Abstract. Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating H-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the/~-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing /3-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the/3-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the cd chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell proliferation and modulates the interactions of cells with their extracellular matrix (ECM), in part by inducing the synthesis of various ECM proteins. Three different isoforms of TGF-beta are synthesized in a defined pattern in specific cell populations in vivo. In the specific case of TGF-beta 1, this well-defined and limited expression stands in sharp contrast to its synthesis by virtually all cells in culture. Using mammary epithelial cells as a model system, we evaluated the substratum dependence of the expression of TGF-beta 1. The level of TGF-beta 1 expression is high in cells on plastic, but is strongly downregulated when cells are cultured on a reconstituted basement membrane matrix. In contrast, TGF-beta 2 mRNA levels in cells on either substratum remain unchanged. Using the chloramphenicol acetyl transferase gene as reporter gene under the control of the TGF-beta 1 promoter, we show that transcription from this promoter is suppressed when the cells are in contact with either endogenously synthesized or exogenously administered basement membrane. TGF-beta 1 promoter activity is strongly induced by the absence of basement membrane, i.e., by direct contact of the cells with plastic. This modulation of transcription from the TGF-beta 1 promoter occurs in the absence of lactogenic hormones which allow full differentiation. Our results thus indicate that basement membrane is an important regulator of TGF-beta 1 synthesis, and explain why most cells in culture on plastic express TGF-beta 1 in contrast with the more restricted TGF-beta 1 synthesis in vivo. We propose that there is a feedback loop whereby TGF-beta 1-induced synthesis of basement membrane components is repressed once a functional basement membrane is present. Finally, these results together with our current knowledge of regulation of TGF-beta 1 and TGF-beta 2 synthesis, suggest that, in vivo, TGF-beta 1 may play a major role in regulating the ECM synthesis and the cell-ECM interactions, whereas TGF-beta 2 may be more important in morphogenetic processes.

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM-and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express 1casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete f-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine ,3casein gene. The expression of CAT in these lines demonstrated a strking matrix and hormone dependency (>150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the (-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous (-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.Mammary epithelial cells provide a prime example for the dramatic effect of extracellular matrix (ECM) on morphology (1-3) as well as milk protein gene expression (2, 4-7). The molecular mechanisms involved in this interaction, however, are far from understood.Two approaches have been taken to address this problem: (i) To determine the nature of ECM-mammary cell interaction at the cell surface (8) and (ii) to determine the nature of cis-and trans-acting sequences and factors involved in transcriptional regulation. The absence offunctional cell lines for stable transfection of chimeric genes has been a major impediment in the latter approach. While transgenic animals have been used for studies ofhormonal regulation (9), the role of the ECM in tissue-specific gene expression cannot be easily studied in such models.With the availability of the functional COMMA-1D mammary epithelial cell strain derived from midpregnant mice (10), a number of laboratories have attempted to study the regulatory sequences of milk protein genes in response to lactogenic hormones. Some earlier attempts to transfect casein constructs failed, as the transfected cell bypassed hormonal regulation (11). In HC11 cells, a cloned derivative of COMMA-1D cells, Doppler et al. (12,13) succeeded in dissecting aspects of the hormone-responsive cis-acting elements of the -3-casein gene. These cells, however, have lost their ability to respond to substrata and theref...

Lactogenic hormones and extracellular matrix (ECM) act synergistically to regulate f-casein expression in culture. We have developed a functional subpopulation of the mouse mammary epithelial cell strain COMMA-1D (designated CID 9), which expresses high level of 3-casein, forms alveolar-like structures when plated onto the EHS tumor-derived matrix, and secretes f-casein unidirectionally into a lumen. We have further shown that ECMand prolactin-dependent regulations of f-casein occur mainly at the transcriptional level and that 5' sequences play an important role in these regulations. To address the question of the nature of the DNA sequence requirements for such regulation, we analyzed the bovine ,B-casein gene promoter in these cells. We now have located a 160-bp transcriptional enhancer (BCE1) within the 5' flanking region of the 3-casein gene. Using functional assays, we show that BCE1 contains responsive elements for prolactin-and ECM-dependent regulation. BCE1 placed upstream of a truncated and inactive f-casein promoter (the shortest extending from -89 to +42 bp with regard to the transcription start site) reconstitutes a promoter even more potent than the intact promoter, which contains BCE1 in its normal context more than 1.5 kb upstream. This small fusion promoter also reconstitutes the normal pattern of regulation, including a requirement for both prolactin and ECM and a synergistic action of prolactin and hydrocortisone. By replacing the milk promoter with a heterologous viral promoter, we show that BCE1 participates in the prolactin-and ECMmediated regulation.