Abstract
Purpose :
Lacrimal gland (LG) is the tear secreting gland which keeps eyes moistened. Dysfunction of the lacrimal gland can lead to the aqueous-deficient dry eye disease and even corneal damage. In this study, we find that C-terminal Src kinase (Csk) plays a key role in the development of lacrimal gland in a YAP dependent manner.
Methods :
Csk was deleted in the lacrimal gland using a LG epithelial specific Cre during embryogenesis. Csk-deleted mouse was sacrificed, and the phenotype of the lacrimal gland was examined at various postnatal stages by fixing the glands with 4% paraformaldehyde followed by immunofluorescence and H&E staining. Primary LG epithelial cells were collected from P1-4 mouse pup and cultured for one week and treated with Csk inhibitor.
Results :
We observed that the Csk-ablated lacrimal gland could not differentiate into a functional LG, instead we found dilated acini and ducts with complete loss of suppressive Src phosphorylation (pSRC-527Y) and the presence of active nuclear YAP. By RNA-seq analysis of the FACS-sorted LG epithelial cells, we also observed a significant increase in Yap target genes. Remarkably we could also rescue the dilated phenotype of the LG by homozygous deletion of YAP in the Csk deletion background, which supports our hypothesis that Csk regulates LG development via regulating YAP activity. Hippo pathway is known to regulate YAP by S-127 phosphorylation on its N terminal domain, which results in the inhibition of YAP function and cytoplasmic retainment. On the other hand, phosphorylation of YAP at Y-357 by Src family of kinase leads to the nuclear localization of YAP and enhanced transcriptional activity. WB analysis shows that treatment of LG epithelial cell with Csk inhibitor did not change the phosphorylation pattern of YAP at the S-127 site, but it led to a significant increase of Y-357 phosphorylation, suggesting that Csk regulates Yap activity independent of Hippo signaling.
Conclusions :
Our results demonstrate that Csk regulates lacrimal gland morphogenesis by controlling the Yap activity. Deletion of Csk results in the activation of Src kinase which activates YAP by phosphorylating Y-357 and results in the dilation of the lacrimal gland.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.