Endocytic prolactin routes to the secretory pathway in lactating mammary epithelial cells

Abstract

Plasma-borne prolactin is carried from blood to milk by transcytosis across the mammary epithelial cell through the endocytic and secretory pathways. To determine the precise route of prolactin endocytosis, intracellular transport of biotinylated prolactin was monitored, in parallel with endocytosis of fluorescein isothiocyanate-conjugated dextran and IgG, by using pulse-chase experiments in lactating mammary fragments and in enzymatically dissociated acini. Biotinylated prolactin was sorted to vesiculo-tubular organelles whereas dextran was very rapidly carried to the lumen and IgG remained accumulated in the basal region of cells. To determine whether prolactin uses routes into and across the Golgi and trans-Golgi network, localisation of biotinylated prolactin was combined with the immunofluorescence detection of caseins and, respectively, p58 and TGN38. Biotinylated prolactin strongly colocalised with caseins during a chase but not all or only very little with p58 and TGN38. To characterise the organelles involved in transcytosis, gold-labelled prolactin, experimentally accumulated in late endosomes and which recovered a normal transport, was localised by electron microscopy. In mammary fragments incubated at low temperature, and in mammary fragments from rats fed with a lipid-deprived diet, transport of gold-labelled prolactin was restored by increasing the temperature and by adding arachidonic acid, respectively. These data demonstrate that a sorting occurs very rapidly between prolactin, dextran and IgG. They suggest that prolactin may reach the biosynthetic pathway after direct fusion between multivesicular bodies and secretory vesicles.

Lipid-deprived diet perturbs O-glycosylation of secretory proteins in rat mammary epithelial cells

“…In parallel, we observed a significant reduction in the intracellular transport of prolactin in the transcytotic pathway of MECs. It was also shown that milk-protein synthesis drops and that the secretagogue effect of prolactin on their secretion disappears in lipid-restricted animals (Ollivier-Bousquet et al, 1993 and1997;Seddiki et al, 2002). These negative effects are reversed, at least in vitro, by the addition of 20:4 n-6 to mammary fragments (Ollivier-Bousquet et al, 1997).…”

Cathepsin D released by lactating rat mammary epithelial cells is involved in prolactin cleavage under physiological conditions

“…Samples were washed and incubated with the appropriate fluorescence-conjugated secondary antibodies. Mammary tissues from lactating rats were fixed, frozen and cut as described (Seddiki et al, 2002). The sections were treated for immunofluorescence as described for acini.…”

“…From blood, PRL is carried through the mammary epithelial cells (MECs) by transcytosis and is released into milk as intact and cleaved forms (Sinha, 1995;Ollivier-Bousquet, 1998). During this intracellular transport very little PRL is sorted to lysosomes (Seddiki and Ollivier-Bousquet, 1991;Seddiki et al, 2002). Consequently, the cleavage of PRL in these organelles might be relatively limited.…”

Milk lipid and protein traffic in mammary epithelial cells: joint and independent pathways

“…The lactating MEC conveys newly synthesised milk proteins by a process involving considerable membrane flow, which is used now as a model of cisternal maturation trafficking [13,20,38]. Another type of endomembrane trafficking involves endocytosis of membrane-associated molecules, and a meeting of the endocytic and exocytic pathway has been described [50,61,62]. Such membrane traffic may be required at each stage of milk lipid droplet transport: formation in the ER, growth and transit, associated or not with secretory vesicles, and secretion after envelopment in the apical plasma membrane.…”

“…(2) An endocytotic and transcytotic pathway involving internalisation of plasmaborne components into the cell and their transport into the milk [50]: during transport, a sorting of internalised molecules strictly controls the destination of the transcytosed products [41,61].…”