PITPβ/βA3/A1-crystallin complex is critical for PIP metabolism in retinal pigment epithelium (RPE)

Abstract

Purpose : Phosphatidylinositol phosphates (or PIPs) have emerged as key regulators of diverse cellular processes including membrane trafficking, receptor tyrosine kinase signaling, and cytoskeleton dynamics. However, the regulation of PIP metabolism in RPE cells has been rarely investigated. We have reported that βA3/A1-crystallin, an ocular moonlighting protein with diverse functions interacts with a phosphatidylinositol transfer protein-PITPβ. In this study, we examined their effect on PIP-metabolism in RPE cells.

Methods : Cryba1 (encoding βA3/A1-crystallin) RPE conditional KO (cKO) mice and Cryba1^fl/fl control mice were used in this study. RPE explant cultures isolated from either wildtype C57BL/6J mice or transgenic mice were utilized for in vitro RPE studies. RNA-seq was performed to examine the changes in transcriptome profile in RPE cells overexpressing βA3/A1-crystallin. Immunoprecipitation studies were utilized for detecting the interaction between βA3/A1-crystallin and PITPβ. The expression of proteins involved in EGFR (epidermal growth factor receptor) activation and PLCγ1 (phospholipases C gamma 1) signaling were detected by western blotting. The PIP fraction was isolated from RPE tissue and PIP levels were determined by ELISA.

Results : RPE cells overexpressing Cryba1 have decreased expression of genes involved in inositol phosphate metabolism, such as phosphatidylinositol phosphatases and kinases, and phospholipases. βA3/A1-crystallin interacts with PITPβ in polarized RPE cells, and the binding was more significant when cells were stimulated with EGF. Both PI(4,5)P2 and PI(3,4,5)P3 were significantly reduced, and the EGFR activation was compromised in RPE cells lacking βA3/A1-crystallin. In Cryba1 cKO RPE cells overexpressing βA3/A1-crystallin, EGFR activation was rescued and the PI(3,4,5)P3 level was upregulated, although the PI (4,5)P2 was not increased. PLCγ1 activity was found to be negatively correlated with βA3/A1-crystallin in RPE cells. Moreover, PITPβ overexpression stimulates PLCγ1/PKC activity and inhibits EGFR activation, but RPE flatmounts overexpressing both βA3/A1-crystallin and PITPβ showed no significant changes of PLCγ1/PKC activity or EGFR activation.

Conclusions : The data suggest an important role for βA3/A1-crystallin and PITPβ in the regulation of PIP metabolism, particularly in regulating PLCγ1 activity in the turnover of PI(4,5)P2 in RPE cells.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.