We recently showed that mammary glands contain a novel class of calcium-binding proteins (CBPs) that bind to membranes in a calcium-dependent manner. We have also established that these mammary CBPs are equivalent to the calelectrins and calpactin I/p36. Since it has been suggested that these proteins might be involved in exocytosis, we examined mammary glands for these CBPs during secretory differentiation. Immunohistochemical examination showed glands from virgin animals to be rich in calelectrins and calpactin I/p36, while glands from lactating animals contained little immunoreactive material. In addition, silver-staining and immunoblot estimation of the CBPs in lysates from collagenase harvested secretory epithelia showed these proteins to be significantly reduced compared to nonsecretory epithelia. Close examination of the CBP immunoreactive cells of the mammary gland shows that ductal cells are prominent in their staining and that the immunoreactive material is associated with the cell surface. Also, in juvenile glands the myoepithelial stem cells (cap cells) of the elongating end bud are devoid of the CBPs. In contrast to the in vivo data, epithelia cultivated on collagen gels demonstrate comparable levels of the CBPs in both nonsecretory and secretory monolayers. The in vivo data indicate that the CBPs are developmentally regulated during mammary gland differentiation such that secretory epithelia are essentially devoid of these novel proteins. Furthermore, a role for calelectrin and calpactin I/p36 in exocytotic casein secretion is questioned.
Amino acid incorporation experiments show that epithelial cells from lactating mouse mammary glands and from collagen gel culture both synthesize and secrete four principal phosphocaseins (p45, p40, p27, and p23 kD). In both cases, however, the casein production is largely dominated by the p27 species. The average percentage distribution of the above casein species in medium from cultured epithelia is approximately 13%, 6%, 68%, and 14%, respectively; for milk the distribution is approximately 23%, 7%, 54%, and 16%. The predominance of the p27 species is not a consequence of extensive extracellular differential degradation of the secreted caseins since no significant casein degradation was observed in culture medium, either in contact or isolated from epithelial cell monolayers. Synthesis and secretion of all the caseins by cultured epithelia is dependent upon insulin, prolactin, and hydrocortisone. Presumably some intracellular events result in the secretion of p27 as the principal casein in mouse milk. Apparently, some selection factor(s) operate to make p27 a major nitrogenous nutritional component for a newborn mouse. In addition, on a quantitative basis, the relative levels of various caseins secreted by epithelia from lactating mammary glands is essentially duplicated by epithelia in collagen gel culture.
Mouse mammary epithelial cells cultivated on collagen gels synthesize and secrete casein in a hormone-dependent manner. Fine-structure electron microscopy of secretory cultures revealed numerous cytoplasmic structures surrounded by membrane that is studded with ribosomes. The structures appear to be distended rough endoplasmic reticulum (RER). Electron microscope protein A-colloidal gold immunolocalization showed casein antiserum-specific deposition of gold particles over the RER cytoplasmic vesicles in cells provided insulin, prolactin, and hydrocortisone (IPF). Nonimmune antiserum showed no gold particle deposition over these cytoplasmic structures. Epithelia provided only insulin showed no such cytoplasmic vesicles nor any specific deposition of gold particles. Immunoblot analysis of cell lysate and culture medium showed casein only in IPF-treated cultures. It appears that the casein secretory pathway in collagen gel cultured mammary epithelia is blocked at the step that fuses RER vesicles to Golgi membrane. The data raise questions regarding the processing and maturation of casein and the mechanism of casein secretion in these cultures.
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