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Sensory denervation modulates eIF-2 alpha kinase expression in the rabbit lacrimal gland.

ABSTRACT: To investigate the hypothesis that sensory denervation of the rabbit lacrimal gland results in dysregulation of protein synthesis. We used differential display of mRNA to identify genes associated with protein synthesis and secretion that may be altered in this situation.New Zealand white rabbits underwent unilateral sensory denervation by the ablation of the trigeminal ganglion. After 7 days, the denervated and contralateral control lacrimal glands were removed. The effects of denervation on gene expression were carried out using differential mRNA display. Northern and Western blot analyses were used to verify differential gene expression.Differential mRNA display identified the gene heme-regulated inhibitor eukaryotic initiation factor-2 alpha kinase (HRI eIF-2a kinase) in the lacrimal gland, the expression of which was reduced in the denervated lacrimal gland. The sequenced fragment from differential display showed 94% identity to rabbit HRI eIF-2a kinase. The decreased expression of HRI eIF-2a kinase was confirmed by Northern and Western blots, and measurement of HRI eIF-2a kinase phosphorylation activity in the lacrimal gland after ablation of sensory neurons showed that it was significantly decreased compared with that of normal and control lacrimal glands.The results suggest that loss of sensory innervation has a role in the lacrimal gland, contributing to the expression of HRI eIF-2a kinase, a pivotal negative regulator of protein synthesis. A reduction in control of protein synthesis may lead to the translation of repressed messages associated with cell stress responses.

SUBMITTER: Nguyen DH

PROVIDER: S-EPMC2835540 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Sensory denervation modulates eIF-2 alpha kinase expression in the rabbit lacrimal gland.

Nguyen Doan H DH Beuerman Roger W RW Meneray Michele M Toshida Hiroshi H

Current eye research 20060401 4

<h4>Purpose</h4>To investigate the hypothesis that sensory denervation of the rabbit lacrimal gland results in dysregulation of protein synthesis. We used differential display of mRNA to identify genes associated with protein synthesis and secretion that may be altered in this situation.<h4>Methods</h4>New Zealand white rabbits underwent unilateral sensory denervation by the ablation of the trigeminal ganglion. After 7 days, the denervated and contralateral control lacrimal glands were removed. ...[more]

PMID: 16603461

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Project description:BackgroundAqueous-deficient dry eye disease (ADDED) resulting from dysfunction of the lacrimal gland (LG) is currently incurable. Although LG stem/progenitor cell-based therapy is considered to be a promising strategy for ADDED patients, the lack of a reliable serum-free culture method to obtain enough lacrimal gland stem cells (LGSCs) and the basic standard of LGSC transplantation are obstacles for further research.MethodsAdult mouse LGSCs were cultured in Matrigel-based 3D culture under serum-free culture condition, which contained EGF, FGF10, Wnt3A, and Y-27632. LGSCs were continuously passaged over 40 times every 7 days, and the morphology and cell numbers were recorded. LGSCs were induced to differentiate to ductal cells by reducing Matrigel rigidity, while fetal bovine serum was used for the induction of acinar cells. RT-PCR or qRT-PCR analysis, RNA-sequence analysis, H&E staining, and immunofluorescence were used for characterization and examining the differentiation of LGSCs. LGSCs were allotransplanted into diseased LGs to examine the ability of repairing the damage. The condition of eye orbits was recorded using a camera, the tear production was measured using phenol red-impregnated cotton threads, and the engraftments of LGSCs were examined by immunohistochemistry.ResultsWe established an efficient 3D serum-free culture for adult mouse LGSCs, in which LGSCs could be continuously passaged for long-term expansion. LGSCs cultured from both the healthy and ADDED mouse LGs expressed stem/progenitor cell markers Krt14, Krt5, P63, and nestin, had the potential to differentiate into acinar or ductal-like cells in vitro and could engraft into diseased LGs and relieve symptoms of ADDED after orthotopic injection of LGSCs.ConclusionWe successfully established an efficient serum-free culture for adult mouse LGSCs aiming at LG repair for the first time. Our approach provides an excellent theoretical and technical reference for future clinical research for ADDED stem cell therapy.

Dataset Information

Sensory denervation modulates eIF-2 alpha kinase expression in the rabbit lacrimal gland.

Publications

Sensory denervation modulates eIF-2 alpha kinase expression in the rabbit lacrimal gland.

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