Dominant-Negative Interference of the TGF-ß Type II Receptor in
Mammary Gland Epithelium
Summary
TGF-ß1 and -ß3 are normally expressed at high levels in the mammary gland during all
stages of development except lactation. Exogenously added TGF-ß1, -ß2, and -ß3 have
been shown to regulate growth and differentiation of mammary epithelial cells in vitro and
in vivo. TGF-ßs signal through a heteromeric complex of type I and type II
serine/threonine kinases. The current model is that TGF-ß ligand binds to the TGF-ß type
II receptor on the cell surface, then recruits the type I receptor to form a heteromeric
complex. The type II receptor, which is a constitutively active kinase, phosphorylates the
GS domain of the type I receptor, activating the type I serine/threonine kinase.
Downstream targets of the type I receptor then transduce the signal to the nucleus. Type
II receptors with mutations or deletions of the kinase domain can compete with endogenous
receptors for ligand and type I receptors thereby acting as dominant-negative mutations to
block signaling by TGF-ß. To evaluate the role of endogenous TGF-ßs in the growth and
differentiation of the mammary gland in vivo, we have targeted expression of a truncated,
kinase defective TGF-ß type II receptor (DNIIR) to mammary epithelial cells in transgenic
mice using the mouse mammary tumor virus promoter/enhancer (MMTV). Transgene expression
was localized to the epithelial cells of terminal ducts and alveolar buds. At
approximately 20 weeks of age, virgin female transgenic mice demonstrated varying degrees
of mammary epithelial hyperplasia. Mammary glands from transgenic, virgin animals
exhibited alveolar development and expression of the milk protein, ß-casein. The data
suggest that impaired responsiveness in the epithelium to endogenous TGF-ßs results in
inappropriate alveolar development and differentiation in the mammary gland. We conclude
that endogenous TGF-ßs signal to the epithelium to maintain quiescence in the mammary
glands of virgin animals.
Citations
Gorska, A.E., Joseph, H. ,Derynck, R., Moses, H.L., Serra, R. Dominant-negative
interference of the TGF beta II receptor in mammary epithelium results in alvelolar
hyperplasia and differentation in virgin mice. Cell Growth and Differentation 9,
229-238, 1998
Background
Members of the TGF-ß superfamily are multifunctional peptides that are involved in the
regulation of growth and differentiation. During puberty, TGF-ß1,2, and 3 are expressed
within the epithelium of quiescent mammary ducts and actively growing end buds. TGF-ß2
and -ß3 levels increase during pregnancy and are localized to the epithelium of ducts and
alveoli. Expression of all three TGF-ß genes is dramatically reduced during lactation. It
has been proposed that TGF-ßs normally act to maintain ductal spacing during active
branching at puberty and to regulate lactogenesis. TGF-ß1, -ß2, or -ß3 administered via
slow release pellets to actively branching mouse mammary glands resulted in reversible
growth inhibition and involution of mammary gland end buds. In addition, transgenic mice
that express a constitutively active TGF-ß1 under the control of the MMTV
promoter/enhancer demonstrated a reduction in total ductal tree volume. TGF-ß1 also
inhibits expression of ß-casein, a differentiation marker for mammary epithelium, in
mammary explant cultures and in the HC11 mouse mammary epithelial cell line. Targeted
expression of active TGF-ß1 to the pregnant mammary gland using a whey acidic protein
(WAP) promoter resulted in inhibition of alveolar development in two out of four mouse
lines.
Transgene
The expression plasmid (MMTV-DNIIR) contained the cDNA sequence encoding a truncated human
TGF-ß type II receptor and the MMTV LTR promoter/ enhancer was used to generate
transgenic mice.
mouse strain
The transgene was introduced into C57BL/6XDBA mouse background.
Mammary development
Expression of the DNIIR resulted in inappropriate differentiation of mammary epithelium
giving an early to mid- pregnant phenotype in virgin animals. Formation of alveolar
structures which expressed ß-casein was observed in virgin animals in all stages of the
estrous cycle. ß casein expression was evaluated by northern blot hybridization and
immunohistochemistry.
Gene expression
Expression of the DNIIR transgene resulted in increased expression of ß-casein mRNA and
protein as evaluated by northern blot hybridization and immunohistochemical analysis.
Mechanistic implications
Based on data from experiments in which TGF-ß was administered exogenously to mammary glands and the observed pattern of expression of the TGF-ß isoforms in the mammary gland, it has been suggested that TGF-ßs may act as a regulator of ductal spacing, maintaining patterning and suppressing adventitious lateral branching in the developing, actively branching mammary gland. Disorganized branching was not detected in mammary glands from MMTV DNIIR transgenic mice relative to wild type mice. This observation suggests TGF-ß does not act on the epithelium to regulate this process. At 20 weeks, MMTV-DNIIR mammary glands from virgin transgenic mice demonstrated the development of alveolar buds. This pattern is similar to what is seen normally at early to mid-gestation in mammary glands and suggest endogenous TGF ßs act on the epithelium to prevent development of alveoli and maintain quiescence in the virgin mammary gland. In the MMTV-DNIIR mice, impaired responsiveness to TGF-ßs resulted in expression of ß-casein supporting a role for endogenous TGF-ß in preventing inappropriate expression of the differentiated phenotype.
The mechanism by which impaired responsiveness to TGF-ß results in alveolar hyperplasia and expression of ß-casein is not clear but several possibilities exist. 1) The TGF-ßs are known to regulate the accumulation of extracellular matrix (ECM) and the integrity of the ECM regulates morphogenesis and differentiation of mammary epithelial cells. 2) TGF-ßs may act to maintain quiescence of the mammary ductal tree by inducing apoptosis in cells that stochastically differentiate in the absence of the appropriate endocrine hormones. 3) TGF-ßs inhibit the growth of many epithelial cell types including mammary epithelial cells. TGF-ßs may act to maintain quiescence in the mammary gland by inhibiting growth of epithelial cells. In this model, impaired responsiveness to TGF-ßs would directly result in increased DNA synthesis in normally quiescent cells sufficient to result in the formation of alveolar buds.
key words
Submitted by
Rosa Serra
Rosa.Serra@mcmail.vanderbilt.edu
last update: July 1998