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We previously identified rat8 in the pathway involved in epithelial cell differentiation that occurs in the rat mammary gland at pregnancy when tubules and alveoli are formed. rat8, which encodes an IFN-inducible membrane protein, is the rat homologue of the mouse gene fragilis. By differential detergent extraction and isopycnic sucrose density gradients, we show that rat8 protein is associated to lipid membrane domains together with Lyn and Fyn, members of the Src tyrosine kinase family. We also show that recruitment of rat8 to lipid membrane domains is a necessary step in mammary epithelial cell differentiation. Immunoprecipitation analysis, performed with an anti-Fyn protein antibody, shows that rat8 was present in the Fyn immunoprecipitate. Antisense oligonucleotides, used to inhibit Fyn protein expression, block mammary cell differentiation. Taken together, these results suggest that the functional interaction, via lipid membrane domains, of rat8 and Fyn proteins is required for mammary cell differentiation. Therefore, rat8, like fragilis, may be involved in developmental decisions and the demarcation of a subset of cells in the mammary gland that cause epithelial cells to develop into a network of tubuloalveolar structures involved in secretion.

The study of the development of the mammary gland at the molecular level in the animal is difficult because of the complex tissue organization of the gland. We have previously developed an in vitro system for genetic analysis of mammary cell differentiation, based on the cell line LA7 clonally derived from a rat mammary adenocarcinoma. This cell line, after induction with DMSO, differentiates forming structures called domes. This process is under strict gene regulation, and we have previously identified several of the genes involved. In the present paper, we have defined the meaning of dome formation in relation to mammary development, by showing that treatment of LA7 cells with the lactogenic hormones hydrocortisone and prolactin induces dome formation; in the animal, these hormones precede and accompany milk production. Moreover, dome formation is accompanied by expression within the cells of the milk protein genes WDMN1 and beta-casein, which are differentiation markers for the gland during pregnancy and lactation. We also show that two proteins, highly expressed in the mammary gland during lactation, HSP90-beta and annexin I, are strongly expressed in DMSO-induced LA7 cells. Both proteins are essential in the formation of domes because when their synthesis is blocked by antisense RNA oligonucleotides, dome formation is abolished. Thus our in vitro system is a model for lobulo-alveolar development, and the genes identified in the pathway of dome formation are likely to be involved in the early differentiation steps occurring in the rat mammary gland during pregnancy and lactation.

In this work we extended the study of genes controlling the formation of specific differentiation structures called "domes" formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the beta-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells. Two differentially expressed proteins were investigated. The first, tropomyosin-5b, strongly expressed in DMSO-induced LA7 cells, is needed for dome formation because its synthesis inhibition by the antisense RNA technology abolished domes. The second protein, maspin, strongly expressed in the uninduced 106A10 cell line, inhibits dome formation because 106A10 cells, transfected with rat8 cDNA (the function of which is required for the organization of these structures), acquired the ability to develop domes when cultured in presence of an antimaspin antibody. Dome formation in these cultures are accompanied by ENaC beta-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC beta-subunit and the rat8 proteins, and is under the negative control of maspin.