PTHrP intracrine actions divergently influence breast cancer growth through p27 and LIFR.

The role of parathyroid hormone (PTH)-related protein (PTHrP) in breast cancer remains controversial, with reports of PTHrP inhibiting or promoting primary tumor growth in preclinical studies. Here, we provide insight into these conflicting findings by assessing the role of specific biological domains of PTHrP in tumor progression through stable expression of PTHrP (-36-139aa) or truncated forms with deletion of the nuclear localization sequence (NLS) alone or in combination with the C-terminus. Although the full-length PTHrP molecule (-36-139aa) did not alter tumorigenesis, PTHrP lacking the NLS alone accelerated primary tumor growth by downregulating p27, while PTHrP lacking the NLS and C-terminus repressed tumor growth through p27 induction driven by the tumor suppressor leukemia inhibitory factor receptor (LIFR). Induction of p27 by PTHrP lacking the NLS and C-terminus persisted in bone disseminated cells, but did not prevent metastatic outgrowth, in contrast to the primary tumor site. These data suggest that the PTHrP NLS functions as a tumor suppressor, while the PTHrP C-terminus may act as an oncogenic switch to promote tumor progression through differential regulation of p27 signaling.

Parathyroid hormone-related protein in breast cancer bone metastasis.

Parathyroid hormone-related protein (PTHrP) was discovered as the tumor product causing the humoral hypercalcemia of malignancy. Its structural similarity to the hormone, PTH, with 8 of the first 13 amino acids identical, was sufficient to explain the sharing by PTHrP and PTH of a common receptor, PTH1R, although the remainder of the sequences are unique. PTHrP has important roles in development of several organs, including breast and bone, and functions as a paracrine factor postnatally in these and other tissues. In addition to its hormonal role in cancer, PTHrP is produced by two thirds of primary breast cancers and 90% of bone metastases from breast cancer, leading to the concept that its production in bone by breast cancer cells promotes bone resorption, thus favoring tumor establishment and expansion, and an exit from tumor dormancy in bone through downregulation of leukemia inducing factor receptor (LIFR). Cancer production of PTHrP is increased by bone-derived growth factors, with particular attention paid to TGFß, as well as by promoter-driven transcriptional effects, such as the hedgehog signaling factor, GLI2, and microenvironment effects including changes in underlying stiffness of substrates for cells. Although interest has been focused on PTHrP-induced bone resorption in bone metastasis, a mechanistically separate, protective effect against tumor progression has been proposed. Although there is conflicting mouse data, there are clinical studies suggesting that increased production of PTHrP by breast cancers confers upon them a less invasive phenotype, an effect distinct from the bone resorption-stimulating action that favors bone metastasis.

G-CSF Receptor Deletion Amplifies Cortical Bone Dysfunction in Mice With STAT3 Hyperactivation in Osteocytes.

Bone strength is determined by the structure and composition of its thickened outer shell (cortical bone), yet the mechanisms controlling cortical consolidation are poorly understood. Cortical bone maturation depends on SOCS3-mediated suppression of IL-6 cytokine-induced STAT3 phosphorylation in osteocytes, the cellular network embedded in bone matrix. Because SOCS3 also suppresses granulocyte-colony-stimulating factor receptor (G-CSFR) signaling, we here tested whether global G-CSFR (Csf3r) ablation altereed bone structure in male and female mice lacking SOCS3 in osteocytes, (Dmp1Cre :Socs3f/f mice). Dmp1Cre :Socs3f/f :Csf3r-/- mice were generated by crossing Dmp1Cre :Socs3f/f mice with Csf3r-/- mice. Although G-CSFR is not expressed in osteocytes, Csf3r deletion further delayed cortical consolidation in Dmp1Cre :Socs3f/f mice. Micro-CT images revealed extensive, highly porous low-density bone, with little true cortex in the diaphysis, even at 26 weeks of age; including more low-density bone and less high-density bone in Dmp1Cre :Socs3f/f :Csf3r-/- mice than controls. By histology, the area where cortical bone would normally be found contained immature compressed trabecular bone in Dmp1Cre :Socs3f/f :Csf3r-/- mice and greater than normal levels of intracortical osteoclasts, extensive new woven bone formation, and the presence of more intracortical blood vessels than the already high levels observed in Dmp1Cre :Socs3f/f controls. qRT-PCR of cortical bone from Dmp1Cre :Socs3f/f :Csf3r-/- mice also showed more than a doubling of mRNA levels for osteoclasts, osteoblasts, RANKL, and angiogenesis markers. The further delay in cortical bone maturation was associated with significantly more phospho-STAT1 and phospho-STAT3-positive osteocytes, and a threefold increase in STAT1 and STAT3 target gene mRNA levels, suggesting G-CSFR deletion further increases STAT signaling beyond that of Dmp1Cre :Socs3f/f bone. G-CSFR deficiency therefore promotes STAT1/3 signaling in osteocytes, and when SOCS3 negative feedback is absent, elevated local angiogenesis, bone resorption, and bone formation delays cortical bone consolidation. This points to a critical role of G-CSF in replacing condensed trabecular bone with lamellar bone during cortical bone formation. © 2022 American Society for Bone and Mineral Research (ASBMR).

