Differential function and regulation of orphan nuclear receptor TR3 isoforms in endothelial cells

Abstract

TR3 has been reported to be an excellent target for angiogenesis therapies. We reported three TR3 transcript variant messenger RNAs (mRNAs) are expressed in human umbilical vein endothelial cell (HUVEC) and are differentially regulated by vascular endothelial growth factor (VEGF). TR3 transcript variant 1 (TR3-TV1) and variant 2 (TR3-TV2) encoding the same TR3 isoform 1 protein (TR3-iso1) that was named TR3 has been extensively studied. However, the function of TR3 isoform 2 protein (TR3-iso2) encoded by TR3 transcript variant 3 (TR3-TV3) is still not known. Here, we clone and express the novel TR3-iso2 protein and find that expression of TR3-iso2, in contrast to TR3-iso1, inhibits en-dothelial cell proliferation induced by VEGF-A, histamine, and phorbol-12-myristate-13-acetate (PMA). The differential function of TR3-iso2 correlates with the down-regulation of cyclin D1. However, TR3-iso2 plays similar roles in endothelial cell migration and monolayer permeability as TR3-iso1. We further demonstrate that several intracellular signaling pathways are involved in histamine-induced TR3 transcript variants, including histamine receptor H1-mediated phospholipase C (PLC)/calcium/calcineurin/protein kinase C (PKC)/ protein kinase D (PKD) pathway and ERK pathway, as well as histamine receptor H3-mediated PKC-ERK pathway. Further, expressions of TR3-TV1, TR3-TV2, and TR3-TV3 by VEGF and histamine are regulated by different promoters, but not by their mRNA stability.

Orphan nuclear receptors in angiogenesis and follicular development

“…Full name Function on ovary Effect on the expression of genes that regulate follicular angiogenesis (Murayama et al 2008) Reduces THBS1 (Smalling et al 2013) NR1D1 REV-ERBα Reverse strand of ERB-alpha Regulates fertility (deletion causes subfertility) and steroid synthesis (Preitner et al 2002, Chen et al 2012 Promotes VEGF (Burgermeister et al 2019); Reduces PGE2 synthesis (Isayama et al 2015); Reduces THBS1 (Li et al 2014 Represses FSH expression but seems to promote steroidogenesis (Xing et al 2002) May promote or inhibit VEGF (Qin et al 2010a, Zhu et al 2016; Inhibits VEGFR1 and sVEGFR1 (Qin et al 2010a) (Zou et al 2014); Reduces VEGFR2 expression (Likhite et al 2019), THBS1 (Wu et al 2017); Reduced by VEGF (Likhite et al 2019) reciprocal sense, ONRs may be regulated by angiogenic factors themselves, as, for example, the expression and activity of the NR4A family (NUR77/TR3/NGFIB, NURR1/RNR-1/TONOR, and NOR1) are regulated by VEGFA, PGE2 and ANGPT1 (Ismail et al 2012, Land et al 2015, Zhou et al 2016. Despite the importance of ONRs in the regulation of angiogenesis and in the control of synthesis and function of ovarian angiogenic factors, the specific roles of these NRs in the ovarian angiogenic context have been not evaluated.…”

“…The importance of the NR4A subfamily as transcription factors involved in the control of angiogenesis is well documented (Mohan et al 2012, Peng et al 2019, Ye et al 2019. The effects may be reciprocal, as several lines of evidence from in vitro and in vivo angiogenic models show that VEGFA and other angiogenic factors increase mRNA and protein concentrations of NR4A1, NR4A2, and NR4A3 (Liu et al 2003, Rius et al 2006, Zeng et al 2006, Martorell et al 2009, Zhao et al 2011, Qin et al 2013, Zhou et al 2016. To date, direct or reciprocal angiogenic effects have not yet been evaluated at the ovarian level.…”