Traffic of endogenous, transduced, and endocytosed prolactin in rabbit lacrimal acinar cells

Abstract

The rabbit lacrimal gland undergoes an immunophysiological transformation during pregnancy, reminiscent of that of the mammary gland as it prepares to deliver secretory IgA into the nascent fluid product. The contents of TGF-beta and prolactin (PRL) within ductal epithelial cells increase, and their primary localizations shift from the apical to the basal cytoplasm, suggesting a transformation from exocrine to paracrine secretion. Studies with ex vivo acinar cell models demonstrated that elevated PRL suppresses traffic of secretory proteins into the regulated exocrine apparatus and directs them into a novel, induced, regulated paracrine apparatus [Wang, Y., Chiu, C.T., Nakamura, T., Walker, A.M., Petridou, B., Trousdale M.D., Hamm-Alvarez S.F., Schechter J.E., Mircheff A.K., 2007. Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells. Am. J. Physiol. Endocrinol. Metab. 292, E1122-E1134]. However, it was not clear whether PRL itself entered the induced paracrine apparatus. In the present study, confocal immunofluorescence microscopy revealed that natively expressed PRL and over-expressed PRL co-localized with PRL receptors (PRLR); rab11, a marker for the recycling endosome; gamma-adaptin, a marker for the Golgi complex and trans-Golgi network; and rab7, a marker for the autophagic lysosomal apparatus. Natively expressed, over-expressed, and endocytosed PRL also co-localized with rab4 and rab5A, markers for the early endosome, and with rab3D, a marker for regulated exocrine secretory vesicles. Endocytosed PRL was stored in intact form and released in response to stimulation with carbachol. Subcellular fractionation analysis detected relative excesses of PRL over PRLR in fractions that contained fragments of the recycling endosome and fractions that contained both secretory vesicle fragments and prelysosomal and autolysosomal fragments. EM-gold microscopy demonstrated PRL within small vesicles, consistent with endosomes or secondary lysosomes, and in large vesicles, consistent with regulated secretory vesicles. The secretory vesicles were preponderantly localized in the apical cytoplasm of control cells, and in the basal cytoplasm of PRL over-expressing cells. These results indicate that when lacrimal epithelial cells synthesize PRL, and when they endocytose it from their ambient medium, they traffic it both into the endosomes that constitute the constitutive transcytotic paracrine apparatus and also into regulated secretory vesicles, which are associated with the exocrine apparatus at low PRL levels and with the induced paracrine apparatus at high PRL levels.

Neural regulation of lacrimal gland secretory processes: Relevance in dry eye diseases

“…In non-pregnant females and in males (low prolactin levels) both synthesized and endocytosed prolactin is localized in the apical cytoplasm consistent with secretion by the regulated exocytotic pathway. Cholinergic agonists can then stimulate prolactin secretion via the exocytotic pathway, consistent cholinergic regulation of exocytosis (Wang, Chiu et al 2007). During pregnancy (high prolactin levels) the localization of prolactin changes to the basal cytoplasm consistent with paracrine secretion using the transcytotic pathway.…”

“…Although specific proteins are secreted by each of these pathways, a single protein can use multiple pathways under different conditions. A complex series of intracellular trafficking pathways elucidated by Mircheff and his collaborators over the years have provided the framework for understanding the complex organization of vesicles in the trafficking pathways of lacrimal gland acinar cells and how these pathways are altered with stimulation for different proteins (Wang, Chiu et al 2007; Wang, Chiu et al 2007). …”

“…As summarized by Wang et al lacrimal gland acinar and duct cells can regulate their local extracellular milieu by secretion of cytokines and growth factors by a paracrine secretory process that has much in common with the transcytosis pathway (Wang, Chiu et al 2007). Newly synthesized cytokines and growth factors emerge from the endoplasmic reticulum, Golgi complex, and trans-Golgi network as do other synthetic proteins.…”

“…When prolactin is secreted from the basal side it appears to enhance the transcytotic paracrine apparatus that trafficks sIgA into the apical secretion and hence the tears. The elevated levels of prolactin suppress traffic of secretory proteins into the regulated exocytic apparatus into the endosomes of a paracrine pathway that intersects the constitutive transcytotic pathway (Wang, Chiu et al 2007; Wang, Chiu et al 2007). …”

Suppression of Lymphocyte Proliferation and Regulation of Dendritic Cell Phenotype by Soluble Mediators from Rat Lacrimal Epithelial Cells

“…Work in several laboratories has demonstrated that epithelial cells in human [33, 34] and rat lacrimal glands [35] express mRNAs for TGF‐β 1 and TGF‐β 2 . Immunohistochemical, physiological [36, 37] and cytophysiological [38, 39] studies indicate that epithelial cells in the interlobular ducts of the rabbit lacrimal gland secrete various cytokines and growth factors to the lumen of the acinus‐duct system, via their apparatus for exocrine protein secretion, and to the underlying stromal space, via, under normal circumstances, the same compartments that secrete autoantigens [38, 39]. Accordingly, TGF‐β might be one of the several factors mediating the anti‐proliferative activity documented by Pockley and Montgomery.…”