 . Some studies have demonstrated that the expression of GADD45g is downregulated in breast cancer 7 , esophageal squamous carcinoma 8 , and nasopharyngeal carcinoma 9 , and the mechanism involves changes in promoter methylation or post-transcriptional modi cations 10 .…”

“…In addition, GADD45g overexpression signi cantly decreases tumor cell proliferation, methylation, or post-transcriptional modi cation 10 . Conversely, exogenous expression of GADD45g activates the JNK pathway, leading to apoptosis, which signi cantly inhibits tumor growth in vitro and in vivo 5 . Thus, GADD45g can be used as a potential prognostic indicator of AML due to its low expression and association with poor prognosis of patients 11 .…”