PMCA2 regulates HER2 protein kinase localization and signaling and promotes HER2-mediated breast cancer.

In the lactating mammary gland, the plasma membrane calcium ATPase2 (PMCA2) transports milk calcium. Its expression is activated in breast cancers, where high tumor levels predict increased mortality. We find that PMCA2 expression correlates with HER2 levels in breast cancers and that PMCA2 interacts with HER2 in specific actin-rich membrane domains. Knocking down PMCA2 increases intracellular calcium, disrupts interactions between HER2 and HSP-90, inhibits HER2 signaling, and results in internalization and degradation of HER2. Manipulating PMCA2 levels regulates the growth of breast cancer cells, and knocking out PMCA2 inhibits the formation of tumors in mouse mammary tumor virus (MMTV)-Neu mice. These data reveal previously unappreciated molecular interactions regulating HER2 localization, membrane retention, and signaling, as well as the ability of HER2 to generate breast tumors, suggesting that interactions between PMCA2 and HER2 may represent therapeutic targets for breast cancer.

The scaffolding protein NHERF1 regulates the stability and activity of the tyrosine kinase HER2.

We examined whether the scaffolding protein sodium-hydrogen exchanger regulatory factor 1 (NHERF1) interacts with the calcium pump PMCA2 and the tyrosine kinase receptor ErbB2/HER2 in normal mammary epithelial cells and breast cancer cells. NHERF1 interacts with the PDZ-binding motif in PMCA2 in both normal and malignant breast cells. NHERF1 expression is increased in HER2-positive breast cancers and correlates with HER2-positive status in human ductal carcinoma in situ (DCIS) lesions and invasive breast cancers as well as with increased mortality in patients. NHERF1 is part of a multiprotein complex that includes PMCA2, HSP90, and HER2 within specific actin-rich and lipid raft-rich membrane signaling domains. Knocking down NHERF1 reduces PMCA2 and HER2 expression, inhibits HER2 signaling, dissociates HER2 from HSP90, and causes the internalization, ubiquitination, and degradation of HER2. These results demonstrate that NHERF1 acts with PMCA2 to regulate HER2 signaling and membrane retention in breast cancers.

Phosphorus-31 MRI of hard and soft solids using quadratic echo line-narrowing.

Magnetic resonance imaging (MRI) of solids is rarely attempted. One of the main reasons is that the broader MR linewidths, compared to the narrow resonance of the hydrogen ((1)H) in free water, limit both the attainable spatial resolution and the signal-to-noise ratio. Basic physics research, stimulated by the quest to build a quantum computer, gave rise to a unique MR pulse sequence that offers a solution to this long-standing problem. The "quadratic echo" significantly narrows the broad MR spectrum of solids. Applying field gradients in sync with this line-narrowing sequence offers a fresh approach to carry out MRI of hard and soft solids with high spatial resolution and with a wide range of potential uses. Here we demonstrate that this method can be used to carry out three-dimensional MRI of the phosphorus ((31)P) in ex vivo bone and soft tissue samples.

The remarkable migration of the medial collateral ligament.

The developing cortical surfaces of long bones are sculpted and modeled by periosteal osteoclasts and osteoblasts. These surfaces also receive the insertions of tendons and ligaments, and these insertion sites too are modeled to form the root systems that anchor them into the cortical bone. The regulatory molecules that control modeling are poorly understood, but recent evidence suggests that parathyroid hormone-related protein (PTHrP) participates in this process. PTHrP functions principally as a paracrine regulatory molecule, and is known to be induced by mechanical loading in a number of sites. The most curious example of developmental modeling of the cortex is the migration of insertion sites such as that of the medial collateral ligament (MCL) along the bone surface during long-bone growth. We report here the mechanisms that mediate MCL migration using a combination of genetic, imaging and histological techniques. We describe a MCL migratory complex that comprises two components. The first is the MCL insertion site itself, which is a prototypical fibrous insertion site with coupled osteoclast and osteoblast activities, and its key feature is that it is anchored early in development, well before initiation of the long-bone growth spurt. Above the insertion site the periosteum is excavated by osteoclasts to form a migratory tract; this is mediated by wholly uncoupled osteoclastic bone resorption and remains as an unmineralized canal on the cortical surface in the adult. Load-induction of PTHrP appears to regulate the osteoclastic activity in both the insertion site and migratory tract.

Periosteal PTHrP regulates cortical bone modeling during linear growth in mice.

