Cyclic AMP regulates NCAM expression and phosphorylation in cultured mouse astrocytes

The neural cell adhesion molecule (NCAM) plays a key morphoregulatory role during neural development. Patterns of NCAM expression were investigated in cultured astrocytes exhibiting distinct states of morphological differentiation. In vitro differentiation of confluent primary cultures of astrocytes was enhanced by a long-term (7 days) treatment with dibutyryl cyclic AMP (dBcAMP). The diversity of NCAM mRNAs arising from alternative splicing of a single primary transcript was analyzed by Northern blotting. Synthetic DNA-oligonucleotide probes, designed to recognize selected exons (exons 7, 15, and VASE) or exon combinations (exons 7/8, exons 12/13, and exons 12/a/AAG/13), revealed NCAM mRNA classes of 6.7, 5.2, and 2.9 kilobases (kb) in both control and dBcAMP-treated astrocytes. Although long-term treatment of astrocytes with dBcAMP did not change the qualitative pattern of NCAM transcripts, the relative abundance of individual mRNA species appeared to be altered: in morphologically differentiated astrocytes, a general elevation of NCAM mRNA as well as preferential accumulation of the 6.7 kb message were found. The quantitative rise within this size class may primarily be accounted for by a dramatic upregulation of exon VASE-containing NCAM transcripts. In dBcAMP-treated astrocytes, immunoblot analysis revealed an unexpected expression of the 190 kDa NCAM-A isoform as well as an intense staining of bands corresponding to NCAM-B (135 kDa) and NCAM-C (115 kDa), as compared to the control pattern of untreated astrocytes. Furthermore, NCAM-A and NCAM-B were shown to be phosphorylated in astrocytes subjected to long-term treatment with dBcAMP, whereas no incorporation of [32P]phosphate into NCAM was demonstrated in untreated astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)