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Qian Chen, Yusuke Takahashi, Kazuhiro Oka, Jian-Xing (Jay) Ma; A novel mechanism for canonical Wnt signaling regulation through ectodomain shedding of very low-density lipoprotein receptor. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
Aberrant Wnt signaling mediates pathological vascular growth in multiple human diseases. However, the mechanisms for Wnt signaling dysregulation remain unclear. Very low-density lipoprotein receptor (VLDLR) negatively regulates Wnt signaling. Two alternative splice variants of VLDLR, VLDLRI and VLDLRII, have tissue-specific distributions and differential ectodomain shedding rates. The present study investigated the differential roles of the VLDLR splice variants in Wnt signaling regulation and whether the shed extracellular domain of VLDLR (sVLDLR-N) functions as an endogenous regulator of Wnt signaling.
The expression of VLDLRI and VLDLRII was analyzed by RT-PCR and Western blot analysis. Wnt signaling activity was evaluated by luciferase assay or Western blot analysis. Levels of sVLDLR-N and full-length VLDLR were measured by Western blot analysis, and the rate of sVLDLR-N to full-length VLDLR was determined by densitometry. Wnt-mediated β-galactosidase activity was evaluated using X-gal staining of retinal sections. Plasma levels of sVLDLR-N in Akita and db/db mice were measured by ELISA
Most tissues expressed both VLDLRI and VLDLRII, while the retina expressed only VLDLRII. sVLDLR-N was detected in conditioned medium of retinal pigment epithelial cells, bovine interphotoreceptor matrix (IPM) and human plasma. VLDLRII displayed a higher ectodomain shedding rate and a more potent inhibitory effect on Wnt signaling than VLDLRI in vitro. Intravitreal delivery of VLDLRII, but not VLDLRI, significantly decreased Wnt signaling activity in the retina in both VLDLR-/- and Wnt reporter (Axin2lacZ/VLDLR-/-) mice. O-glycosylation, which is present in VLDLRI but not VLDLRII, determined the differential rates of VLDLR ectodomain shedding. Moreover, release of sVLDLR-N was inhibited by TAPI-1, a metalloproteinases inhibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA). Further, release of sVLDLR-N was decreased under hypoxia. In addition, plasma levels of sVLDLR-N were significantly reduced in Akita mice, a type 1 diabetes model and db/db mice, a type 2 diabetes model.
VLDLRI and VLDLRII have differential roles in Wnt signaling regulation. Shedding of sVLDLR-N may represent a novel mechanism for intercellular inhibition of Wnt signaling, and reduced sVLDLR-N levels in diabetes are associated with diabetic complications.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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