The modeling of long bone surfaces during linear growth is a key developmental process, but its regulation is poorly understood. We report here that parathyroid hormone-related peptide (PTHrP) expressed in the fibrous layer of the periosteum (PO) drives the osteoclastic (OC) resorption that models the metaphyseal-diaphyseal junction (MDJ) in the proximal tibia and fibula during linear growth. PTHrP was conditionally deleted (cKO) in the PO via Scleraxis gene targeting (Scx-Cre). In the lateral tibia, cKO of PTHrP led to a failure of modeling, such that the normal concave MDJ was replaced by a mound-like deformity. This was accompanied by a failure to induce receptor activator of NF-kB ligand (RANKL) and a 75% reduction in OC number (P ≤ 0.001) on the cortical surface. The MDJ also displayed a curious threefold increase in endocortical osteoblast mineral apposition rate (P ≤ 0.001) and a thickened cortex, suggesting some form of coupling of endocortical bone formation to events on the PO surface. Because it fuses distally, the fibula is modeled only proximally and does so at an extraordinary rate, with an anteromedial cortex in CD-1 mice that was so moth-eaten that a clear PO surface could not be identified. The cKO fibula displayed a remarkable phenotype, with a misshapen club-like metaphysis and an enlargement in the 3D size of the entire bone, manifest as a 40-45% increase in the PO circumference at the MDJ (P ≤ 0.001) as well as the mid-diaphysis (P ≤ 0.001). These tibial and fibular phenotypes were reproduced in a Scx-Cre-driven RANKL cKO mouse. We conclude that PTHrP in the fibrous PO mediates the modeling of the MDJ of long bones during linear growth, and that in a highly susceptible system such as the fibula this surface modeling defines the size and shape of the entire bone.

PMCA2 regulates apoptosis during mammary gland involution and predicts outcome in breast cancer.

After lactation, weaning causes mammary epithelial cell (MEC) apoptosis. MECs express the plasma membrane calcium-ATPase 2 (PMCA2), which transports calcium across the apical surface of the cells into milk. Here we show that PMCA2 is down-regulated early in mammary involution associated with changes in MEC shape. We demonstrate that loss of PMCA2 expression raises intracellular calcium levels and sensitizes MECs to apoptosis. In contrast, overexpression of PMCA2 in T47D breast cancer cells lowers intracellular calcium and protects them from apoptosis. Finally, we show that high PMCA2 expression in breast cancers is associated with poor outcome. We conclude that loss of PMCA2 expression at weaning triggers apoptosis by causing cellular calcium crisis. PMCA2 overexpression, on the other hand, may play a role in breast cancer progression by conferring resistance to apoptosis.

Lactation and neonatal nutrition: defining and refining the critical questions.

This paper resulted from a conference entitled "Lactation and Milk: Defining and refining the critical questions" held at the University of Colorado School of Medicine from January 18-20, 2012. The mission of the conference was to identify unresolved questions and set future goals for research into human milk composition, mammary development and lactation. We first outline the unanswered questions regarding the composition of human milk (Section I) and the mechanisms by which milk components affect neonatal development, growth and health and recommend models for future research. Emerging questions about how milk components affect cognitive development and behavioral phenotype of the offspring are presented in Section II. In Section III we outline the important unanswered questions about regulation of mammary gland development, the heritability of defects, the effects of maternal nutrition, disease, metabolic status, and therapeutic drugs upon the subsequent lactation. Questions surrounding breastfeeding practice are also highlighted. In Section IV we describe the specific nutritional challenges faced by three different populations, namely preterm infants, infants born to obese mothers who may or may not have gestational diabetes, and infants born to undernourished mothers. The recognition that multidisciplinary training is critical to advancing the field led us to formulate specific training recommendations in Section V. Our recommendations for research emphasis are summarized in Section VI. In sum, we present a roadmap for multidisciplinary research into all aspects of human lactation, milk and its role in infant nutrition for the next decade and beyond.

Cerebral arterial and ventricular morphology of the dogfish (Squalus acanthias), American bullfrog (Rana catesbeiana), and green iguana (Iguana iguana): Arterial high-resolution micro-CT, dissection, and radiography study.

