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Camk2b protects neurons from homocysteine-induced apoptosis with the involvement of HIF-1? signal pathway.

ABSTRACT: In our previous study using iTRAQ technique we found that the level of calmodulin-dependent protein kinase 2b (Camk2b) was lower in rats with hyperhomocysteinemia. We presumed that Camk2b might be involved in homocysteine-induced apoptosis and tried to explore its role in this study through the transfection with Camk2b gene. Results showed that neurons of HHcy group had lower activity measured by MTT, higher percentage of apoptotic neurons, lower expression levels of Camk2b mRNA and protein than those in normal group. Neurons with overexpression of Camk2b (Camk2b group) had lower percentage of apoptosis and higher activity than those in control group. After exposure to 2-Methoxyestradiol, the activity of neurons with overexpression of Camk2b was suppressed with more apoptotic cells observed. The expressions of BCL2, eNOS, EP300 and EPO were all elevated at both mRNA and protein levels in neurons of CamK2b group compared with other three groups. Thus, Camk2b protects neurons from Homocysteine-induced apoptosis with the involvement of HIF-1? signal pathway.

SUBMITTER: Fang M

PROVIDER: S-EPMC4132126 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Camk2b protects neurons from homocysteine-induced apoptosis with the involvement of HIF-1α signal pathway.

Fang Min M Feng Chao C Zhao Yan-Xin YX Liu Xue-Yuan XY

International journal of clinical and experimental medicine 20140715 7

In our previous study using iTRAQ technique we found that the level of calmodulin-dependent protein kinase 2b (Camk2b) was lower in rats with hyperhomocysteinemia. We presumed that Camk2b might be involved in homocysteine-induced apoptosis and tried to explore its role in this study through the transfection with Camk2b gene. Results showed that neurons of HHcy group had lower activity measured by MTT, higher percentage of apoptotic neurons, lower expression levels of Camk2b mRNA and protein than ...[more]

PMID: 25126162

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Project description:BackgroundNickel nanoparticles (Nano-Ni) are increasingly used in industry and biomedicine with the development of nanotechnology. However, the genotoxic and carcinogenic effects of Nano-Ni and the underlying mechanisms are still unclear.MethodsAt first, dose-response (0, 10, 20, and 30 μg/mL) and time-response (0, 3, 6, 12, and 24 h) studies were performed in immortalized normal human bronchial epithelial cells BEAS-2B to observe the effects of Nano-Ni on DNA damage response (DDR)-associated proteins and the HIF-1α/miR-210/Rad52 pathway by real-time PCR or Western blot. Then, a Hsp90 inhibitor (1 µM of 17-AAG, an indirect HIF-1α inhibitor), HIF-1α knock-out (KO) cells, and a miR-210 inhibitor (20 nM) were used to determine whether Nano-Ni-induced Rad52 down-regulation was through HIF-1α nuclear accumulation and miR-210 up-regulation. In the long-term experiments, cells were treated with 0.25 and 0.5 µg/mL of Nano-Ni for 21 cycles (~ 150 days), and the level of anchorage-independent growth was determined by plating the cells in soft agar. Transduction of lentiviral particles containing human Rad52 ORF into BEAS-2B cells was used to observe the role of Rad52 in Nano-Ni-induced cell transformation. Nano-Ni-induced DNA damage and dysregulation of HIF-1α/miR-210/Rad52 pathway were also investigated in vivo by intratracheal instillation of 50 µg per mouse of Nano-Ni. gpt delta transgenic mice were used to analyze mutant frequency and mutation spectrum in mouse lungs after Nano-Ni exposure.ResultsNano-Ni exposure caused DNA damage at both in vitro and in vivo settings, which was reflected by increased phosphorylation of DDR-associated proteins such as ATM at Ser1981, p53 at Ser15, and H2AX. Nano-Ni exposure also induced HIF-1α nuclear accumulation, miR-210 up-regulation, and down-regulation of homologous recombination repair (HRR) gene Rad52. Inhibition of or knocking-out HIF-1α or miR-210 ameliorated Nano-Ni-induced Rad52 down-regulation. Long-term low-dose Nano-Ni exposure led to cell malignant transformation, and augmentation of Rad52 expression significantly reduced Nano-Ni-induced cell transformation. In addition, increased immunostaining of cell proliferation markers, Ki-67 and PCNA, was observed in bronchiolar epithelial cells and hyperplastic pneumocytes in mouse lungs at day 7 and day 42 after Nano-Ni exposure. Finally, using gpt delta transgenic mice revealed that Nano-Ni exposure did not cause increased gpt mutant frequency and certain DNA mutations, such as base substitution and small base insertions/deletions, are not the main types of Nano-Ni-induced DNA damage.ConclusionsThis study unraveled the mechanisms underlying Nano-Ni-induced cell malignant transformation; the combined effects of Nano-Ni-induced DNA damage and DNA repair defects through HIF-1α/miR-210/Rad52 pathway likely contribute to Nano-Ni-induced genomic instability and ultimately cell transformation. Our findings will provide information to further elucidate the molecular mechanisms of Nano-Ni-induced genotoxicity and carcinogenicity.

Dataset Information

Camk2b protects neurons from homocysteine-induced apoptosis with the involvement of HIF-1? signal pathway.

Publications

Camk2b protects neurons from homocysteine-induced apoptosis with the involvement of HIF-1α signal pathway.

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