Abstract
Abstract: :
Purpose:Researchers have long speculated that prolactin (PRL), as well as sex steroids, influences lacrimal gland function. The possible role of PRL is especially intriguing, since this pituitary protein hormone is also expressed by lacrimal epithelial cells, and lacrimal PRL expression levels appear to be modulated by the sex steroids. We are using a new adenovirus vector for rabbit PRL (AdPRL) to evaluate possible autocrine/intracrine effects of over–expressed PRL on rabbit lacrimal acinar cells (LGCs) ex vivo. Methods:LGCs were isolated and maintained 4 days in a Matrigel–containing system favoring reconstitution of acinus–like structures. On day 2 they were incubated for 1 hour with either AdPRL, AdGFP, or no vector. On day 4 they were lysed and analyzed by subcellular fractionation or fixed and examined by confocal immunofluorescence. Western blots were probed with appropriate antibodies and analyzed by infrared fluorescence imaging. Results:Immunoblotting of LGC lysates revealed significantly increased tyrosine phosphorylation and increased abundance of 40kDa PRL receptors in AdPRL–transduced samples compared to the two controls. Confocal microscopy revealed increased intensity of phospho–tyrosine staining in PRL–overexpressing cells. Immunoblotting specifically demonstrated increased STAT5 phosphorylation. Confocal microscopy demonstrated that PRL was partially co–localized with PRL receptors both in control and in PRL–overexpressing cells. Subcellular fractionation analysis revealed that the abundances of PRL and PRL receptors in endomembrane compartments increased from the Golgi complex through the TGN to the pre–lysosome and lysosome and that PRL overexpression caused PRL receptors to shift from the pre–lysosome and lysosome to the trans–Golgi network. Conclusions:Overexpression of PRL leads to a persistently altered intracellular signaling environment. The partial co–localization of PRL and PRL receptors demonstrated by confocal microscopy and fractionation analysis indicates a likely role for intracrine PRL signaling. Increased PRL receptor abundance might result from decreased flux of PRL receptors to degradative compartments. Thus, there may be a positive feedback loop between PRL receptor activation and PRL receptor abundance.
Keywords: autoimmune disease • lacrimal gland • signal transduction