Stromal Cells Are Critical Targets in the Regulation of Mammary Ductal Morphogenesis by Parathyroid Hormone-Related Protein

AbstractParathyroid hormone-related protein (PTHrP) was originally identified as the tumor product responsible for humoral hypercalcemia of malignancy. It is now known that PTHrP is produced by many normal tissues in which it appears to play a role as a developmental regulatory molecule. PTHrP is a normal product of mammary epithelial cells, and recent experiments in our laboratory have demonstrated that overexpression or underexpression of PTHrP in the murine mammary gland leads to severe disruptions in its development. The nature of these phenotypes suggests that PTHrP acts to modulate branching growth during mammary development by regulating mammary stromal cell function. We now demonstrate that throughout mammary development, during periods of active ductal-branching morphogenesis, PTHrP is produced by epithelial cells, whereas the PTH/PTHrP receptor is expressed on stromal cells. In addition, we show that mammary stromal cells in culture contain specific binding sites for amino terminal PTHrP and respond with an increase in intracellular cAMP. Finally, we demonstrate that the mammary mesenchyme must express the PTH/PTHrP receptor in order to support mammary epithelial cell morphogenesis. These results demonstrate that PTHrP and the PTH/PTHrP receptor represent an epithelial/mesenchymal signaling circuit that is necessary for mammary morphogenesis and that stromal cells are a critical target for PTHrP's action in the mammary gland

Key stages of mammary gland development: Molecular mechanisms involved in the formation of the embryonic mammary gland

The development of the embryonic mammary gland involves communication between the epidermis and mesenchyme and is coordinated temporally and spatially by various signaling pathways. Although many more genes are likely to control mammary gland development, functional roles have been identified for Wnt, fibroblast growth factor, and parathyroid hormone-related protein signaling. This review describes what is known about the molecular mechanisms that regulate embryonic mammary gland development

Calcium-sensing receptor in breast physiology and cancer

The calcium-sensing receptor (CaSR) is expressed in normal breast epithelial cells and in breast cancer cells. During lactation, activation of the CaSR in mammary epithelial cells increases calcium transport into milk and inhibits parathyroid hormone-related protein (PTHrP) secretion into milk and into the circulation. The ability to sense changes in extracellular calcium allows the lactating breast to actively participate in the regulation of systemic calcium and bone metabolism, and to coordinate calcium usage with calcium availability during milk production. Interestingly, as compared to normal breast cells, in breast cancer cells, the regulation of PTHrP secretion by the CaSR becomes rewired due to a switch in its G-protein usage such that activation of the CaSR increases instead of decreases PTHrP production. In normal cells the CaSR couples to Gi to inhibit cAMP and PTHrP production, whereas in breast cancer cells, it couples to Gs to stimulate cAMP and PTHrP production. Activation of the CaSR on breast cancer cells regulates breast cancer cell proliferation, death and migration, in part, by stimulating PTHrP production. In this article, we discuss the biology of the CaSR in the normal breast and in breast cancer, and review recent findings suggesting that the CaSR activates a nuclear pathway of PTHrP action that stimulates cellular proliferation and inhibits cell death, helping cancer cells adapt to elevated extracellular calcium levels. Understanding the diverse actions mediated by the CaSR may help us better understand lactation physiology, breast cancer progression and osteolytic bone metastases

HER2 regulates intracellular calcium concentration in SKBR3 cells.

<p>A) PMCA2 levels in control versus HER2KD-SKBR3 cells as assessed by immunoblot. B) Intracellular calcium measurements in HER2KD-SKBR3 cells relative to control. Numbers indicate the mean calcium concentrations estimated by FURA2 measurements. Each bar represents the mean ± SEM of 3 separate experiments. Asterisk denotes statistically significant difference. C) Expression of a NFAT-luciferase indicator construct in control SKBR3 cells, HER2KD-SKBR3 cells and PMCA2KD-SKBR3 cells. Each bar represents the mean ± SEM of 3 separate experiments. Asterisks denote statistically significant differences. D) Con-focal images of immunofluorescence for NFATc1 (red) or DAPI (blue) in control (top row), HER2KD-SKBR3 (middle row) or PMCA2KD-SKBR3 (bottom row) cells. Scale bars represent 10μm. E) Apoptosis as assessed by TUNEL assay in HER2KD-SKBR3 cells relative to controls exposed to differing concentrations of extracellular calcium ± ionomycin. Each bar represents the mean ± SEM of 3 separate experiments. F) Con-focal images of immunofluorescence for NFATc1 (red) or DAPI (blue) in control (top row), HER2KD-SKBR3 (middle row) or PMCA2KD-SKBR3 (bottom row) cells treated with cyclosporine A to block calcineurin activity. Scale bars represent 10μm.</p

Activation of HER2 signaling promotes the formation of membrane protrusions.

<p>A) Con-focal images of immunofluorescence for actin (phalloidin, left 2 panels), phsopho-HER2 (middle 2 panels) and phospho-AKT (right 2 panels) in SKBR3 cells in serum-free media. B) Con-focal images of immunofluorescence for actin (phalloidin, left 2 panels), phsopho-HER2 (middle 2 panels) and phospho-AKT (right 2 panels) in SKBR3 cells treated with EGF for 2 hours. C) Con-focal images of immunofluorescence for actin (phalloidin, left 2 panels), phsopho-HER2 (middle 2 panels) and phospho-AKT (right 2 panels) in SKBR3 cells treated with NRG1 for 2 hours. For each pair of panels, the image on the right represents an enlargement of the boxed area from the left panel and the insets represent Z-stack images in 2 different orientations. White arrows point to membrane protrusions. Confocal settings for capturing immunofluorescence images were adjusted to detect membrane structure and should not be interpreted as quantitative. All scale bars represent 10μm.</p