Cystatin C induces apoptosis and tyrosine hydroxylase gene expression through JNK-dependent pathway in neuronal cells

doi:10.1016/j.neulet.2011.03.091

Neuroscience Letters

Volume 496, Issue 2, 1 June 2011, Pages 100-105

https://doi.org/10.1016/j.neulet.2011.03.091 Get rights and content

Abstract

Cystatin C (CysC), an endogenous cysteine protease inhibitor, has been implicated in the apoptosis and differentiation processes of neuronal cells. In this study, we have investigated the pathway involved in the process. A human neuronal hybridoma cell line (A1 cell) was treated with CysC in both undifferentiated and retinoic acid (RA)-induced differentiated conditions, which decreased overall process length in both conditions. Also, CysC increased apoptotic cell number time-dependently, as revealed by TUNEL assay. Western blot analysis demonstrated that in differentiated A1 cells, CysC treatment decreased Bcl-2 and increased active caspase-9 protein level time-dependently. Immunocytochemistry results revealed that, CysC treatment significantly increased active form of Bax expressing cell number, which co-localized with mitochondria. Mitogen activated protein (MAP) kinase inhibition experiments showed that Bax mRNA induction and Bcl-2 mRNA inhibition by CysC treatment were c-Jun N-terminal kinase (JNK)-dependent. After RA-induced differentiation, choline acetyltransferase (ChAT) and neurofilament (NF) mRNA levels were increased in A1 cells. CysC treatment inhibited NF mRNA level in both undifferentiated and RA-differentiated, and increased TH mRNA in differentiated A1 neurons. Analysis of signal transduction pathway demonstrated that TH gene induction was also JNK-dependent. Thus, our results demonstrated the significance of JNK-dependent pathways on CysC-induced apoptosis and TH gene expression in neuronal cells, which might be an important target in the management of CysC dependent neurodegenerative processes.

Highlights

► CysC treatment increased the protein level of cleaved active caspase 9. ► CysC-induced Bax mRNA level was decreased by JNK inhibitor. ► CysC increased active form of Bax expressing cell number, which co-localized with mitochondria. ► CysC treatment increased TH mRNA expression. ► JNK inhibitor inhibited the effects of CysC induced TH mRNA expression.

Section snippets

Acknowledgment

This work was supported by Grants from Society for Catecholamines and Neurologic Diseases (AN).

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Recent reports suggest that functions of the Cystatin C are not limited regulating proinflammation cytokines expression, numerous studies have demonstrated the crucial regulation roles of the Cystatin C in apoptosis, such as human colorectal adenocarcinoma HCT116, human esophageal carcinomas EC9706 cells, and rat CNS neurons (Xu et al., 2015; Mori et al., 2016; Yan et al., 2015). Cystatin C has different specific regulation mechanisms among cells in apoptosis, including down-regulate cysteine proteinases, JNK-dependent pathways, Mitochondria and granzyme induced pathway (Boya et al., 2003; Guicciardi et al., 2000; Mullbacher et al., 1999; Liang et al., 2011). Previous investigation reported that SGIV induce apoptosis upon SGIV infection of FHM cells (Huang et al., 2011b).
Cystatin C is an endogenous inhibitor of cysteine proteases and widely exist in organisms. Several studies in mammals have showed that Cystatin C plays critical role in the immune defense against microorganisms. It is also well known that some fish Cystatin C have important immune regulation functions in inflammatory responses. However, the function of fish Cystatin C in virus infection as well as its underlying molecular mechanisms remain to be elucidated. In the present study, a Cystatin C gene termed Ec-CysC was identified from orange-spotted grouper, Epinephelus coioides. The full-length of Ec-CysC cDNA was 817 bp with a 387 bp open reading frame (ORF) that encoded a 129-amino acid (aa) protein, including 18-aa signal peptide and 111-aa mature polypeptide. The deduced amino acid of Ec-CysC shared three conserved domains containing Glycine at the N-terminus region, QVVAG motif in the middle and PW motif near the C-terminus region. Transcription analysis of the Ec-CysC gene showed its expression in all twelve examined tissues including liver, spleen, kidney, brain, intestine, heart, skin, muscle, fin, stomach, gill and head kidney. Its expression following stimulation with Singapore grouper iridovirus (SGIV) was further tested in spleen, the relative expression of Ec-CysC was significantly up-regulated at 12 h post-infection. The subcellular localization experiment revealed that Ec-CysC was mainly distributed in the cytoplasm in Grouper Spleen (GS) cells. In vitro, Overexpression of Ec-CysC in GS cells significantly reduced the expression of viral genes, namely, ORF162, ORF049 and ORF072. Meanwhile, we found that overexpression of Ec-CysC resulted in upward trend of expression of inflammatory cytokines TNF-a, IL-1β and IL8 during SGIV infection. Further, SGIV-inducible apoptosis and Caspase-3 activity were also weakened by overexpression Ec-CysC in fathead minnow (FHM) cells. These results indicated that Ec-CysC might have a deeper involvement in fish immune defense, and played important roles in inflammation and apoptosis induced by SGIV.
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Cystatin C induces apoptosis and tyrosine hydroxylase gene expression through JNK-dependent pathway in neuronal cells

Abstract

Highlights

Section snippets

Acknowledgment

J. Biol. Chem.

J. Biol. Chem.

Brain Res.

Neurobiol. Dis.

Clin. Chim. Acta

Brain Res.

J. Mol. Cell. Cardiol.

Brain Res.

Neurobiol. Dis.

Neuron

Neurobiol. Dis.