Expression of prosalusin in human neuroblastoma cells

Salusins, which are derived from the prosalusin precursor molecule, regulate hemodynamics, mitogenesis and atherogenesis. The preprosalusin gene is ubiquitously expressed, while the salusin-beta peptide is present in systemic endocrine cells and the neuroendocrine system. However, the regulatory mechanisms for the preprosalusin gene and prosalusin expression remain to be investigated. Real-time quantitative RT-PCR and salusin-alpha radioimmunoassay revealed that the neuroblastoma cell line, SK-N-SH, exhibited marked upregulation of preprosalusin mRNA and salusin-alpha-like immunoreactivity (LI) when incubated under 2% serum condition. However, SK-N-SH cells released a limited amount of salusin-alpha-LI into the culture supernatant. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay after extraction of proteins from the conditioned media using an octyl-silica column did not reveal a component that co-eluted with authentic salusin-alpha. Western blotting of the nuclear extracts from SK-N-SH showed the expression of prosalusin and its cleaved fragments, but not authentic salusin-alpha. Addition of Jak-2 inhibitors to growing SK-N-SH cells cultured under 10% serum condition resulted in increased salusin-alpha-LI expression. Suppression of Jak-2 mRNA using siRNAs upregulated intracellular salusin-alpha-LI, as detected by immunofluorescence. In summary, the preprosalusin gene and prosalusin protein are expressed in a neuroblastoma cell line and upregulated by reduced serum. The Jak-2 pathway may be involved in the regulation of salusin expression. (C) 2009 Elsevier Inc. All rights reserved.