Summary
Transgenic mice over expressing the human TIMP-1 gene (TO) under the control of B-Actin
showed a 70% reduction in the number of ductal buds at 7.5days p.c. compared to non
transgenic controls.
Transgenic mice have been generated in which stromelysin-1 has been expressed in mammary tissue under control of the WAP gene promoter (Sympson et al). and under control of the MMTV-LTR (Witty et al.).
Citation
Sympson, C.J., R.S. Talhouk, C.M. Alexander, J.R. Chin, S.M. Clift, M.J. Bissell & Z.
Werb (1994). Targeted expression of stromelysin-1 in mammary gland provides evidence for a
role of proteinases in branching morphogenesis and the requirement for an intact basement
membrane for tissue-specific gene expression. J. Cell Biol. 125: 681-693
Alexander, C.M., E.J. Hansell, O. Behrendtsen, M.L. Flannery, N.S. Kishnani, S.P. Hawkes & Z. Werb (1996). Expression and function of matrixmetalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation. Development. In press.
Alexander, C.M., E.W. Howard, M.J. Bissell & Z. Werb (1996). Rescue of mammary epithelial cell apoptosis and entactin degradation by a TIMP-1 transgene. J. Cell Biol. In press.
Background
TIMP-1 is one of the principle inhibitors of matrix metalloproteinases (MMP). In addition
to the expression of MMPs during mammary gland development , it has been shown that
ectopic over-expression of activated Stromelysin-1 produces hyperplasia of the virgin
gland to the mid-pregnant stage, which suggests that MMPs can be bioactive in gland
development (Sympson et al., 1994). Therefore, transgenic mice over expressing the human
TIMP-1 gene under the ubiquitous control of b-actin promoter/enhancer are useful for
evaluating the in vivo effect of altering the balance between MMPs and inhibitors of MMPs
on the development of the mammary gland. Further understanding of MMP function in vivo is
possible by crossing these transgenic mice with other mice which have altered MMP
expression.
Transgene
4.3 kb of 5' sequence from human b-actin gene (including 3 kb of flanking sequence, 78 bp
of non coding sequence, and 832 bp of intron 1) was placed upstream of a Hind III cloning
site and 877 bp of 3π untranslated sequence from the same gene in Bluescript
vector. A full length human TIMP-1 (an EcoRI fragment tailed with Hind III linkers) ws
inserted and the whole construct removed with Kpn I and Xba I for microinjection into
pronuclei of CD-1 mouse eggs
mouse strain
CD-1
Mammary development
A delay in budding/alveolar development was seen as early as 6.5 days p.c. and continued
through 9.5 days p.c. in the TO mammary gland. By 16.5 days p.c. the lobulo-alveolar
development of the TO mammary glands seemed to have caught up to control glands.
Lactational development was similar to control glands, but fatty infiltration was
accelerated during involution. The TO transgenic mouse has been crossed with the
transgenic mouse over expressing active Stromelysin-1 under the control of WAP (Alexander
et al., 1996, J. Cell Bio.). This cross reverts the phenotype of the Stromelysin -1
transgenic animals; unscheduled apoptosis of the alveolar epithelial cells is
extinguished, the cleavage of basement membrane entactin is reduced, and alveolar volume
is increased to normal.
Gene expression
Human TIMP-1 mRNA was expressed in every tissue examined. It was present in the serum at
50ng/ml. Endogenous TIMP-1 expression was unaffected. The expression of human TIMP-1
protein in mammary gland increased through pregnancy then decreased in lactating glands
(in parallel with endogenous actin) increasing again after weaning.
Mechanistic implications
TIMP-1 over expression inhibits early growth and development of mammary glands,
implicating MMPs in this process.
key words
TIMP, matrix metalloproteinases, alveolar development, ductal development
Contributed By
Zena Werb
UCSF
Laboratory of Radiobiology
3rd and Parnassus
S.F., CA 94143-0750
USA
Phone: 415-476-4622/4758
Fax: 415-476-0721
E-mail: (Zena@radlab.ucsf.edu)