Increased ferritin gene expression is both promoted by cAMP and a marker of growth arrest in rabbit vascular smooth muscle cells.

To facilitate an understanding of the molecular events associated with vascular smooth muscle cells (SMC) growth arrest and differentiation, we have isolated a number of cDNAs encoding mRNAs that are more abundantly expressed in high density growth-arrested SMC. From 40,000 recombinant plaques, we identified 6 cDNA clones which encoded genes more highly expressed in density-arrested SMC. DNA sequence analysis of a cDNA clone which hybridized to a 5.5-kilobase mRNA revealed a 96% sequence homology to the carboxyl-terminal propeptide region of the human alpha 2(I) collagen gene. Sequence analysis of 3 other cDNA clones which all recognized a 1-kilobase mRNA, indicated that they encode the ferritin H-chain subunit gene. The large increase in ferritin H-chain mRNA expression was both growth arrest and high density-dependent, but did not appear to be cell-cell contact-dependent. The increase in H-chain was partially accounted for by an increase in the transcription of this gene, but a post-transcriptional mechanism likely accounts for the majority of the observed difference in mRNA level. A greater than 3-fold higher expression of ferritin H-chain mRNA was also observed in BC3H1s (a nonfusing myogenic cell line) during the process of differentiation, but no elevation in ferritin H-chain mRNA was associated with density-dependent growth arrest in fibroblasts. Hemin (an intracellular iron donor) induced an elevation in ferritin H-chain mRNA expression in both preconfluent and post-confluent SMC, but was unable to elevate the preconfluent mRNA level to an untreated postconfluent level. In addition, deferoxamine (an intracellular iron chelator) was unable to reduce the ferritin mRNA level in postconfluent SMC. These results suggest that high density growth arrest-mediated elevation in ferritin H-chain occurs by a mechanism distinct from that induced by an increase in intracellular iron. cAMP was also a powerful modulator of the expression of ferritin H-chain gene since cells treated with forskolin or dibutyryl cAMP had a 5-7-fold higher level of ferritin H-chain mRNA. We propose that the increase in the ferritin H-chain mRNA level in postconfluent SMC may occur through a cAMP-dependent pathway and may be associated with an enhanced differentiated phenotype.