FGFR signaling is critical to development and disease pathogenesis, initiating phosphorylation-driven signaling cascades, notably the RAS-RAF-MEK-ERK and PI3 K-AKT cascades. PTEN antagonizes FGFR signaling by reducing AKT and ERK activation. Mouse lenses lacking FGFR2 exhibit microphakia and reduced ERK and AKT phosphorylation, widespread apoptosis, and defective lens fiber cell differentiation. In contrast, simultaneous deletion of both Fgfr2 and Pten restores ERK and AKT activation levels as well as lens size, cell survival and aspects of fiber cell differentiation; however, the molecular basis of this “rescue” remains undefined. We performed transcriptomic analysis by RNA sequencing of mouse lenses with conditional deletion of Fgfr2, Pten or both Fgfr2 and Pten, which reveal new molecular mechanisms that uncover how FGFR2 and PTEN signaling interact during development. The FGFR2-deficient lens transcriptome demonstrates overall loss of fiber cell identity with deregulated expression of 1448 genes. We find that ~ 60% of deregulated genes return to normal expression levels in lenses lacking both Fgfr2 and Pten. Further, application of customized filtering parameters to these RNA-seq data sets identified 68 high-priority candidate genes. Bioinformatics analyses showed that the cis-binding motif of a high-priority homeodomain transcription factor, NKX6-1, was present in the putative promoters of ~ 78% of these candidates. Finally, biochemical reporter assays demonstrate that NKX6-1 activated the expression of the high-priority candidate Rasgrp1, a RAS-activating protein. Together, these data define a novel regulatory module in which NKX6-1 directly activates Rasgrp1 expression to restore the balance of ERK and AKT activation, thus providing new insights into alternate regulation of FGFR downstream events.
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21EM15 cells were generously provided by Dr. John R. Reddan (Professor Emeritus, Department of Biological Sciences, Oakland University, Rochester, MI). RNA sequencing was completed at the Sequencing Core Laboratory at the University of Cincinnati. The raw and processes data files are deposited to NCBI GEO (accession number GSE132945). We acknowledge and thank the staff (Dr. Andor Kiss & Ms. Xiaoyun Deng) of the Center for Bioinformatics and Functional Genomics (CBFG) at Miami University for instrumentation and computational support.
This work was supported by an National Institute of Health (NIH) grant R01 EY012995 awarded to M.L.R. S.A.L. was supported by NIH grant R01 EY021505.
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All procedures were approved by the Miami University Institutional Animal Care and Use Committee and complied with the ARVO Statement for the Use of Animals in Research and consistent with those published by the Institute for Laboratory Animal Research (Guide for the Care and Use of Laboratory Animals).
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Padula, S.L., Anand, D., Hoang, T.V. et al. High-throughput transcriptome analysis reveals that the loss of Pten activates a novel NKX6-1/RASGRP1 regulatory module to rescue microphthalmia caused by Fgfr2-deficient lenses. Hum Genet 138, 1391–1407 (2019). https://doi.org/10.1007/s00439-019-02084-8