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Na Luo, Xingjuan Chen, Cathleen Wallmuth, Alexander G Obukhov, Ryan M. Anderson, Karen M Joos, Yang Sun; The Role of TRPV4 and OCRL in Primary Cilia of Trabecular Meshwork . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1989.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the role of TRPV4 and OCRL in primary cilia of trabecular meshwork cells. Oculocerebrorenal syndrome of Lowe (Lowe syndrome) is a rare X-linked recessive disease that affects multiple organ systems including eye, brain and kidney. The defect gene of Lowe syndrome is OCRL, which encodes an inositol polyphosphate 5-phosphatase, converting phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol 4-phosphate (PI4P). It still remains unknown for the molecular mechanisms of OCRL in ciliogenesis and in Lowe syndrome. Previous works have shown that the activation of transient receptor potential vanilloid 4 (TRPV4) is regulated by phosphoinositides; we thus proposed whether OCRL regulates cilia function by interactions with TRPV4.
Wild type or mutant OCRL were transfected in HEK293T or HTM cells together with Flag-TRPV4. Immunoprecipitation and immunoblotting were performed to detect interactions between OCRL and TRPV4. Immunofluorescence was performed to investigate the co-localization of TRPV4 with OCRL or ciliary markers in primary cilia. Laminar flow-induced Ca2+ signals were examined to measure the channel activity of TRPV4 in Lowe syndrome patient cells. Ciliary phenotypes were examined in TRPV4 or OCRL knockdown zebrafish morphants injected with antisense morpholino oligonucleotides.
TRPV4 interacts with OCRL and they co-localized in primary cilia in HTM cells after 48 hours serum starvation. Decreased co-localization of TRPV4 was detected in HTM cells transfected with Lowe syndrome causing- OCRL mutations. TRPV4 knockdown morphants presented a series of phenotypes such as microphthalmia, smaller brain, kinked tail, pronephros cysts, heart left-right asymmetry and edema, which are consistent with cilia-associated defects in OCRL morphants. Shorter primary cilia were observed in trabecular meshwork cells of TPRV4-/- mice. Compared to normal control cells, Lowe syndrome patient cells had decreased calcium flux responses when treated with TRPV4 agonists.
This study showed that TRPV4 interacts with OCRL, and that the defects of OCRL in Lowe syndrome effected the distribution of TRPV4 in primary cilia, which provides a new insight for the role of TRPV4 in primary cilia signaling in Lowe syndrome.
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