Overexpression of parathyroid hormone-related protein or parathyroid
hormone in transgenic mice impairs branching morphogenesis during mammary gland
development
Summary
Overexpression of PTHrP targeted to myoepithelial cells in the mammary glands of
transgenic mice resulted in a form of hypoplasia characterized by a profound defect in
branching morphogenesis of the developing mammary duct system. Also, these mice displayed
a deficiency in lobuloalveolar development during pregnancy that appeared to be, in part,
the consequence of an impaired ability to form terminal ducts in response to estrogen and
progesterone stimulation. The effects of PTHrP on branching morphogenesis during mammary
development are apparently mediated by amino-terminal PTH-like sequences that signal
through the PTH/PTHrP receptor, since overexpression of parathyroid hormone itself in the
mammary glands of transgenic mice caused a similar developmental phenotype, and delivery
of PTHrP (residues 1-36) via locally-implanted slow-release pellets impaired mammary
development in normal mice. These results suggest that PTHrP, which is a native product of
mammary epithelial and myoepithelial cells, plays a role in normal mammary development as
a locally-secreted inhibitor of differentiation.
Citations
Wysolmerski, J.J., Broadus, A.E., Zhou, J., Fuchs, E., Milstone, L.M. and Philbrick, W.M.
Overexpression of parathyroid hormone-related protein in the skin of transgenic mice
interferes with hair follicle development. Proc. Natl. Acad. Sci. USA. 91, 1133-1137
(1994).
Wysolmerski, J.J., McCaughern-Carucci, J.F., Daifotis, A.G., Broadus, A.E., and Philbrick, W. M. Overexpression of parathyroid hormone-related protein or parathyroid hormone in transgenic mice impairs branching morphogenesis during mammary gland development. Development 121, 3539-3547 (1995).
Note: The role of PTHrP in ductal morphogenesis has been studied in knockout
mice
Rescue of the Parathyroid
Hormone-related Protein Knockout Mouse Demonstrates that Parathyroid Hormone-related
Protein is Essential for Mammary Gland Development
Background
Parathyroid hormone-related protein (PTHrP) was originally discovered as the tumor product
that is responsible for most instances of the syndrome of humoral hypercalcemia of
malignancy. Subsequently, PTHrP has been found to be produced by a wide variety of normal
adult and fetal tissues and has been implicated in a variety of biological processes such
as cell growth and differentiation, the regulation of smooth muscle tone, placental
calcium transport and fetal development. Recently, the PTH receptor has also been shown to
be widely expressed in many tissues not involved in systemic calcium homeostasis. PTHrP
mRNA was found to be expressed in lactating mammary tissue, and large amounts of the
peptide were detected in milk. Immunohistochemical studies have localized PTHrP to both
ductal epithelial cells and myoepithelial cells of the human and canine non-pregnant
mammary gland and biochemical responses to PTHrP have been detected in myoepithelial
cells. Overexpression of PTHrP in the myoepithelial cells of transgenic mice directed by
the keratin 14 (K14) promoter was found to result in mammary hypoplasia characterized by a
severe defect in branching morphogenesis.
Transgene
The K14-PTHrP transgene was constructed by joining a 2 kb fragment of the human keratin 14
promoter region to a 568 bp EcoRI-StyI cDNA encoding the human PTHrP 1-141 isoform. Human
growth hormone sequences were included downstream to provide termination /polyadenylation
sites. The K14-PTH transgene was constructed by replacing the PTHrP cDNA fragment with a
539 bp DdeI-HinfI cDNA fragment encoding human PTH (residues 1-84).
mouse strain
(C57BL/6 X SJL)F2 transgenics have been crossed to CD1 outbred mice
In mature nulliparous transgenic animals at six weeks of age, the overall growth of the duct system into the fat pad appeared to be delayed, and the transgenic gland displayed a much simpler, sparser duct structure than the control, with a dramatic reduction in the degree of side branching and many fewer small tertiary and quaternary ducts. Although by 10 - 12 weeks of age the transgenic duct system had grown nearly to the borders of the fat pad, the defect in side branching persisted. By day 17 of pregnancy, transgenic glands appeared to have many fewer, less well-developed alveoli. On examination of H&E-stained sections, the individual alveoli in the transgenic gland appeared normal in their overall architecture, but there were fewer clusters of alveoli, and the individual acini were smaller and less well expanded as compared to the normal gland. These differences persisted into lactation.
Mammary development
Overexpression of PTHrP in myoepithelial cells of the murine mammary gland results in an
impairment of mammary development in adolescence and early pregnancy. During sexual
maturation, K14-PTHrP mice manifest a slower rate of ductal elongation, as well as a
marked inhibition of side branching. There also appear to be changes in lobuloalveolar
development in the transgenic mice characterized by the delayed formation of a reduced
number of alveoli. It is difficult to discern whether this latter defect is simply the
result of a reduced ductal mass or whether overexpression of PTHrP also impairs
lobuloalveolar development per se, especially since keratin 14 expression has been
detected in developing alveolar cells in early pregnancy. Previous studies have suggested
that mammary epithelial cells elaborate some growth inhibiting factor(s) that helps to
maintain the proper spacing of ducts within the gland, and it is possible that as a
locally-active inhibitor of mammary development, PTHrP might normally play such a role.
Gene expression
The levels of transgene expression as measured by RNase protection mirrored those of the
native murine K14 gene during the stages of mammary gland development. PTHrP
immunoreactivity appeared to be located in both myoepithelial cells (as defined by a-actin
staining) and the basement membrane of the transgenic ducts. Levels of bioactive PTHrP
were measured in acid-urea extracts of mammary tissue using a PTH-sensitive rat
osteosarcoma cell assay. Transgenic glands contained 100 fmol of PTHrP per mg total
protein, while normal mammary tissue was below the detection limit of the assay.
Mechanistic implications
PTH and PTHrP share a short stretch of amino-terminal homology that allows each protein to
bind and to activate a common PTH/PTHrP receptor. Although PTHrP may exert many of its
physiological effects through the actions of this receptor, there is also evidence that
PTHrP may be processed to give rise to several biologically active peptides that would
presumably signal through other than the conventional PTH/PTHrP receptor. To resolve this
issue, K14-PTH transgenic mice were created and were found to exhibit a profound delay in
the overall growth of the transgenic duct system into the mammary fat pad, as well as a
deficiency in ductular side branching. These defects were essentially identical to those
seen in the K14-PTHrP mice, implicating amino-terminal PTHrP and the PTH/PTHrP receptor as
the main participants in the genesis of the defects in branching morphogenesis in the
K14-PTHrP mice. Furthermore, surgically implanted, slow-release pellets containing PTHrP
(residues 1-36), retarded the penetration of the ducts into the fat pad in 3-4 week-old
normal mice receiving systemic estrogen and progesterone. This suggests that PTHrP
antagonizes the growth-promoting effects of these hormones on ductular development.
key words
epithelial-mesenchymal interaction, myoepithelial cells, differentiation inhibitor.
Submitted
by William Philbrick and John Wysolmerski
John Wysolmerski
Department of Internal Medicine
Yale University School of Medicine
New Haven, CT, USA
John-Wysolmerski@Yale.edu
tel: (203) 785-5488
FAX: (203) 785-6015