This study's objective was to investigate obtaining high-resolution micro-computed tomography (CT) imaging of the injected arterial circulation of the brains of the dogfish (Squalus acanthias), American bullfrog (Rana catesbeiana), and green iguana (Iguana iguana). No micro-CT images of the arterial morphology of the brains of these vertebrates were previously published. Micro-CT imaging was performed on brains that had the cerebral arterial and ventricular systems injected with a radiopaque barium-gelatin compound in the early 1970s. These specimens were dissected and placed in a preservative fluid for 35 years, until imaged with micro-CT. The obtained micro-CT images were processed with a software program that provided 3D rotational motion rendering, and sequential display of 2D renderings of the micro-CT data. The anatomic information provided by the high-resolution micro-CT is not reproducible by any other radiopaque contrast currently available, without tissue removal corrosion, and enhanced the dissection information. The digital videos of the micro-CT 3D rotational motion rendering and sequential display of 2D renderings of the dogfish, bullfrog, and green iguana, demonstrate the extent of the arterial network within the brain, the arterial segments obscured by overlying structures such as nerves, and identified in the bullfrog the venous cerebral circulation resulting from the centrifugal leptomeningeal arterial capillaries. The rotational 3D images separated superimposed arterial structures, and the sequential display of the 2D renderings clarifies the relationship of cut or overlapped arterial branches. Comparing the brain and arterial morphology of the dogfish, bullfrog, and green iguana demonstrates some of the evolutionary modifications in these vertebrates.

An atypical degenerative osteoarthropathy in Hyp mice is characterized by a loss in the mineralized zone of articular cartilage.

Patients with X-linked hypophosphatemia (XLH) develop enthesophytes and osteophytes secondary to articular cartilage degeneration and together are the primary cause of morbidity in adult patients so afflicted. We have previously characterized the enthesopathy in Hyp mice, a murine model of XLH. We now extend these studies to the synovial joint in order to characterize potential cellular changes in articular cartilage that may predispose patients to the osteoarthropathy of XLH. We report that, despite highly elevated levels of alkaline phosphatase activity throughout articular cartilage, there is a complete loss in the mineralized zone of articular cartilage as assessed by von Kossa staining of mineral and as quantified by EPIC-microCT analysis and evidence of vascular invasion. We also identify the downregulation of extracellular matrix (ECM) factors identified as regulators of terminally differentiated mineralizing articular chondrocytes. There is also a striking increase in the histochemical staining of sulfated proteoglycans, a change that may reflect the loss of a transitional tissue that reduces mechanical stress at the interface between cartilage and subchondral bone. The failure of mineralizing articular chondrocytes to develop in the hypophosphatemic state suggests that phosphate may be a key regulator of chondrocyte mineralization. Accordingly, we find that the appropriate zonal arrangement and phenotypic markers of articular cartilage are significantly reestablished by phosphate-replacement therapy. Given the turnover and maintenance of articular cartilage ECM, the identification of early and abnormal cellular changes unique to XLH will undoubtedly aid in a more effective management of this disease to minimize the onset of degenerative osteoarthropathy.

The calcium-sensing receptor regulates plasma membrane calcium adenosine triphosphatase isoform 2 activity in mammary epithelial cells: a mechanism for calcium-regulated calcium transport into milk.

The calcium-sensing receptor (CaR) regulates transepithelial calcium transport into milk by mammary epithelial cells. Using a genome-wide screening strategy, we identified the plasma membrane calcium ATPase isoform 2 (PMCA2) as a potential downstream target of the CaR. We show that PMCA2 expression in the mouse mammary gland increases during lactation and that PMCA2 is localized solely to the apical plasma membrane of mammary epithelial cells. In milk from deafwaddler mice, which have mutations in the gene encoding PMCA2, calcium concentrations were reduced, confirming its importance in calcium transport into milk. Furthermore, in cultured primary and EpH4 mouse mammary epithelial cells, CaR stimulation up-regulated calcium-dependent ATPase activity in plasma membrane preparations. By small interfering RNA-mediated gene knockdown of PMCA2, we show that PMCA2 accounts for the preponderance of calcium-ATPase activity. We also show that reduction of CaR expression with small interfering RNA eliminates the ability of extracellular calcium to elicit an increase in calcium-dependent ATPase activity in EpH4 cell membranes. These results demonstrate that activation of the CaR increases PMCA2 activity in mouse mammary epithelial cells, providing a mechanism for the regulation of transepithelial calcium transport by calcium in the lactating mouse mammary gland.

Weaning triggers a decrease in receptor activator of nuclear factor-kappaB ligand expression, widespread osteoclast apoptosis, and rapid recovery of bone mass after lactation in mice.

