ETS-1 transcription factor binds cooperatively to the palindromic head to head ETS-binding sites of the stromelysin-1 promoter by counteracting autoinhibition

Stromelysin-1 (matrix metalloproteinase-3) is a member of the matrix metalloproteinase family. Regulation of its gene expression is critical for tissue homeostasis. Patterns of increased co-expression of stromelysin-1 and ETS-1 genes have been observed in pathological processes. Stromelysin-1 promoter is transactivated by ETS proteins through two palindromic head to head ETS-binding sites, an unusual configuration among metalloproteinase promoters. By using surface plasmon resonance, electrophoretic mobility shift assay, and photo-cross-linking, we showed that full-length human ETS-1 (p51) binds cooperatively to the ETS-binding site palindrome of the human stromelysin-1 promoter, with facilitated binding of the second ETS-1 molecule to form an ETS-1.DNA.ETS-1 ternary complex. The study of N-terminal deletion mutants allowed us to conclude that cooperative binding implied autoinhibition counteraction, requiring the 245-330-residue region of the protein that is encoded by exon VII of the gene. This region was deleted in the natural p42 isoform of ETS-1, which was unable to bind cooperatively to the palindrome. Transient transfection experiments showed a good correlation between DNA binding and promoter transactivation for p51. In contrast, p42 showed a poorer transactivation, reinforcing the significance of cooperative binding for full transactivation. It is the first time that ETS-1 was shown to be able to counteract its own autoinhibition.     

Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.

The matrix-degrading metalloproteinases stromelysin-1, stromelysin-3, and gelatinase A are expressed during ductal branching morphogenesis of the murine mammary gland. Stromelysin-1 expression in particular correlates with ductal elongation, and in situ hybridization and three-dimensional reconstruction studies revealed that stromelysin-1 mRNA was concentrated in stromal fibroblasts along the length of advancing ducts. Transgenic mice expressing an activated form of stromelysin-1 under the control of the MMTV promoter/enhancer exhibited inappropriate alveolar development in virgin females. Ultrastructural analysis demonstrated that the basement membrane underlying epithelial and myoepithelial cells was amorphous and discontinuous compared with the highly ordered basal lamina in control mammary glands. Transgenic mammary glands had at least a twofold increase in the number of cells/unit area and a 1.4-fold increase in the percent of cycling cells by 13 wk of age compared with nontransgenic littermates. In addition, transgenic glands expressed beta-casein mRNA, but not protein, and resembled the proliferative and differentiated state of an animal between 8 and 10 days pregnant. An analysis of metalloproteinase expression in the glands of normal pregnant females demonstrated that the same matrix metalloproteinase family members, including stromelysin-1, were expressed in connective tissue cells surrounding epithelial clusters during the time of lobuloalveolar development. These results suggest that metalloproteinases may assist in remodeling ECM during normal ductal and alveolar branching morphogenesis, and that disruption of the basement membrane by an activated metalloproteinase can affect basic cellular processes of proliferation and differentiation.     

Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene

We have used transgenic mice overexpressing the human tissue inhibitor of metalloproteinases (TIMP)-1 gene under the control of the ubiquitous beta-actin promoter/enhancer to evaluate matrix metalloproteinase (MMP) function in vivo in mammary gland growth and development. By crossing the TIMP-1 transgenic animals with mice expressing an autoactivating stromelysin-1 transgene targeted to mammary epithelial cells, we obtained a range of mice with genetically engineered proteolytic levels. The alveolar epithelial cells of mice expressing autoactivating stromelysin-1 underwent unscheduled apoptosis during late pregnancy. When stromelysin-1 transgenic mice were crossed with mice overexpressing TIMP-1, apoptosis was extinguished. Entactin (nidogen) was a specific target for stromelysin-1 in the extracellular matrix. The enhanced cleavage of basement membrane entactin to above-normal levels was directly related to the apoptosis of overlying mammary epithelial cells and paralleled the extracellular MMP activity. These results provide direct evidence for cleavage of an extracellular matrix molecule by an MMP in vivo.