Summary
Cell surface b1,4-galactosyltransferase (GalTase) serves as a receptor in a variety of
cell-cell and cell-matrix interactions during fertilization and development, including
mammary epithelial cell-matrix interactions. To analyze GalTase function during mammary
gland morphogenesis in vivo, we created transgenic animals that overexpress cell
surface-localized GalTase. Mammary epithelial cells from transgenic animals had 2.3-times
more GalTase activity on their cell surface than did wild-type cells, and homozygous
transgenic females from multiple independent lines failed to lactate. Transgenic animals
expressing the purely biosynthetic form of GalTase lactated normally. Glands from
transgenic females were characterized by abnormal and reduced ductal development with a
concomitant reduction in alveolar expansion during pregnancy. The phenotype was not due to
a defect in cell proliferation. Morphological changes were accompanied by a dramatic
reduction in the expression of milk-specific proteins. Cultures derived from transgenic
mammary glands were unable to form anastomosing networks of epithelial cells and failed to
express milk-specific proteins, unlike wild-type mammary cultures that formed epithelial
tubules and expressed milk proteins. Our results suggest that cell surface GalTase is an
important mediator of mammary cell interaction with the extracellular matrix. Furthermore,
perturbing surface GalTase levels inhibits the expression of mammary-specific gene
products, implicating GalTase as a component of a receptor-mediated signal transduction
pathway required for normal mammary gland differentiation.
Citation
Hathaway, H.J., and Shur, B.D. (1996). Mammary gland morphogenesis is inhibited in
transgenic mice that overexpress cell surface b1,4-galactosyltransferase. Development
122:2859-2872.
Barcellos-Hoff, M.H. (1992). Mammary epithelial reorganization on extracellular matrix is mediated by cell surface galactosyltransferase. Exp. Cell Res. 201:225-234.
Background
In somatic cells, GalTase is expressed in two isoforms, termed long and short GalTase, due
to differential transcription initiation from a single GalTase gene. The two proteins
differ in that the long form contains an additional 13 amino acids within the cytoplasmic
domain not found in the short form. Results show that the long GalTase isoform represents
the biologically relevant isoform on the cell surface where it mediates cellular
interactions. Recent studies suggest that cell surface GalTase participates as a receptor
in a variety of cellular interactions by associating with the cytoskeleton and specific
signal transduction pathways, including the heterotrimeric G proteins. One cell-matrix
interaction in which GalTase participates is mammary epithelial cell migration on
laminin-containing extracellular matrices. We therefore created mice that overexpress
surface GalTase to ask how this aberrant expression might affect mammary gland
morphogenesis as well as other morphogenetic events.
Transgene
The transgene consists of the complete coding region of long form GalTase cDNA and
contains intron 4 derived from genomic GalTase. This construct was inserted into the
EV-142 vector, containing the mouse metallothionein (MT-1) promoter and the human growth
hormone polyadenylation signal (hGH).
mouse strain
C57/Bl6 X DBA
Females homozygous for GalTase do not lactate. Heterozygotes do lactate normally (with one or two exceptions) probably reflecting different levels of transgene expression. All studies were therefore done comparing homozygous females and age-matched wild-type females. In 5-week-old transgenic virgins, the ductal tree showed reduced development and TEB1s were smaller than wild-type. Histologically, ductal structure and TEB epithelia appeared relatively normal. By 10 weeks, virgin mammary ducts appeared irregular and dilated, and secondary branching was reduced. Duct termini were enlarged; histologically these enlarged termini were distinct from TEBs. The duct epithelia of transgenic 10-week-old virgins was detached from the surrounding basement membrane. During pregnancy, transgenic mammary glands had dramatically reduced alveolar development, and alveolar epithelial cells did not appear to be secretory, compared to wild-type pregnant glands. On the day of parturition, alveolar development in transgenic glands remained low, and little to no milk was produced. Milk protein gene expression was concommitantly reduced in transgenic glands. Cultures of mammary epithelial cells derived from pregnant transgenic animals displayed abnormal interactions with a laminin-containing extracellular matrix.
Mammary development
Mammary gland development was abnormal in the transgenic mice from the onset of puberty (earlier stages have not been examined). The reduced ductal development and branching, as well as the abnormal morphology of the ducts and TEB's, became quite pronounced by 10 weeks of age in the virgin female. The defects were not due to a delay of development, but were accompanied by morphogenetic defects in the ductal epithelia, particularly in the TEB's. Proliferation rates were similar in wild-type and transgenic glands, suggesting that another mechanism, such as apoptosis, is responsible for the reduced ductal development. Morphogenetic abnormalities persist into pregnancy and at parturition, but it is not clear if these defects are secondary, and due to the reduced ductal density.
Gene expression
Since the long GalTase transgene is under the control of the metallothionein promoter,
expression is seen in multiple tissues. In fact, homozygous animals display a high rate of
neonatal lethality (up to 95%). Those animals that do survive appear to have a normal life
span, and can reproduce normally, with the exception of the lactation defect. In the
mammary gland, transgene expression, assessed by RNase protection, was high at all stages
examined (5-week-old virgin, through parturition). Western blotting detected higher levels
of GalTase protein, and GalTase enzyme assays on intact mammary epithelial cells
demonstrated a 2.3-fold increase in surface GalTase activity in transgenics compared to
wild-type.
Mechanistic implications
Cell surface GalTase has been shown to be a receptor for laminin in a variety of cell
types, and is specifically involved in cellular spreading and migration on (but not
attachment to) laminin. The cumulative in vivo and in vitro observations in this study are
consistent with the hypothesis that surface GalTase participates during normal mammary
epithelial cell interactions with the laminin-rich basal lamina, and that overexpression
of surface GalTase interferes with ductal morphogenesis. Although mammary gland
morphogenesis is reduced, we saw no evidence for a decrease in cell proliferation.
Interestingly, recent work in the Bissell and Werb labs has demonstrated that the loss of
cell-basement membrane interactions results in an increase in mammary epithelial cell
apoptosis during the normal process of involution. One possiblity to account for the
GalTase transgenic phenotype is that GalTase overexpression disturbs normal cell-basement
membrane interactions, resulting in an increase in apoptosis at an inappropriate stage of
development. Experiments to address this question and others is ongoing.
key words
galactosyltransferase, growth factors, mammary development, basement membrane, cell-matrix
interactions, transgene, metallothionein
Submitted by : Helen Hathaway on November 19, 1996
Emory University School of Medicine
Dept. of Anatomy & Cell Biology
1648 Pierce Dr.
Atlanta, GA 30322
USA
Phone: (404) 727-8158
Fax: (404) 727-6256
Contact: hhath@anatomy.emory.edu