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Han Peng, Nihal Kaplan, Jong Kook Park, Ying Dong, Wending Yang, Robert M Lavker; FIH-1 regulates ΔNp63α: a novel insight on the control of proliferation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2609.
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© ARVO (1962-2015); The Authors (2016-present)
ΔNp63α functions to promote proliferation in stem and early transient amplifying cells. Whereas much is known about the genes regulated by ΔNp63α in cornea/limbal keratinocytes, how ΔNp63α is regulated is less clear. We have shown that in limbal epithelium from mice null for the hydroxylase, FIH-1, ΔNp63α is attenuated, indicative that FIH-1 might play a role in ΔNp63α regulation.
Genetic approaches to alter FIH-1 expression in vitro and in vivo, in conjunction with immunostaining and immunoblotting were used. BrdU labeling and ki67 immunostaining were conducted to assess proliferative status. To investigate the downstream pathway(s) involved in FIH-1-induced ΔNp63α expression, gene expression profiling was combined with bioinformatic analysis. To determine the interaction of FIH-1 with its substrates, the BioID assay was performed. To confirm the downstream target pathway(s) modulated by FIH-1, we conducted rescue experiments using pharmacological and genetic approaches.
Microarray combined with bioinformatic analysis suggested that FIH-1 regulated ΔNp63α signaling. In support of this idea, loss of FIH-1 decreased ΔNp63α expression during limbal epithelial development. Such a decrease in ΔNp63α expression is accompanied by decreased proliferation and increased differentiation. Knockdown of FIH-1 attenuated ΔNp63α expression in vitro. Overexpression of FIH-1 increased ΔNp63α levels, which can be reversed by inhibiting hydroxylase activity, suggestive that hydroxylation is the means by which FIH-1 regulates ΔNp63α. Bioinformatic analysis of microarray data indicated that overexpression of FIH-1 attenuates GADD45, which is a negative regulator of ΔNp63. Rescue experiments demonstrated that the decrease of ΔNp63α in keratinocytes lacking FIH-1 was reversed by knocking down GADD45α. Interestingly, ASPP2, which is a substrate of FIH-1, has also been shown to regulate ΔNp63α via NF-kappaB signaling. The BioID assay revealed that ASPP2 specifically interacted with FIH-1 in limbal keratinocytes. Knockdown of ASPP2 upregulated ΔNp63α and reversed the decrease of ΔNp63α by FIH-1 depletion, which may be via negatively regulating NF-kappaB signaling.
Our findings indicate that FIH-1 can positively regulate ΔNp63α via two distinct signaling pathways and helps to explain mechanistically the association of FIH-1 overexpression with a more proliferative phenotype.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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