Expression of neurofilament proteins during retinoic acid-induced differentiation of P19 embryonal carcinoma cells.

Retinoic acid (RA) induces P19 embryonal carcinoma cells to differentiate into neurons with the extension of neuritic processes. We used the P19 cell as a model system to elucidate the regulation of neurofilament (NF) expression. Four mammalian NF proteins, NF-66 (alpha-internexin), peripherin, NF-L and NF-M, and the neural-specific, growth-associated gene, GAP-43, were studied during the RA treatment of P19 cells in vitro. As controls, untreated P19 cells were maintained in parallel. Indirect immunofluorescent staining showed that in RA-treated, morphologically differentiated P19 cells NF-66 was expressed in neuron-like cells characterized by phase bright cell bodies and long neuritic processes. At various times P19 cells were harvested for protein analysis by immunoblotting with antibodies to individual NF proteins or for total RNA extraction and Northern blotting with cDNA probes for NF-66, -L, -M, peripherin and GAP-43. During induction, both NF-66 and NF-L were expressed but in distinct patterns. NF-66 mRNA and protein were detected after 6 days of induction. In contrast, NF-L mRNA, but not protein, was expressed in both induced and control cells. Neither NF-M nor peripherin were expressed during induction. During differentiation of P19 cells, NF-66 mRNA levels rose markedly by the 1st day, reached a plateau between the 3rd-5th days and declined by the 7th day. NF-66 protein accumulation lagged slightly, reaching maximum abundance about the 5th day. The kinetics of NF-66 expression were similar to that of GAP-43. However, the pattern of NF-L expression was distinct from that of NF-66. NF-L mRNA, and some protein, was expressed in both RA-treated and control cells within 6 h after plating, but was down-regulated to baseline level thereafter in both populations. Neither NF-M or peripherin expression was detected during the differentiation. In summary, NF-66 was up-regulated most robustly among the four NF proteins during differentiation in P19 cells and was the major NF protein correlated with neurite extension.