A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression.

Abstract

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.

Activation of STAT Factors by Prolactin, Interferon-γ, Growth Hormones, and a Tyrosine Phosphatase Inhibitor in Rabbit Primary Mammary Epithelial Cells

“…A promoter element, BCE-1, which is thought to respond to ECM, has been described in the distal region of bovine ␤-casein gene (49). This enhancer is able to respond to PRL in the absence of ECM.…”

“…Although ECM and PRL can activate the enhancer independently, the ECM response has not been differentiated from the PRL response in terms of sequence requirements (48,49). Curiously, this DNA fragment contains MGF consensus sequence TTC TCA GAA, raising the possibility of the involvement of MGF in ECM response.…”

A hierarchy of ECM-mediated signalling regulates tissue-specific gene expression

“…When ECM is added, however, insulin does not interfere with second tier β-casein signalling [86,87], suggesting that signals generated by the ECM may impinge upon insulin-dependent signal transduction. Recently, it has been demonstrated that integrinmediated signals can influence events that occur downstream of the insulin receptor [88].…”

“…For example, a 160 bp enhancer, designated BCE-1, which was isolated from the bovine β-casein gene is activated when mammary epithelial cells are placed on reconstituted basement membrane gels [86,87]. Like the promoter of the albumin gene, BCE-1 contains a C/EBPbinding element that is required for ECM-dependent activation.…”

“…Second tier signal processing also affects the loading of transcription factors that respond to signals generated by prolactin, a lactogenic hormone that is absolutely required for ECMdependent β-casein expression and BCE-1 activation [87]. Although signal transduction mediated by the prolactin receptor in general is not cell shape or ECM dependent (J Ashkenas, Z Werb, MJ Bissell, unpublished data), its ability to induce milk protein gene expression may be.…”