Cyclin-dependent kinase inhibitor p27(Kip1) is required for mouse mammary gland morphogenesis and function.

We have studied the role of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) in postnatal mammary gland morphogenesis. Based on its ability to negatively regulate cyclin/Cdk function, loss of p27 may result in unrestrained cellular proliferation. However, recent evidence about the stabilizing effect of p27 on cyclin D1-Cdk4 complexes suggests that p27 deficiency might recapitulate the hypoplastic mammary phenotype of cyclin D1-deficient animals. These hypotheses were investigated in postnatal p27-deficient (p27(-/-)), hemizygous (p27(+/)-), or wild-type (p27(+/+)) mammary glands. Mammary glands from p27(+/)- mice displayed increased ductal branching and proliferation with delayed postlactational involution. In contrast, p27(-/-) mammary glands or wild-type mammary fat pads reconstituted with p27(-/-) epithelium produced the opposite phenotype: hypoplasia, low proliferation, decreased ductal branching, impaired lobuloalveolar differentiation, and inability to lactate. The association of cyclin D1 with Cdk4, the kinase activity of Cdk4 against pRb in vitro, the nuclear localization of cyclin D1, and the stability of cyclin D1 were all severely impaired in p27(-/-) mammary epithelial cells compared with p27(+/+) and p27(+/-) mammary epithelial cells. Therefore, p27 is required for mammary gland development in a dose-dependent fashion and positively regulates cyclin D-Cdk4 function in the mammary gland.

The Ron/STK receptor tyrosine kinase is essential for peri-implantation development in the mouse.

The Ron/STK receptor tyrosine kinase is a member of the c-Met family of receptors and is activated by hepatocyte growth factor-like protein (HGFL). Ron activation results in a variety of cellular responses in vitro, such as activation of macrophages, proliferation, migration, and invasion, suggesting a broad biologic role in vivo. Nevertheless, HGFL-deficient mice grow to adulthood with few appreciable phenotypic abnormalities. We report here that in striking contrast to the loss of its only known ligand, complete loss of Ron leads to early embryonic death. Embryos that are devoid of Ron (Ron-/-) are viable through the blastocyst stage of development but fail to survive past the peri-implantation period. In situ hybridization analysis demonstrates that Ron is expressed in the trophectoderm at embryonic day (E) 3.5 and is maintained in extraembryonic tissue through E7.5, compatible with an essential function at this stage of development. Hemizygous mice (Ron+/-) grow to adulthood; however, these mice are highly susceptible to endotoxic shock and appear to be compromised in their ability to downregulate nitric oxide production. These results demonstrate a novel role for Ron in early mouse development and suggest that Ron plays a limiting role in the inflammatory response.

Nuclear factor-kappaB (NF-kappaB) regulates proliferation and branching in mouse mammary epithelium.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors has been shown to regulate proliferation in several cell types. Although recent studies have demonstrated aberrant expression or activity of NF-kappaB in human breast cancer cell lines and tumors, little is known regarding the precise role of NF-kappaB in normal proliferation and development of the mammary epithelium. We investigated the function of NF-kappaB during murine early postnatal mammary gland development by observing the consequences of increased NF-kappaB activity in mouse mammary epithelium lacking the gene encoding IkappaBalpha, a major inhibitor of NF-kappaB. Mammary tissue containing epithelium from inhibitor kappaBalpha (IkappaBalpha)-deficient female donors was transplanted into the gland-free mammary stroma of wild-type mice, resulting in an increase in lateral ductal branching and pervasive intraductal hyperplasia. A two- to threefold increase in epithelial cell number was observed in IkappaBalpha-deficient epithelium compared with controls. Epithelial cell proliferation was strikingly increased in IkappaBalpha-deficient epithelium, and no alteration in apoptosis was detected. The extracellular matrix adjacent to IkappaBalpha-deficient epithelium was reduced. Consistent with in vivo data, a fourfold increase in epithelial branching was also observed in purified IkappaBalpha-deficient primary epithelial cells in three-dimensional culture. These data demonstrate that NF-kappaB positively regulates mammary epithelial proliferation, branching, and functions in maintenance of normal epithelial architecture during early postnatal development.

Characterization of the mouse Ron/Stk receptor tyrosine kinase gene.

In an effort to understand the mechanisms governing the regulation of the mouse Ron receptor gene, a mouse genomic library was screened and overlapping clones coding for the Ron gene and flanking DNA were identified. Continuous DNA sequence was obtained for approximately 16.4 kilobases. The gene, from the initiator methionine to the polyadenylation site, is contained within 13 244 basepairs and contains 19 exons. Primer extension analyses were performed to determine the transcription start site of the mouse Ron transcript. Multiple transcription start sites were found which also appear to be used in transfected reporter constructs containing Ron 5' flanking DNA. To determine the location of sites which may be critical for the function of the Ron gene promoter, a series of chimeric genes containing serial deletions of the Ron gene promoter fused to the coding sequences for the chloramphenicol acetyl-transferase gene were constructed. Transient transfection analyses of these hybrid genes into various cell lines demonstrated that two regions of the Ron gene promoter, encompassing nucleotides -585 to -465 and from -465 to -285, are important for expression of this transcript in CMT-93 cells. Further analysis of the Ron promoter utilizing gel mobility shift analyses suggests that regions encompassing nucleotides -585 to - 508 and nucleotides -375 to -285 appear to bind specific proteins which may be involved in the negative and positive regulation, respectively, of the mouse Ron gene.

