Differential expression of the heavy-chain ferritin gene in non-adhered and adhered oligodendrocytes.
Oligodendrocytes (OLGs) synthesize and maintain central nervous system myelin. Little is known about the molecules and pathways involved in signaling the commencement of myelination; yet myelination is spatially and temporally controlled. We are interested in deciphering the signaling events that control the on and off switch of myelination. To address these questions we are using an in vitro model system consisting of pure cultures of OLGs, isolated from postmyelination brains. We have shown that in vitro, these OLGs regenerate and reenact the ontogenic development of myelin upon adhesion to a substratum; we have also shown that when transplanted in vivo, they can form myelin. Hence, identifying the genes that are turned on upon OLG adhesion seemed a worthwhile approach to singling out those genes that are critical to the differentiation process. For this we adopted a novel technique-differential display-that permits the comparison of gene expression under two sets of conditions; in our case between non-adhered and adhered OLGs. Using the differential display method in conjunction with a set of five arbitrary primers, we have identified five cDNAs that are activated upon OLG substratum adhesion. Four of these cDNAs proved to be fragments of the heavy (H)-chain ferritin cDNA. H-chain ferritin is involved in the rapid sequestration and delivery of iron; it is also a cytoprotectant. Brain iron is localized predominantly in OLGs. Our finding that the expression of H-chain ferritin is upregulated upon OLG regeneration/differentiation agrees with reports, in other cell types, that H-chain ferritin transcription is modulated by factors that control cell growth and differentiation. The enhanced transcription results in a twofold augmentation in ferritin synthesis. This is the first demonstration of an adherence-mediated activation of the H-chain ferritin gene. The observation has interesting implications. The transcription of the H-chain ferritin gene is associated with tissue stress such as might occur during rapid cell growth. The fact that this transcription takes place upon OLG-substratum adhesion can be viewed as another manifestation of the anchorage-induced signal that drives OLGs toward a differentiation program. This entails the synthesis of all the necessary components for the assembly of large quantities of membranes, hence constituting a period of rapid growth and considerable cell stress. We have sequenced the full-length ferritin cDNA of ovine OLGs; the cDNA is 775 bp long. The coding sequence starts at residue 57 with the ATG codon and terminates at residue 602, with the stop codon TAA. The cDNA codes for 181 amino acids with a predicted size for the protein of 22 kD. The deduced amino acid sequence is 93% identical to human ferritin. We postulate that OLG H-chain ferritin may function at more than one level: 1) it provides the iron required to sustain an oxidative metabolism; and 2) it acts as a cytoprotectant against oxidant-mediated injury.
Department of Neurology, University of Chicago, IL 60637, USA.
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Sanyal
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Polak
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Szuchet
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002448:Cell Adhesion / Q000502:physiology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D005293:Ferritins / Q000096:biosynthesis* / Q000235:genetics*; D005784:Gene Amplification; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009836:Oligodendroglia / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012756:Sheep; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005293:Ferritins