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L. Huang, M. A. Walter; The Protein Inhibitor of Activated Stat 3 (PIAS3) Interacts With the Forkhead Transcription Factor FOXC2 in HTM Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3076.
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Mutations in FOXC2 cause hereditary lymphedema with distichiasis (LD), a rare disease that affects the formation of the lymphatic vasculature system. Patient with LD also typically exhibit distichiasis, an extra row of eyelashes. FOXC2 missense mutations and nonsense mutations in the forkhead domain are associated with mild anterior segment defects. Moreover during eye development, Foxc2+/- mice display anterior segment malformations. This study aimed to identify novel FOXC2 interacting partners and the cellular pathways through which FOXC2 mutations lead to defect in ocular development.
A human trabecular meshwork (HTM) yeast two hybrid (Y2H) system was used to identify the interacting proteins of FOXC2. mRNA from a HTM primary cell line was used for cDNA synthesis. Full length FOXC2 was cloned into the pDEST32 vector containing the GAL4 DNA binding domain which was used as "bait" for the Y2H screen. Approximately 2x10e5 clones from the TM Y2H cDNA library were subjected to a preliminary screen with FOXC2.
Two putative positive colonies were isolated that presented interaction phenotypes and were able to activate the 3 reporter genes HIS3, URA3 and lacZ . These cDNA clones were recovered from yeast cells and sequenced. Comparison of the DNA sequence to the publicly available genome database revealed that both clones encode the Protein Inhibitor of Activated STAT 3 (PIAS3). Retransformation of these clones and the bait construct into yeast cells further validated the interaction in the Y2H system. Interactions between FOXC2 and PIAS3 are being confirmed in other experimental systems including co-immunoprecipitation, Ni-pull down assays, co-immunofluorenscence.
PIAS3 interacts with FOXC2 in human trabecular meshwork cells. As PIAS3 has been proven to function in cytokine signaling, including the STAT-, NF-ΚB- and SMAD-signaling pathways, investigation of the connection of FOXC2 to these cytokine signaling pathways and the ramification of this association in the human ocular development and disease, are underway.
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