A significant portion of milk calcium comes from the mother's skeleton, and lactation is characterized by rapid bone loss. The most remarkable aspect of this bone loss is its complete reversibility, and the time after weaning is the most rapid period of skeletal anabolism in adults. Despite this, little is known of the mechanisms by which the skeleton repairs itself after lactation. We examined changes in bone and calcium metabolism defining the transition from bone loss to bone recovery at weaning in mice. Bone mass decreases during lactation and recovers rapidly after weaning. Lactation causes changes in bone microarchitecture, including thinning and perforation of trabecular plates that are quickly repaired after weaning. Weaning causes a rapid decline in urinary C-telopeptide levels and stimulates an increase in circulating levels of osteocalcin. Bone histomorphometry documented a significant reduction in the numbers of osteoclasts on d 3 after weaning caused by a coordinated wave of osteoclast apoptosis beginning 48 h after pup removal. In contrast, osteoblast numbers and bone formation rates, which are elevated during lactation, remain so 3 d after weaning. The cessation of lactation stimulates an increase in circulating calcium levels and a reciprocal decrease in PTH levels. Finally, weaning is associated with a decrease in levels of receptor activator of nuclear factor-kappaB ligand mRNA in bone. In conclusion, during lactation, bone turnover is elevated, and bone loss is rapid. Weaning causes selective apoptosis of osteoclasts halting bone resorption. The sudden shift in bone turnover favoring bone formation subsequently contributes to the rapid recovery of bone mass.

The calcium-sensing receptor regulates mammary gland parathyroid hormone-related protein production and calcium transport.

The transfer of calcium from mother to milk during lactation is poorly understood. In this report, we demonstrate that parathyroid hormone-related protein (PTHrP) production and calcium transport in mammary epithelial cells are regulated by extracellular calcium acting through the calcium-sensing receptor (CaR). The CaR becomes expressed on mammary epithelial cells at the transition from pregnancy to lactation. Increasing concentrations of calcium, neomycin, and a calcimimetic compound suppress PTHrP secretion by mammary epithelial cells in vitro, whereas in vivo, systemic hypocalcemia increases PTHrP production, an effect that can be prevented by treatment with a calcimimetic. Hypocalcemia also reduces overall milk production and calcium content, while increasing milk osmolality and protein concentrations. The changes in milk calcium content, milk osmolality, and milk protein concentration were mitigated by calcimimetic infusions. Finally, in a three-dimensional culture system that recapitulates the lactating alveolus, activation of the basolateral CaR increases transcellular calcium transport independent of its effect on PTHrP. We conclude that the lactating mammary gland can sense calcium and adjusts its secretion of calcium, PTHrP, and perhaps water in response to changes in extracellular calcium concentration. We believe this defines a homeostatic system that helps to match milk production to the availability of calcium.

Mammary-specific deletion of parathyroid hormone-related protein preserves bone mass during lactation.

Large amounts of calcium are transferred to offspring by milk. This demand results in negative calcium balance in lactating mothers and is associated with rapid bone loss. The mechanisms of bone loss during lactation are only partly understood. Several studies have suggested that parathyroid hormone-related protein (PTHrP) might be secreted into the circulation by the lactating mammary gland and regulate bone turnover during lactation. Because mammary development fails in the absence of PTHrP, conventional PTHrP knockout mice cannot be used to address this possibility. To examine this hypothesis, we therefore used mice carrying a beta-lactoglobulin promoter-driven Cre transgene, one null PTHrP allele, and one floxed PTHrP allele. Expression of Cre specifically in mammary epithelial cells during late pregnancy and lactation resulted in efficient deletion of the PTHrP gene; mammary gland PTHrP mRNA and milk PTHrP protein were almost completely absent. Removal of PTHrP from the lactating mammary glands resulted in reductions in levels of circulating PTHrP and 1,25-dihydroxy vitamin D and urinary cAMP. In addition, bone turnover was reduced and bone loss during lactation was attenuated. We conclude that during lactation mammary epithelial cells are a source of circulating PTHrP that promotes bone loss by increasing rates of bone resorption.

HER2 signaling regulates HER2 localization and membrane retention.

