Epithelial differentiation has been widely studied, and the process can be divided into two general steps: generation of proliferating progenitors from resident stem cells, and the subsequent arrest and differentiation of these progenitors. Oscillation of the transcription factor TCF3 between activator and repressor is key to both maintaining a resident stem cell population and generating pulses of progenitor cells for these stem cells. These progenitors proliferate to fill the tissue field, and then the Notch pathway triggers their cell cycle arrest and differentiation. Accordingly when Notch1 is mutated, progenitors in the cornea, meibomian gland and hair follicles fail to differentiate and they proliferate beyond the tissue fields leading to pathologic loss of function. The timing of Notch1 signal initiation in proliferating progenitors is critical to define the precise number of cells and thus tissue topology. Insight into to how Notch1 signaling is initiated in this process is only now emerging. Epithelial tissue fields are established by cell-cell contact inhibition as proliferating progenitors come in contact. Mutations in cell contact inhibition signaling lead to progenitor outgrowth beyond normal tissue fields (as with Notch1 mutation), loss of tissue function and ultimately cancer. But, how might onset of Notch1 signaling be linked to such cell-cell contact? Cadherin-initiated junctional complexes not only mediate apical polarity in epithelial cells, they also initiate a kinase cascade known as Hippo which phosphorylates a set of transcription factors that regulate both the cell cycle as well as differentiation programs. This phosphorylation provides a binding site for 14-3-3, which in turn sequesters the factors in an inactive form in the cytoplasm. Recent results demonstrate that mutations in components of the cell contact inhibition kinase cascade lead to loss of Notch 1 activity, and likewise we have found that mutation of 14-3-3s in this pathway also leads to loss of Notch1 expression. Taken together, such results imply that as progenitors expand in tissues, their eventual cell contact signals 14-3-3s -dependent expression of Notch1 thereby signaling arrest and differentiation. In support of this hypothesis, we have found that mutation of 14-3-3s in mice leads to accumulation of proliferating progenitor cells and a phenotype indistinguishable from that of Notch 1 in the cornea, hair follicle and ductal structures such as those in the meibomian gland. This loss of 14-3-3s or Notch1 causes severe corneal defects, ductal obstruction leading to loss of terminal ascini and abnormal hair follicles leading to hair loss. Importantly, we demonstrate that expression of activated Notch1 rescues the differentiation defect in 14-3-3s mutant epithelial progenitors in culture. Here, we propose a series of studies designed to further establish linkage between 14-3-3s and the Notch1 pathway in epithelial differentiation in the cornea, meibomian gland and hair follicles and to development mouse model systems which can be used as a basis for a future R01 proposal examining the molecular details of the pathway through which 14-3-3s and Notch1 regulate the onset of epithelial differentiation.

Public Health Relevance

The integrity of the cornea, the most anterior part of the eye, is indispensable for vision, as evident by the facts that more than forty-five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of the loss of corneal transparency. Our proposed experiments aim to uncover the essential role of 14-3-3s and the signaling network in controlling the corneal epithelial homeostasis. Our studies will elucidate the molecular mechanism underlying the corneal disease development such as corneal intraepithelial neoplasia and squamous cell carcinoma;and provide new strategies for detection and treatment.

National Institute of Health (NIH)
National Eye Institute (NEI)
Exploratory/Developmental Grants (R21)
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Anterior Eye Disease Study Section (AED)
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Mckie, George Ann
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University of Louisville
Schools of Medicine
United States
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Sambandam, Sumitha A T; Kasetti, Ramesh B; Xue, Lei et al. (2015) 14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization. J Invest Dermatol 135:1621-8
Lu, Huayi; Lu, Qingxian; Gaddipati, Subhash et al. (2014) IKK2 inhibition attenuates laser-induced choroidal neovascularization. PLoS One 9:e87530