Calreticulin Regulates VEGF-A in Neuroblastoma Cells

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.

The family of 14‐3‐3 proteins and specifically 14‐3‐3σ are up‐regulated during the development of renal pathologies

“…Although the exact mechanism for this phenomenon is not known, we speculate that it may be mediated by the transcription factor HIF1α, since there is recent evidence from a neuroblastoma cell line that the HIF1α transcript is up‐regulated or down‐regulated following calreticulin up‐regulation or down‐regulation . We have confirmed this finding in our renal tubular epithelial cell line, where we have observed a 10‐fold increase in the HIF1α transcript when calreticulin is overexpressed (data not shown).…”

A sketch of known and novel MYCN-associated miRNA networks in neuroblastoma

“…Indeed, the expression levels of TGFBR2, SMAD2 and SMAD4, three important components along the TGF-β cascade, were found to be downregulated in NB cells (48). VEGF, Wnt, JAK-STAT and GnRH pathways were found among the targets of the underexpressed miRNAs in accordance with their growth-inhibitory potential on NB (49–51). Moreover, some biological processes linked to cell cycle, adherents junction and focal adhesion could have also been altered.…”

Identification of potential targets for differentiation in human leukemia cells induced by diallyl disulfide

“…Upregulated CRT expression may have a significant impact on cancer progression. Regulation of CRT levels has deep effects on cancer cell proliferation and angiogenesis as well as differentiation in neuroblastoma cells; the mechanism that CRT suppresses cell proliferation, and enhances cell differentiation is related to upregulating VeGF expression (31). Lu et al verified that CRT plays a critical role in the control of cell adhesion and migration via various mechanisms (32).…”