Dynamic expression and activity of NF-kappaB during post-natal mammary gland morphogenesis.

The Rel/NF-kappaB family of transcription factors has been implicated in such diverse cellular processes as proliferation, differentiation, and apoptosis. As each of these processes occurs during post-natal mammary gland morphogenesis, the expression and activity of NF-kappaB factors in the murine mammary gland were examined. Immunohistochemical and immunoblot analyses revealed expression of the p105/p50 and RelA subunits of NF-kappaB, as well as the major inhibitor, IkappaBalpha, in the mammary epithelium during pregnancy, lactation, and involution. Electrophoretic mobility shift assay (EMSA) demonstrated that DNA-binding complexes containing p50 and RelA were abundant during pregnancy and involution, but not during lactation. Activity of an NF-kappaB-dependent luciferase reporter in transgenic mice was highest during pregnancy, decreased to near undetectable levels during lactation, and was elevated during involution. This highly regulated pattern of activity was consistent with the modulated expression of p105/p50, RelA, and IkappaBalpha.

Expression of hepatocyte growth factor-like protein is repressed by retinoic acid and enhanced by cyclic adenosine 3',5'-monophosphate response element-binding protein (CREB)-binding protein (CBP).

In an effort to understand the molecular mechanisms involved in the regulation of expression of the gene encoding hepatocyte growth factor-like protein (HGFL), it was found that all-trans-retinoic acid dramatically represses expression of the endogenous HGFL gene in HepG2 cells, a human hepatocyte-derived cell line. This repression requires the sequence between nucleotides -135 and -105 in the 5'-flanking sequence of the HGFL gene, a site that has previously been shown to bind the transcription factor hepatocyte nuclear factor-4 (HNF-4). Electrophoretic mobility shift analysis suggests that the retinoic acid receptor does not bind to this site, and that retinoic acid does not alter binding of HNF-4 to this DNA site. However, the transcriptional coactivator, CREB-binding protein (CBP) coactivates expression of this gene through an indirect interaction with the HNF-4-binding site, and overexpression of CBP in HepG2 cells eliminates retinoic acid repression of reporter gene expression driven by the HGFL promoter. Overexpression of CBP also protects the endogenous HGFL gene from down-regulation by retinoic acid. These results suggest that HGFL gene expression requires CBP, and competition for limiting amounts of CBP by retinoic acid receptor may be a means of modifying the activity of HNF-4 at the HGFL gene promoter.

Mesenchymal expression of nuclear factor-kappaB inhibits epithelial growth and branching in the embryonic chick lung.

It is becoming increasingly recognized that the ubiquitous, inducible transcription factor nuclear factor-kappaB (NF-kappaB) is involved in developmental processes. For example, NF-kappaB acts as a mediator of epithelial-mesenchymal interactions in the developing chick limb. We investigated the role of NF-kappaB in directing the branching morphogenesis of the developing chick lung, a process which relies on epithelial-mesenchymal communication. High level expression of relA was found in the mesenchyme surrounding the nonbranching structures of the lung but was not detected either in the mesenchyme surrounding the branching structures of the distal lung or in the developing lung epithelium. Specific inhibition of mesenchymal NF-kappaB in lung cultures resulted in increased epithelial budding. Conversely, expression of a trans-dominant activator of NF-kappaB in the lung mesenchyme repressed budding. Ectopic expression of RelA was sufficient to inhibit the ability of the distal mesenchyme to induce epithelial bud formation. Cellular proliferation in the mesenchyme was inhibited by hyperactivation of NF-kappaB in the mesenchyme of lung cultures. Interestingly, increased NF-kappaB activity in the mesenchyme also decreased the proliferation of the associated epithelium, while inhibition of NF-kappaB activity increased cellular proliferation in lung cultures. Expression patterns of several genes which are known to influence lung branching morphogenesis were altered in response to changes in mesenchymal NF-kappaB activity, including fgf10, bmp-4, and tgf-beta1. Thus NF-kappaB represents the first transcription factor reported to function within the lung mesenchyme to limit growth and branching of the adjacent epithelium.

Functional characterization of domains contained in hepatocyte growth factor-like protein.

To delineate the functional protein domains necessary for the biological activity of hepatocyte growth factor-like protein (HGFL), we created various site-directed and deletion mutated cDNAs coding for this protein. Wild-type and mutated versions of HGFL were produced after transfection of the corresponding cDNAs into tissue culture cells. The biological importance of the domains within HGFL was then examined by addition of recombinant wild-type or mutant forms of HGFL to assays aimed at elucidating regions involved in the stimulation of DNA synthesis, the induction of shape changes in macrophages, and the ability to stimulate cell scattering. Mutant proteins lacking the serine protease-like domain (light chain) were not biologically active in any of the assays tested and could not compete with wild-type HGFL in cell scattering experiments. These data, in addition to direct enzyme-linked immunosorbent assay analyses, suggest that the light chain may play an important role in the interaction of HGFL with its receptor, Ron. Elimination of the proposed protease cleavage site between the heavy and light chains (by mutation of Arg-483 to Glu) produced a protein with activity comparable to wild-type HGFL. Further studies with this mutated protein uncovered an additional proteolytic cleavage site that produces biologically active protein. Deletion of the various kringle domains or the amino-terminal hairpin loop had various effects in the multiple assays. These data suggest that the heavy chain may play a pivotal role in determining the functional aspects of HGFL.