THE LIM HOMEOBOX PROTEIN MLIM3 LHX3 INDUCES EXPRESSION OF THE PROLACTIN GENE BY A PIT-1/GHF-1-INDEPENDENT PATHWAY IN CORTICOTROPH ATT20 CELLS/

mLIM3, a member of the LIM homeobox family, was recently demonstrated to be critical for proliferation and differentiation of the pituitary cell Lineage, Using a pool of degenerate oligonucleotides we determine d the DNA sequence ANNAGGAAA(T/C)GA(C/G)AA as the set preferentially r ecognized by mLIM3. A nearly identical sequence is found in the prolac tin (PRL) promoter, within a 15-mer stretch from nucleotides (nts) -21 8 to -204 which is highly conserved between human, rat, and bovine, In order to test the hypothesis of a transcriptional effect of mLIM3 on the prolactin promoter, stable transfectants of mLIM3 cDNA in AtT20 tu rner cells revealed that PRL mRNA expression was induced in 3 separate stable clones. Gel retardation experiments performed using nuclear ex tracts isolated from one of the AtT20/mLIM3 stable transfectants revea led affinity towards the 15-mer element of the PRL promoter. From thes e results, we propose that the PRL promoter element (nts -218 to -204) could be functional in vivo. Finally, me demonstrate that in AtT20 ce lls prolactin mRNA expression is not induced by the Pit-1/GHF-1 pathwa y and that growth hormone mRNA is not detected concomitantly with prol actin, We conclude that mLIM3 may play a key role in inducing PRL gene expression in lactotrophs by binding to a conserved motif close to a Pit-1/GHF-1 site within the proximal PRL promoter. (C) 1998 Federation of European Biochemical Societies.