Dmp1Cre-directed knockdown of parathyroid hormone-related protein (PTHrP) in murine decidua is associated with a life-long increase in bone mass, width, and strength in male progeny.

Parathyroid hormone-related protein (PTHrP, gene name Pthlh) is a pleiotropic regulator of tissue homeostasis. In bone, Dmp1Cre-targeted PTHrP deletion in osteocytes causes osteopenia and impaired cortical strength. We report here that this outcome depends on parental genotype. In contrast to our previous report using mice bred from heterozygous (flox/wild type) Dmp1Cre.Pthlhf/w parents, adult (16-week-old and 26-week-old) flox/flox (f/f) Dmp1Cre.Pthlhf/f mice from homozygous parents (Dmp1Cre.Pthlhf/f(hom) ) have stronger bones, with 40% more trabecular bone mass and 30% greater femoral width than controls. This greater bone size was observed in Dmp1Cre.Pthlhf/f(hom) mice as early as 12 days of age, when greater bone width was also found in male and female Dmp1Cre.Pthlhf/f(hom) mice compared to controls, but not in gene-matched mice from heterozygous parents. This suggested a maternal influence on skeletal size prior to weaning. Although Dmp1Cre has previously been reported to cause gene recombination in mammary gland, milk PTHrP protein levels were normal. The wide-bone phenotype was also noted in utero: Dmp1Cre.Pthlhf/f(hom) embryonic femurs were more mineralized and wider than controls. Closer examination revealed that Dmp1Cre caused PTHrP recombination in placenta, and in the maternal-derived decidual layer that resides between the placenta and the uterus. Decidua from mothers of Dmp1Cre.Pthlhf/f(hom) mice also exhibited lower PTHrP levels by immunohistochemistry and were smaller than controls. We conclude that Dmp1Cre leads to gene recombination in decidua, and that decidual PTHrP might, through an influence on decidual cells, limit embryonic bone radial growth. This suggests a maternal-derived developmental origin of adult bone strength. © 2021 American Society for Bone and Mineral Research (ASBMR).

Bone Geometry Is Altered by Follistatin-Induced Muscle Growth in Young Adult Male Mice.

The development of the musculoskeletal system and its maintenance depends on the reciprocal relationship between muscle and bone. The size of skeletal muscles and the forces generated during muscle contraction are potent sources of mechanical stress on the developing skeleton, and they shape bone structure during growth. This is particularly evident in hypermuscular global myostatin (Mstn)-null mice, where larger muscles during development increase bone mass and alter bone shape. However, whether muscle hypertrophy can similarly influence the shape of bones after the embryonic and prepubertal period is unknown. To address this issue, bone structure was assessed after inducing muscle hypertrophy in the lower hindlimbs of young-adult C57BL/6J male mice by administering intramuscular injections of recombinant adeno-associated viral vectors expressing follistatin (FST), a potent antagonist of Mstn. Two FST isoforms were used: the full-length 315 amino acid isoform (FST-315) and a truncated 288 amino acid isoform (FST-288). In both FST-treated cohorts, muscle hypertrophy was observed, and the anterior crest of the tibia, adjacent to the tibialis anterior muscle, was lengthened. Hypertrophy of the muscles surrounding the tibia caused the adjacent cortical shell to recede inward toward the central axis: an event driven by bone resorption adjacent to the hypertrophic muscle. The findings reveal that inducing muscle hypertrophy in mice can confer changes in bone shape in early adulthood. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Physiological and Pharmacological Roles of PTH and PTHrP in Bone Using Their Shared Receptor, PTH1R.

