EXPRESSION OF NEUROFILAMENT PROTEINS DURING RETINOIC ACID-INDUCED DIFFERENTIATION OF P19 EMBRYONAL CARCINOMA-CELLS

Retinoic acid (RA) induces P19 embryonal carcinoma cells to differenti ate into neurons with the extension of neuritic processes. We used the P19 cell as a model system to elucidate the regulation of neurofilame nt (NF) expression. Four mammalian NF proteins, NF-66 (alpha-internexi n), peripherin, NF-L and NF-M, and the neural-specific, growth-associa ted 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, morphol ogicaIly differentiated P19 cells NF-66 was expressed in neuron-like c ells characterized by phase bright cell bodies and long neuritic proce sses. At various times P19 cells were harvested for protein analysis b y immunoblotting with antibodies to individual NF proteins or for tota l 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 detec ted 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 per ipherin were expressed during induction. During differentiation of P19 cells, NF-66 mRNA levels rose markedly by the Ist day, reached a plat eau between the 3rd-5th days and declined by the 7th day. NF-66 protei n accumulation lagged slightly, reaching maximum abundance about the 5 th day, The kinetics of NF-66 expression were similar to that of GAP-4 3. However, the pattern of NF-L expression was distinct from that of N F-66. NF-L mRNA, and some protein, was expressed in both RA-treated an d control cells within 6 h after plating, but was down-regulated to ba seline level thereafter in both populations. Neither NF-M or peripheri n expression was detected during the differentiation, In summary, NF-6 6 was up-regulated most robustly among the four NF proteins during dif ferentiation in P19 cells and was the major NF protein correlated with neurite extension.