ErbB2/HER2/Neu is a receptor tyrosine kinase that is overexpressed in 25-30% of human breast cancers, usually associated with amplification of the ERBB2 gene. HER2 has no recognized ligands and heterodimers between HER2 and EGFR (ErbB1/HER1) or HER2 and ErbB3/HER3 are important in breast cancer. Unlike other ErbB family members, HER2 is resistant to internalization and degradation, and remains at the cell surface to signal for prolonged periods after it is activated. Although the mechanisms underlying retention of HER2 at the cell surface are not fully understood, prior studies have shown that, in order to avoid internalization, HER2 must interact with the chaperone, HSP90, and the calcium pump, PMCA2, within specific plasma membrane domains that protrude from the cell surface. In this report, we demonstrate that HER2 signaling, itself, is important for the formation and maintenance of membrane protrusions, at least in part, by maintaining PMCA2 expression and preventing increased intracellular calcium concentrations. Partial genetic knockdown of HER2 expression or pharmacologic inhibition of HER2 signaling causes the depletion of membrane protrusions and disruption of the interactions between HER2 and HSP90. This is associated with the ubiquitination of HER2, its internalization with EGFR or HER3, and its degradation. These results suggest a model by which some threshold of HER2 signaling is required for the formation and/or maintenance of multi-protein signaling complexes that reinforce and prolong HER2/EGFR or HER2/HER3 signaling by inhibiting HER2 ubiquitination and internalization.

Hypercalcemia of malignancy due to ectopic transactivation of the parathyroid hormone gene.

CONTEXT: The physiology of PTH is well described, but regulation of PTH gene expression remains enigmatic. This is, at least in part, because of a lack of suitable cell culture systems. OBJECTIVE, DESIGN, SETTING, PATIENTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: We report a case of severe hyperparathyroidism resulting from the ectopic production of PTH by a pancreatic malignancy. Cells from the primary tumor (PEPP1 cells) were established in culture to examine the etiology of ectopic PTH gene expression in this patient. RESULTS AND CONCLUSIONS: We failed to find amplification or rearrangement of the PTH gene but documented hypomethylation of the PTH promoter in tumor tissue. We found that PEPP1 cells support expression of a reporter gene containing regulatory sequences from the human PTH gene promoter. Therefore, this is the first report documenting ectopic PTH production by a tumor as the result of transactivation of the PTH gene. PEPP1 cells may be useful for future studies aimed at elucidating the details of PTH gene regulation.

The calcium-sensing receptor regulates PTHrP production and calcium transport in the lactating mammary gland.

Lactating mammals must supply large amounts of calcium to the mammary gland where it is transported across mammary epithelial cells and into milk. This demand for calcium is associated with transient loss of bone mass, triggered, in part, by the secretion of parathyroid hormone-related protein (PTHrP) from the mammary gland into the circulation. The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium ions. It is responsible for coordinating calcium homeostasis by regulating parathyroid hormone secretion in the parathyroid glands and by regulating calcium handling in the renal tubules. Previous studies had shown that the CaR is expressed on mammary epithelial cells during lactation, and it had been suggested that CaR signaling in the mammary gland helps to coordinate its production of PTHrP and calcium transport into milk. In this study, we examined mammary gland PTHrP production and calcium transport in CaR(+/-) mice, a genetic model of CaR insufficiency. We found that haploinsufficiency for the CaR resulted in increased PTHrP production both in vivo and in vitro. In contrast, CaR haploinsufficiency impaired calcium transport into milk in vivo and transepithelial calcium transport by mammary epithelial cells in vitro. These data provide genetic confirmation that the CaR regulates PTHrP production and calcium transport in the lactating mammary gland. This allows the mammary gland to become a calcium-sensing organ and to participate in systemic calcium homeostasis during lactation.

Calcium-Sensing Receptor Promotes Breast Cancer by Stimulating Intracrine Actions of Parathyroid Hormone-Related Protein.

Parathyroid hormone-related protein (PTHrP) contributes to the development and metastatic progression of breast cancer by promoting hypercalcemia, tumor growth, and osteolytic bone metastases, but it is not known how PTHrP is upregulated in breast tumors. Here we report a central role in this process for the calcium-sensing receptor, CaSR, which enables cellular responses to changes in extracellular calcium, through studies of CaSR-PTHrP interactions in the MMTV-PymT transgenic mouse model of breast cancer and in human breast cancer cells. CaSR activation stimulated PTHrP production by breast cancer cells in vitro and in vivo Tissue-specific disruption of the casr gene in mammary epithelial cells in MMTV-PymT mice reduced tumor PTHrP expression and inhibited tumor cell proliferation and tumor outgrowth. CaSR signaling promoted the proliferation of human breast cancer cell lines and tumor cells cultured from MMTV-PyMT mice. Further, CaSR activation inhibited cell death triggered by high extracellular concentrations of calcium. The actions of the CaSR appeared to be mediated by nuclear actions of PTHrP that decreased p27(kip1) levels and prevented nuclear accumulation of the proapoptotic factor apoptosis inducing factor. Taken together, our findings suggest that CaSR-PTHrP interactions might be a promising target for the development of therapeutic agents to limit tumor cell growth in bone metastases and in other microenvironments in which elevated calcium and/or PTHrP levels contribute to breast cancer progression. Cancer Res; 76(18); 5348-60. ©2016 AACR.

