Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.

Abstract

When primary mouse mammary epithelial cells are cultured on plastic, they rapidly lose their ability to synthesize and secrete most milk proteins even in the presence of lactogenic hormones, whereas cells cultured on released type I collagen gels show greatly enhanced mRNA levels and secretion rates of ,B-casein and of some other milk proteins. We show here that culture on a reconstituted basement membrane from Engelbreth-Holm-Swarm tumor (EHS) allows >90% of cells to produce high levels of 13-casein. By 1-3). Until a decade ago, most investigators cultured cells on plastic surfaces, which led to drastic alterations of morphology and function from the parent tissue (4). Following the example of Emerman and Pitelka (5), we and others have shown that several aspects of functional epithelium can be maintained when primary mouse mammary epithelial cells (PMME) are cultured on "released" (RG or "floating") collagen type I gels instead of plastic. These include polarization of organelles, appearance of apical microvilli, formation of a basal lamina (5, 6), changes in glucose metabolite pattern (7), lumina formation (8), altered synthesis and compartmentalization of extracellular matrix (ECM) components (9, 10), enhancement ofmost milk protein synthesis and secretion (11), and increases in p-casein (12) and transferrin (13,14) mRNA levels. These results signify the importance of cell-substratum interactions in regulating tissue-specific functions and point to a possible regulatory role for ECM in vivo.Cells on released gels (and not on plastic or flat gels) were shown to synthesize an intact basement membrane (5) containing high levels of heparan sulfate and other sulfated glycosaminoglycans (9), type IV collagen, and laminin (10). We reasoned that the released gel may act through the influence of newly synthesized basement membrane components. To test the mechanism of cell-ECM interaction more directly, we have analyzed the consequences of culturing PMME cells on a reconstituted basal lamina derived from Engelbreth-Holm-Swarm (EHS) tumor (15) and on some of its individual components.MATERIALS AND METHODS PMME from 14-to 17-day pregnant BALB/c mice and collagen from rat tail tendon were prepared as described (5, 10, 11). Tumors (EHS) from normal or lathyritic mice were extracted with high salt and 2 M urea as described by Kleinman et al. (15). Dialyzed EHS extract (100-200 1.d) was spread either directly on 35-mm dishes or on top of rat-tail collagen gels. Collagen gels were released 3-4 days after seeding. Matrigel, an EHS preparation from Collaborative Research (Waltham, MA), was used for some experiments. Laminin and type IV collagen (Bethesda Research Laboratories) were spread at 2.5-10 pug/cm2. Heparan sulfate proteoglycan (HSPG; low density form) was prepared from EHS tumors as described by Hassell et al. (16) and was spread at 1-5 ,ug/cm2 or was added to the medium at 5 ,ug/ml every other day. The latter was less toxic but also less effective. EHS (10 ,ug/ml) and individual substrata other than HSPG we...

Differentiation of Monkey Embryonic Stem Cells into Hepatocytes and mRNA Expression of Cytochrome P450 Enzymes Responsible for Drug Metabolism: Comparison of Embryoid Body Formation Conditions and Matrices

“…5). Matrigel influenced gene expression in adult rat hepatocytes 45,46) as well as in three-dimensional culture of mouse [47][48][49] and human 50,51) mammary epithelial cells. Three-dimensional culture is important not only in accommodating an organ-like mass but also in enhancing physical cell-to-cell contact, accumulation of extracellular matrices, and local growth factor delivery, with a much greater biochemical effect on the maturation of fetal mouse and human hepatocytes than those in monolayer cultures.…”

Integrins in regulation of tissue development and function

“…Adhesion to basement membrane components stimulates the synthesis of milk proteins by increasing the phosphorylation of the prolactin receptor in cultured mammary epithelial cells [48,49]. Another example of regulation of differentiation by integrins in vitro is the inhibition by blocking integrin antibodies of the formation of contracting myotubes and expression of meromyosin by embryonic myoblasts [50].…”

Effects of extracellular matrix analogues on primary human fibroblast behavior

“…A more complete suite of matricellular proteins and growth factors is provided by Matrigel ™ , an ECM preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma [14]. Matrigel ™ has been successfully utilized for a variety of applications, such as cell growth and differentiation, angiogenesis and invasion assays, and promotes a natural cell morphology and behavior [14][15][16][17][18]. However, limited availability, batch-to-batch variability, pathogen transmission, immunogenicity, technical challenges in handling, and the inability to experimentally vary composition and compliance suggested the need for a more versatile ECM equivalent.…”