Abstract
Abstract: :
Purpose: Ocular surface trauma may elicit increased paracrine epidermal growth factor (EGF) signaling in the lacrimal gland. In the course of studies of the traffic of endocytosed [125I]-EGF in lacrimal acini (Xie et al., Cornea 19: S137, 2000), we noted that EGF stimulation altered the subcellular distributions of a number of endomembrane proteins. Since the consistency of endomembrane protein sorting is likely to be critical to the maintenance of self-tolerance, we sought to document the EGF-induced traffic changes. Methods: Reconstituted rabbit lacrimal gland acini were maintained in primary culture for 40 hours before being incubated at 37º for 20 min or 120 min with and without 200 ng/ml [125I]-EGF. Cells were then lysed and analyzed by isopycnic centrifugation on sorbitol density gradients. Aliquots of the initial post-nuclear fraction were subjected to 10% trichloroacetic acid (TCA) precipitation to separate intact [125I]-EGF from small proteolytic products. Results: [125I]-EGF content decreased by about 40% between 20 min and 120 min of loading. The decrease was accompanied by increased proteolytic degradation in lysosomal fractions. The accumulation phase was accompanied by increased Na+, K+-ATPase and acid phosphatase without change in the overall distribution patterns; increased ß-hexosaminidase in fractions containing Golgi, endosomes, and blm; and increased EGF receptors, Rab5, and Rab6 in fractions containing Golgi and endosomes. Most changes reversed by 120 min of loading; EGF receptors content even fell below control level. Conclusions: EGF initially stimulates recruitment of Rab5 and Rab6 from the soluble phase to the membrane phase, leading to net increase of traffic within the endosomes and Golgi, and a net decrease in traffic into the lysosomal pathway for degradation. Decreased degradation results in increased accumulation. Accumulated ß-hexosaminidase is poorly retained in the Golgi and then inappropriately targeted to the blm-communicating compartments. We suggest that increased blm-expression and/or secretion to interstitium of ß-hexosaminidase and similarly behaving proteins could challenge peripheral tolerance mechanisms.
Keywords: 452 lacrimal gland • 423 growth factors/growth factor receptors • 342 cell membrane/membrane specializations