Oligodendrocytes as glucocorticoids target cells: Functional analysis of the glycerol phosphate dehydrogenase gene

Previous research has established that the development and function of olig odendrocytes are influenced by glucocorticoids. The enzyme glycerol phospha te dehydrogenase (E.C.1.1.1.8) has been used as a model to study glucocorti coid regulation of gene expression in oligodendrocytes and the C6 glial cel l line. In the rat brain this enzyme is exclusively localized to oligodendr ocytes. The sequence of the 5' flanking region for the rat gene encoding Gl ycerol Phosphate Dehydrogenase (GPDH; EC 1.1.1.8) was determined. 4 kb of s equence from the 5' flanking region, exon 1, and part of intron I of the ra t GPDH gene was compared to the corresponding mouse sequence. Dotplot matri x comparison revealed that the rat sequence is more than 80% similar to the mouse sequence, but differs from the mouse sequence in two regions: the ra t sequence is devoid of 200 bp of B1 repeat sequence that is present in the mouse, and the rat sequence has an excess 700 bp of B2 repeat sequence ins erted between -0.7 kb and -1.4 kb that is absent in the mouse. To determine the regulatory activity of the rat GPDH 5' flanking region, various portio ns of the rat GPDH 5' flanking region were placed in luciferase reporter co nstructs and tested for transcriptional activity. Transient transfection of reporter constructs into the C6 glial cell line revealed that the distal e nd of the 5' flanking region was glucocorticoid-inducible. A 385 bp Glucoco rticoid Response Unit (GRU) was identified whose glucocorticoid induction w as enhanced by dibutyryl-cAMP and reduced by phorbol esters. Sequence analy sis of the GRU revealed the presence of four consensus GRE sequences and ot her putative consensus elements. Results here suggest that the 5' flanking region of the GPDH gene mediates the ligand-inducible regulation of GPDH, a nd that multiple signaling pathways converge at the 5' regulatory sequence to modulate GPDH gene expression in oligodendrocytes. (C) 2000 Wiley-Liss, Inc.