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Calreticulin Regulates VEGF-A in Neuroblastoma Cells

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Abstract

Calreticulin (CRT) has been previously correlated with the differentiation of neuroblastoma (NB), implying a favorable prognostic factor. Vascular endothelial growth factor (VEGF) has been reported to participate in the behavior of NB. This study investigated the association of CRT and VEGF-A in NB cells. The expressions of VEGF-A and HIF-1α, with overexpression or knockdown of CRT, were measured in three NB cells (SH-SY5Y, SK-N-DZ, and stNB-V1). An inducible CRT NB cell line and knockdown CRT stable cell lines were also established. The impacts of CRT overexpression on NB cell apoptosis, proliferation, and differentiation were also evaluated. We further examined the role of VEGF-A in the NB cell differentiation via VEGF receptor blockade. Constitutive overexpression of CRT led to NB cell differentiation without proliferation. Thus, an inducible CRT stNB-V1 cell line was generated by a tetracycline-regulated gene system. CRT overexpression increased VEGF-A and HIF-1α messenger RNA (mRNA) expressions in SH-SY5Y, SK-N-DZ, and stNB-V1 cells. CRT overexpression also enhanced VEGF-A protein expression and secretion level in conditioned media in different NB cell lines. Knockdown of CRT decreased VEGF-A and HIF-1α mRNA expressions and lowered VEGF-A protein expression and secretion level in conditioned media in different NB cell lines. We further demonstrated that NB cell apoptosis was not affected by CRT overexpression in stNB-V1 cells. Nevertheless, overexpression of CRT suppressed cell proliferation and enhanced cell differentiation in stNB-V1 cells, whereas blockage of VEGFR-1 markedly suppressed the expression of neuron-specific markers including GAP43, NSE2, and NFH, as well as TrkA, a molecular marker indicative of NB cell differentiation. Our findings suggest that VEGF-A is involved in CRT-related neuronal differentiation in NB. Our work may provide important information for developing a new therapeutic strategy to improve the outcome of NB patients.

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Acknowledgments

The authors thank the financial support of the Cutting-Edge Steering Research Project of the National Taiwan University (grant NTU-CESRP-102R76263A) and the National Health Research Institutes of Taiwan (grant NHRI-EX101-10130BI (to H.L.)).

Conflict of Interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Pediatrics, College of Medicine, National Taiwan University Hospital and National Taiwan University, Taipei, Taiwan

    Wen-Chin Weng & Wang-Tso Lee

  2. Department of Life Science, National Taiwan University, Taipei, Taiwan

    Wen-Chin Weng, Kuan-Hung Lin, Pei-Yi Wu, Yi-Chien Lu, Yi-Cheng Weng & Hsinyu Lee

  3. Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan

    Bo-Jeng Wang & Yung-Feng Liao

  4. Department of Surgery, College of Medicine, National Taiwan University Hospital and National Taiwan University, Taipei, Taiwan

    Wen-Ming Hsu

  5. Department of Electrical Engineering, National Taiwan University, Taipei, Taiwan

    Hsinyu Lee

  6. Angiogenesis Research Center, National Taiwan University, Taipei, Taiwan

    Hsinyu Lee

  7. Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan

    Hsinyu Lee

  8. Center for Biotechnology, National Taiwan University, Taipei, Taiwan

    Hsinyu Lee

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  1. Wen-Chin Weng
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  2. Kuan-Hung Lin
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  10. Hsinyu Lee
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Correspondence to Hsinyu Lee.

Additional information

Drs. Wen-Chin Weng and Kuan-Hung Lin contributed equally to this work.

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Weng, WC., Lin, KH., Wu, PY. et al. Calreticulin Regulates VEGF-A in Neuroblastoma Cells. Mol Neurobiol 52, 758–770 (2015). https://doi.org/10.1007/s12035-014-8901-8

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