Abstract
The nerve growth factor (NGF)/TrkA-signaling is necessary for neural development, and its abnormality has been tightly associated with the tumorigenesis of various cancers originated from the nervous system. The characterization of key molecules involved in the NGF/TrkA-mediated neuronal differentiation could pave the way for the development of novel therapeutic strategies against neural malignancy. We have previously demonstrated that calreticulin (CRT) is a favorable prognostic factor highly expressed in primary neuroblastomas (NBs) with a more differentiated histology. In the present study, we found that the level of CRT was enhanced in NGF-stimulated differentiation of PC-12 cells through the extracellular signal-regulated kinase (ERK)-dependent mitogen-activated protein kinase (MAPK) pathway. A deficiency of CRT significantly decreased NGF-elicited neuronal differentiation. Furthermore, overexpression of CRT enhanced neuronal differentiation via simultaneously activating the ERK-dependent MAPK pathway. The Ca2+-regulating capacity of CRT was demonstrated to be indispensable for NGF-elicited neuronal differentiation. Intriguingly, the expression levels of CRT and NGF receptor TrkA were highly correlated in NBs with differentiated histology, and the coexistence of CRT and TrkA in NB tumors synergistically predicted a better 5-year survival rate. Together, our present findings delineate a CRT-dependent regulation of NGF-induced neuronal differentiation.
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Acknowledgements
We thank the Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica, for technical support. The shRNA lentiviral vectors were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, supported by the National Research Program for Genomic Medicine Grants of National Science Council, Taiwan (NSC 97-3112-B-001-016). This study was supported by the National Health Research Institutes (NHRI-EX96-9620NI to W.-M.H.), National Science Council, Taiwan (NSC 95-2314-B-002-155-MY2 to W.-M.H.), and Academia Sinica (to Y.-F.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Fig. S1
The effects of the ERK MAPK pathway on the expression of CRT and GAP43 in the absence of NGF. a PC-12 cells were treated with vehicle alone (0.1% DMSO), 20 μM U0126, or 20 μM PD98059 for 24 h. Cells were lysed and subjected to SDS–PAGE and Western blot analysis with the indicated antibodies. The levels of p-ERK, t-ERK, GAP43, CRT, and GAPDH (protein loading control) were quantitated by densitomtry. The relative levels of p-ERK/t-ERK, CRT (CRT versus GAPDH), and GAP43 (GAP43 versus GAPDH) were determined and compared with those of DMSO-treated cells that are referred to as 1-fold relative expression. *p < 0.05, compared to the DMSO-treated control. b PC-12 cells were transfected with the wild-type (WT) MEK1, a constitutively active MEK1 (MEK1-EE), a dominant-negative MEK1 (MEK-AA), or an empty vector (Mock) for 48 h. The expression levels of CRT were determined by SDS–PAGE and immunoblotting. The relative levels of p-ERK/t-ERK and CRT (CRT versus GAPDH) were determined and compared with those of Mock-transfected cells that are referred to as 1-fold relative expression. Data are shown as the mean (± SEM) of at least three independent experiments. *p < 0.05, compared to the mock-transfected control. The arrowhead indicates the endogenous expression of MEK1. c Cells transiently transfected with siRNA targeting ERK1/2 or scrambled sequences (NS) for 24 h. The relative levels of t-ERK (t-ERK versus GAPDH) and CRT (CRT versus GAPDH) were determined and compared with those of NS siRNA-transfected cells that are referred to as 1-fold relative expression. Results are presented as the mean (± SEM) of three independent experiments. *p < 0.05, compared to nonspecific siRNA (NS)-transfected control (DOCX 361 kb)
Fig. S2
The expression of GAP43 in PC-12 cells is not affected by cytosolic concentration of calcium in the absence of NGF. PC-12 cells were treated with 0.1% DMSO (control), BAPTA-AM (20 μM), or ionomycin (1 μM) for 24 h. Clarified lysates with equivalent amounts of proteins were analyzed by Western blotting. Normalized level of GAP43 in controls was referred to as 1-fold of relative expression. Data are shown as the mean (± SEM) of at least three independent experiments (DOC 113 kb)
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Shih, YY., Nakagawara, A., Lee, H. et al. Calreticulin Mediates Nerve Growth Factor-Induced Neuronal Differentiation. J Mol Neurosci 47, 571–581 (2012). https://doi.org/10.1007/s12031-011-9683-3
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DOI: https://doi.org/10.1007/s12031-011-9683-3