abstract

• The Wnt-1 proto-oncogene encodes a member of a class of secreted glycoproteins which function in various developmental processes from Drosophila to humans. Although initial studies implicated Wnt-1 in mammary oncogenesis, the restriction of expression of Wnt-1 to developing mammalian brain as well as phenotypes of Wnt null mutants strongly suggest a role for Wnt-1 in midbrain development. To develop a mammalian cell culture assay for Wnt-1, Varmus and colleagues introduced the Wnt-1 cDNA into the rat pheochromocytoma cell line, PC12. PC12 cells, which arise from the neural crest lineage, demonstrate dramatic morphological and biochemical alterations in response to ectopic Wnt-1 expression.

  To understand the mechanism underlying the flat morphology induced by Wnt-1 in stably transfected PC12 cells, we initially surveyed the expression patterns of several growth regulatory genes, predominantly of the HLH class of neuroregulatory transcription factors, cyclins, and cyclin-dependent kinases. We observed a significant increase in the expression of the mRNA for the transcription factor HES-1 (an inhibitor of neuronal differentiation) and a decrease in expression of MASH-1 (an inducer of neuronal differentiation) mRNA and protein in Wnt-1/PC12 cells relative to control cells. We also observed a contraction in the G1 cell cycle population as well as cyclin D1 elevation in Wnt/PC12 cells relative to control cells. These changes in Wnt-1/PC12 cells were observed under conditions of prolonged Wnt-1 expression.

  HES-1 expression correlates directly with cell proliferation and inversely with neuronal differentiation. Understanding the induction of HES-1 expression in Wnt-1/PC12 cells may provide clues to Wnt-1 dependent regulation of the cell differentiation state. We therefore studied regulatory regions of the HES-1 promoter in order to determine the elements required for transcriptional activation in Wnt/PC12 cells. We have identified two such elements one of which binds Wnt-1-induced protein complexes in a sequence specific manner. Our results suggest a model in which Wnt-1 dependent alteration of the differentiation state is mediated by transcriptional regulation of growth regulatory HLH transcription factors and cyclin D1.