PROP1 Directs Pituitary Differentiation through Protein Interaction and for Regulation of Gene Expression Involved in an EMT-Like Process

MERCOGLIANO, MARÍA FLORENCIA; PÉREZ MILLÁN, MARÍA INÉS; DE PAUL, ANA; MARTÍ, MARCELO

New Orleans

Congreso; ENDO 2019 Annual Meeting and Expo; 2019

Endocrine Society

Multiple factors participate in the differentiation process of the pituitary. In particular, PROP1 is a member of the paired-like family of homeodomain transcription factors, is expressed early in Rathke?s pouch and is key in the developing pituitary. In humans, PROP1 mutations are the leading causes of Combined Pituitary Hormone Deficiency (CPHD). We previously have determined that Prop1 is essential for stimulating stem cells to undergo an epithelial to mesenchymal transition-like (EMT) process, but it is not well understood how Prop1 regulates this process. In this work, we explored the role of Prop1 in pituitary differentiation, and the proteins with which interacts to complete its functions. To this end, we genetically engineered the GHFT1 mouse pituitary cell line to express biotin-tagged Prop1. We used this cell line and its control counterpart, that does not express Prop1, to identify PROP1-mediated changes in gene expression by RNA-seq and Prop1-interacting proteins by mass spectrometry. Gene expression profiling revealed that Prop1 upregulates genes involved in the migration process, either dispersion or adhesion, and genes implicated in degrading extracellular matrix proteins, i.e. matrix metalloproteinases (MMP2, MMP15 and MMP19). On the other hand, Prop1 downregulates tissue inhibitor of metalloproteinases like TIMP-3. Specifically, MMPs play a crucial role in embryonic development and have been implicated in EMT in the morphogenesis of many tissues. We examined MMP2 protein expression by immunofluorescence during pituitary development in control and Prop1df/df mice. At e12.5, e14.5 and e16.5, MMP2 is normally expressed in the pituitary gland in normal mice and it is almost absent in Prop1 mutant pituitaries. Furthermore, we observed an increased in cell-cell junctions by TEM in P1 pituitaries from Prop1df/df mice. Using immunoprecipitation coupled to mass spectrometry, we identified 93 proteins that interact specifically with Prop1. These proteins were tested for GO biological process enrichment and two predominant terms were determined: cell-cell adherents? junction and cell-cell adhesion. Several genes from these groups were previously described for their involvement in migration and EMT. Said genes fail in Prop1 mutants.Altogether, these results unveil the different actors in the Prop1 network, either by direct interaction with the transcription factor or by regulation of target gene expression. This knowledge could pinpoint candidate genes that could explain cases of hypopituitarism with unknown etiology and thereby, yield an improved diagnosis. And as a future perspective it could lead to the development of a targeted therapy that could overcome the disease.