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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.

Lactation is associated with an increased demand for calcium and is accompanied by a remarkable cycle of bone loss and recovery that helps to supply calcium and phosphorus for milk production. Bone loss is the result of increased bone resorption that is due, in part, to increased levels of PTHrP and decreased levels of estrogen. However, the regulation of bone turnover during this time is not fully understood. In the 1960’s and 1970’s many observations were made to suggest that osteocytes could resorb bone and increase the size of their lacunae. This concept became know as osteocytic osteolysis and studies suggested that it occurred in response to parathyroid hormone and/or an increased systemic demand for calcium. However, this concept fell out of favor in the late 1970’s when it was established that osteoclasts were the principal bone-resorbing cells. Given that lactation is associated with increased PTHrP levels and negative calcium balance, we recently examined whether osteocytes contribute to bone loss during this time. Our findings suggest that osteocytes can remodel their perilacunar and pericanalicular matrix and that they participate in the liberation of skeletal calcium stores during reproductive cycles. These findings raise new questions about the role of osteocytes in coordinating bone and mineral metabolism during lactation as well as the recovery of bone mass after weaning. It is also interesting to consider whether osteocyte lacunar and canalicular remodeling contributes more broadly to the maintenance of skeletal and mineral homeostasis.

Normal breast epithelial cells and breast cancer cells express the calcium-sensing receptor (CaSR), the master regulator of systemic calcium metabolism. During lactation, activation of the CaSR in mammary epithelial cells downregulates parathyroid hormone-related protein (PTHrP) levels in milk and in the circulation, and increases calcium transport into milk. In contrast, in breast cancer cells the CaSR upregulates PTHrP production. A switch in G-protein usage underlies the opposing effects of the CaSR on PTHrP expression in normal and malignant breast cells. During lactation, the CaSR in normal breast cells coordinates a feedback loop that matches the transport of calcium into milk and maternal calcium metabolism to the supply of calcium. A switch in CaSR G-protein usage during malignant transformation converts this feedback loop into a feed-forward cycle in breast cancer cells that may promote the growth of osteolytic skeletal metastases.

PTHrP gene expression was evaluated in a murine keratinocyte line, Pam 212K, transformed with E1A and ras. We found that the 12S-E1A oncogene, with or without ras transformation, markedly reduced PTHrP mRNA expression. Using transient transfection assays, we found that the 12S isoform repressed activity from a 5′PTHrP-driven reporter gene. E1A-induced repression of PTHrP reporter constructs appears to be mediated by sequences within minimal promoter region. The 13S-E1A isoform did not repress PTHrP reporter gene activity, and a 13S-deletion mutant that lacked the repressor domains activated a subset of reporter constructs. Mutation of an Ets-1 binding site upstream of the basal promoter substantially decreased activation of reporter constructs by this 13S-deletion mutant. These findings suggest that the E1A oncoprotein may serve as a model for both activation and repression of PTHrP gene expression.

Parathyroid 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.

Parathyroid hormone-related protein (PTHrP) was originally discovered because of its role in humoral hypercalcemia of malignancy (HHM), a common metabolic complication of many types of cancer. In HHM, PTHrP is released into the circulation by malignant cells and cross reacts with parathyroid hormone (PTH) receptors in bone and kidney, which results in hypercalcemia. In recent years, it has become clear that PTHrP is a normal product of many adult and fetal tissues where it appears to act in an autocrine and/or paracrine fashion to regulate organogenesis. This article explores the molecular evolution of PTHrP and how this understanding has begun to shed some light on the molecular mechanisms responsible for the biochemical manifestations of HHM. In addition, the normal biological function of PTHrP is discussed, with an emphasis on its role as a developmental regulatory molecule.

Humoral hypercalcemia of malignancy is a common paraneoplastic syndrome which is characterized by hypercalcemia resulting from secretion by tumors of a circulating bone-resorbing factor. Evidence suggests that in many instances this factor is an adenylate cyclase-stimulating protein which shares features with, but is distinct from, parathyroid hormone (PTH). The current report describes the purification to homogeneity from a humoral hypercalcemia of malignancy-associated tumor of a novel, basic, highly potent PTH-like adenylate cyclase-stimulating protein. This factor differs from previously described PTH-like factors with respect to size, amino acid composition, and specific activity.