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Neuronal differentiation in SH‐SY5Y human neuroblastoma cells induces synthesis and secretion of tenascin and upregulation of α v integrin receptors
Author(s) -
Linnala Auli,
Lehto VeliPekka,
Virtanen Ismo
Publication year - 1997
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/(sici)1097-4547(19970701)49:1<53::aid-jnr6>3.0.co;2-c
Subject(s) - fibronectin , microbiology and biotechnology , neuroblastoma , immunocytochemistry , neurite , immunoprecipitation , secretion , laminin , cell culture , retinoic acid , biology , cellular differentiation , downregulation and upregulation , protein subunit , integrin , extracellular matrix , receptor , biochemistry , endocrinology , in vitro , gene , genetics
Human SH‐SY5Y neuroblastoma cells were induced to neuronal differentiation by using 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) and retinoic acid (RA). Both treatments rapidly induced long neurites and increased the content of neurofilaments as shown by immunocytochemistry and immunoblotting. Immunoprecipitation and immunoblotting of the culture medium with monoclonal antibodies demonstrated a rapid onset of synthesis and secretion of M r 280,000 tenascin (Tn) polypeptide with TPA and both M r 280,000 and 190,000 Tn polypeptides with RA and an increased secretion of extradomain A cellular fibronectin (EDA‐Fn) upon both treatments. Upon RA treatment both Tn polypeptides were also found in extracellular matrix preparations of the differentiated cells. A diffuse extracellular Tn immunoreactivity and a distinct cytoplasmic reaction were seen in differentiated cells especially after exposure to monensin to inhibit cellular secretion. Instead, immunoprecipitation experiments suggested that laminin was synthesized by the cells but was not upregulated upon differentiation. Experiments with purified Tn, used to coat the culture substratum, demonstrated that the undifferentiated cells were unable to adhere or spread on Tn but rapidly acquired the spreading capacity upon differentiation with the inducing agents. In immunofluorescence and immunoblotting the undifferentiated cells presented only a faint heterogenous reaction for β 1 integrin (Int) subunit, whereas cells exposed to RA presented a strong reaction for the Int α 1 and β 1 subunits, hence suggestive of Int α 1 β 1 , and for Int α v subunit. Cells exposed to TPA showed an enhanced immunoreaction for Int α 2 and β 1 subunits, suggestive of Int α 2 β 1 , and for Int α v subunit. Immunoreactivity for Int α v located to distinct punctate plaques in the differentiated cells after both inducing agents. The results suggest that Tn is produced by cultured neuronally differentiating cells, and it is accompanied by the acquitance of an adhesion receptor for Tn. J. Neurosci. Res. 49:53–63, 1997. © 1997 Wiley‐Liss Inc.