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Dylan Scott Audette, Deepti Anand, Tammy So, Troy Rubenstein, Frank J Lovicu, Salil Anil Lachke, Melinda K Duncan; Prox1 and fibroblast growth factor receptors function in a novel regulatory loop to control lens fiber differentiation and gene expression. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4836.
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© ARVO (1962-2015); The Authors (2016-present)
Lens epithelial cells differentiate into lens fibers in response to FGF and other growth factors during development and throughout life. Lens fiber differentiation also requires the action of several transcription factors including Prox1. However, the mechanisms of cross-talk between FGF receptor (FGFr) signaling and transcription factor function in the lens are unclear.
RNAseq was performed to identify differentially expressed genes (DEGs) in lenses from mice lacking Prox1 due to Cre/Lox mediated deletion. Key DEGs were validated by qRT-PCR and direct Prox1 promoter interactions were assayed by chromatin immunoprecipitation (ChIP). Primary chicken lens cells were transfected to determine whether Prox1 expression was sufficient to increase expression of targets. LEC to LFC differentiation was induced in rat lens epithelial cells by the addition of FGF, and the effect of FGF on Prox1 expression was assessed.
Deletion of Prox1 from the lens resulted in a failure in fiber cell morphogenesis and was associated with the downregulation of crystallins and lens fiber membrane proteins, including essentially every known marker of lens fiber cell differentiation. Notably, the downregulated DEGs included three FGFrs and ChIP indicated that the promoters of these genes support direct binding of Prox1 in the lens. Notably, two of the differentially expressed FGFrs had not been previously studied in the lens. LCTL is a klotho adapter protein that is required for the interaction of endocrine FGFs with FGFrs, and FGFRL-1 is an atypical FGFr that can signal through the MAPK pathway in both a ligand independent and dependent manner. We find that FGFr signaling resulted in the upregulation of nuclear Prox1 in lens epithelial cells, an effect blocked by inhibition of the MAPK pathway.
These data support the hypothesis that lens epithelial to fiber differentiation initiates when FGFr signaling via basally expressed FGFRs results in an increase in Prox1 expression. Prox1 then transcriptionally upregulates the expression of FGFRs, which in turn further upregulate Prox1 expression. This feed-forward loop ramps up both Prox1 and FGFr expression which allows for the induction of lens fiber cell elongation and the upregulated expression of nearly all genes presently recognized to be important for lens fiber cell function.
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