Pitx factors are involved in basal and hormone-regulated activity of the human prolactin promoter.
The pituitary-specific POU homeodomain factor Pit-1 likely interacts with other factors for cell-specific expression of prolactin. Here we identify the paired-like homeobox transcription factors Pitx1 and Pitx2 as factors functionally activating the proximal human prolactin promoter (hPRL-164luc). Using in vitro binding assays and a series of site-specific mutations of the proximal hPRL promoter, we mapped the B1 and B2 bicoid sites involved in Pitx-mediated transactivation of the hPRL-164luc construct. In somatolactotroph GH4C1 cells, basal proximal hPRL promoter activity was inhibited by a Pitx2 dominant-negative form in a dose-dependent manner, whereas binding disruptive mutations in the Pitx sites significantly reduced basal activity of the promoter. We also show that synergistic activation of hPRL-164luc by Pitx2 and Pit-1 requires the integrity of the B2 Pitx binding site, and at least one of the P1 and P2 Pit-1 response elements. In addition, mutation in the B2 Pitx site results in attenuation of the promoter's responsiveness to forskolin, thyrotropin-releasing hormone, and epidermal growth factor. Conversely, Pitx1 or Pitx2 overexpression in GH4C1 cells leads to an enhancement of the drugs stimulatory effects. Altogether, these results suggest that full responsiveness to several signaling pathways regulating the hPRL promoter requires the B2 Pitx binding site and that Pitx factors may be part of the proteic complex involved in these regulations. Finally, in situ hybridization analysis showing coexpression of the PRL and Pitx2 genes in rat and human lactotroph cells corroborates the physiological relevance of these results.
Quentien
Marie-Hélène
MH
Laboratoire ICNE, CNRS UMR 6544-Université de la Méditerranée, Marseille, France.
Manfroid
Isabelle
I
Moncet
Daniel
D
Gunz
Ginette
G
Muller
Marc
M
Grino
Michel
M
Enjalbert
Alain
A
Pellegrini
Isabelle
I
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D018398:Homeodomain Proteins / Q000502:physiology*; D006728:Hormones / Q000378:metabolism*; D006801:Humans; D017403:In Situ Hybridization; D008156:Luciferases / Q000378:metabolism; D009687:Nuclear Proteins; D051761:Paired Box Transcription Factors; D010902:Pituitary Gland / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism* / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism; D000097577:Homeobox Protein PITX2
D004247:DNA; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D006728:Hormones; D008156:Luciferases; D009687:Nuclear Proteins; D051761:Paired Box Transcription Factors; D011388:Prolactin; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D001616:beta-Galactosidase; D000097577:Homeobox Protein PITX2; C494187:POU1F1 protein, human; C494186:Pou1f1 protein, rat; C101485:homeobox protein PITX1