OPG Treatment Prevents Bone Loss During Lactation But Does Not Affect Milk Production or Maternal Calcium Metabolism.

Lactation is associated with increased bone turnover and rapid bone loss, which liberates skeletal calcium used for milk production. Previous studies suggested that an increase in the skeletal expression of receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells ligand (RANKL) coupled with a decrease in osteoprotegerin (OPG) levels likely triggered bone loss during lactation. In this study, we treated lactating mice with recombinant OPG to determine whether bone loss during lactation was dependent on RANKL signaling and whether resorption of the maternal skeleton was required to support milk production. OPG treatment lowered bone resorption rates and completely prevented bone loss during lactation but, surprisingly, did not decrease osteoclast numbers. In contrast, OPG was quite effective at lowering osteoblast numbers and inhibiting bone formation in lactating mice. Furthermore, treatment with OPG during lactation prevented the usual anabolic response associated with reversal of lactational bone loss after weaning. Preventing bone loss had no appreciable effect on milk production, milk calcium levels, or maternal calcium homeostasis when mice were on a standard diet. However, when dietary calcium was restricted, treatment with OPG caused maternal hypocalcemia, maternal death, and decreased milk production. These studies demonstrate that RANKL signaling is a requirement for bone loss during lactation, and suggest that osteoclast activity may be required to increase osteoblast numbers during lactation in preparation for the recovery of bone mass after weaning. These data also demonstrate that maternal bone loss is not absolutely required to supply calcium for milk production unless dietary calcium intake is inadequate.

Low estrogen and high parathyroid hormone-related peptide levels contribute to accelerated bone resorption and bone loss in lactating mice.

Providing enough calcium for milk production stresses calcium homeostasis in lactating mammals. A universal response to these demands for calcium appears to be the mobilization of maternal skeletal reserves, and bone loss during lactation has been well documented. However, the regulation of calcium and skeletal metabolism during lactation remains enigmatic. Our study was designed to examine mineral and bone metabolism in lactating mice. We found that mice lose bone rapidly at all sites during lactation. Bone mineral density as determined by dual-energy x-ray absorptiometry was 20 to 30% lower at the spine, femur, and total body in lactating compared with either age-matched virgin or pregnant mice. The decrease in bone mineral density was accompanied by dramatic reductions in bone volume and changes in trabecular architecture. Bone loss was also accompanied by increases in bone turnover as determined by biochemical markers and histomorphometry. PTHrP levels were elevated during lactation and correlated positively with markers of bone resorption and negatively with bone mass at all sites. Estrogen levels were low during lactation and correlated negatively with bone resorption markers. Finally, estrogen and pamidronate treatment lowered rates of bone resorption to baseline virgin levels and mitigated, but did not prevent, bone loss. These data suggest that the combination of estrogen deficiency and elevations in circulating PTHrP during lactation act to stimulate bone resorption and promote bone loss.

Deletion of the nuclear localization sequences and C-terminus of PTHrP impairs embryonic mammary development but also inhibits PTHrP production.

Parathyroid hormone-related protein (PTHrP) can be secreted from cells and interact with its receptor, the Type 1 PTH/PTHrP Receptor (PTHR1) in an autocrine, paracrine or endocrine fashion. PTHrP can also remain inside cells and be transported into the nucleus, where its functions are unclear, although recent experiments suggest that it may broadly regulate cell survival and senescence. Disruption of either the PTHrP or PTHR1 gene results in many abnormalities including a failure of embryonic mammary gland development in mice and in humans. In order to examine the potential functions of nuclear PTHrP in the breast, we examined mammary gland development in PTHrP (1-84) knock-in mice, which express a mutant form of PTHrP that lacks the C-terminus and nuclear localization signals and which can be secreted but cannot enter the nucleus. Interestingly, we found that PTHrP (1-84) knock-in mice had defects in mammary mesenchyme differentiation and mammary duct outgrowth that were nearly identical to those previously described in PTHrP-/- and PTHR1-/- mice. However, the mammary buds in PTHrP (1-84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function. Examination of the effects of nuclear PTHrP in the mammary gland in vivo will require the development of alternative animal models.