Parathyroid hormone (PTH) and the paracrine factor, PTH-related protein (PTHrP), have preserved in evolution sufficient identities in their amino-terminal domains to share equivalent actions upon a common G protein-coupled receptor, PTH1R, that predominantly uses the cyclic adenosine monophosphate-protein kinase A signaling pathway. Such a relationship between a hormone and local factor poses questions about how their common receptor mediates pharmacological and physiological actions of the two. Mouse genetic studies show that PTHrP is essential for endochondral bone lengthening in the fetus and is essential for bone remodeling. In contrast, the main postnatal function of PTH is hormonal control of calcium homeostasis, with no evidence that PTHrP contributes. Pharmacologically, amino-terminal PTH and PTHrP peptides (teriparatide and abaloparatide) promote bone formation when administered by intermittent (daily) injection. This anabolic effect is remodeling-based with a lesser contribution from modeling. The apparent lesser potency of PTHrP than PTH peptides as skeletal anabolic agents could be explained by lesser bioavailability to PTH1R. By contrast, prolongation of PTH1R stimulation by excessive dosing or infusion, converts the response to a predominantly resorptive one by stimulating osteoclast formation. Physiologically, locally generated PTHrP is better equipped than the circulating hormone to regulate bone remodeling, which occurs asynchronously at widely distributed sites throughout the skeleton where it is needed to replace old or damaged bone. While it remains possible that PTH, circulating within a narrow concentration range, could contribute in some way to remodeling and modeling, its main physiological role is in regulating calcium homeostasis.

PTH1R Actions on Bone Using the cAMP/Protein Kinase A Pathway.

After the initial signaling action of parathyroid hormone (PTH) on bone was shown to be activation of adenylyl cyclase, its target was found to be cells of the osteoblast lineage, to the exclusion of osteoclasts and their precursors. This led to the view that the osteoblast lineage regulated osteoclast formation, a proposal that was established when the molecular mechanisms of osteoclast formation were discovered. This is in addition to the effect of PTH1Rv signaling throughout the osteoblast differentiation process to favour the formation of bone-forming osteoblasts. Initial signaling in the PTH target cells through cAMP and protein kinase A (PKA) activation is extremely rapid, and marked by an amplification process in which the later event, PKA activation, precedes cAMP accumulation in time and is achieved at lower concentrations. All of this is consistent with the existence of "spare receptors", as is the case with several other peptide hormones. PTH-related protein (PTHrP), that was discovered as a cancer product, shares structural similarity with PTH in the amino-terminal domain that allows the hormone, PTH, and the autocrine/paracrine agent, PTHrP, to share actions upon a common G protein coupled receptor, PTH1R, through which they activate adenylyl cyclase with equivalent potencies. Studies of ligand-receptor kinetics have revealed that the PTH/PTH1R ligand-receptor complex, after initial binding and adenylyl cyclase activation at the plasma membrane, is translocated to the endosome, where adenylyl cyclase activation persists for a further short period. This behavior of the PTH1R resembles that of a number of hormones and other agonists that undergo such endosomal translocation. It remains to be determined whether and to what extent the cellular effects through the PTH1R might be influenced when endosomal is added to plasma membrane activation.

Multiple actions of parathyroid hormone-related protein in breast cancer bone metastasis.

The sequence similarity within the amino-terminal regions of parathyroid hormone (PTH) and PTH-related protein (PTHrP) allows the two to share actions at a common site, the PTH1 receptor. A number of biological activities have been ascribed to actions of other domains within PTHrP. PTHrP production by late stage breast cancer has been shown to contribute to bone metastasis formation through promotion of osteoclast formation and bone resorption by action through PTH1 receptors. There is evidence also for a role for PTHrP early in breast cancer that is protective against tumour progression. No signalling pathway has been identified for this effect. PTHrP has also been identified as a factor promoting the emergence of breast cancer cells from dormancy in bone. In that case, PTHrP does not function through activation of PTH1 receptors, despite having very substantial effects on transcriptional activity of the breast cancer cells. This indicates actions of PTHrP that are non-canonical, that is, mediated through domains other than the amino-terminal. It is concluded that PTHrP has several distinct paracrine, autocrine, and intracrine actions in the course of breast cancer pathophysiology. Some are mediated through action at PTH1 receptors and others are controlled by other domains within PTHrP. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

Cortical bone maturation in mice requires SOCS3 suppression of gp130/STAT3 signalling in osteocytes.

Bone strength is determined by its dense cortical shell, generated by unknown mechanisms. Here we use the Dmp1Cre:Socs3f/f mouse, with delayed cortical bone consolidation, to characterise cortical maturation and identify control signals. We show that cortical maturation requires a reduction in cortical porosity, and a transition from low to high density bone, which continues even after cortical shape is established. Both processes were delayed in Dmp1Cre:Socs3f/f mice. SOCS3 (suppressor of cytokine signalling 3) inhibits signalling by leptin, G-CSF, and IL-6 family cytokines (gp130). In Dmp1Cre:Socs3f/f bone, STAT3 phosphorylation was prolonged in response to gp130-signalling cytokines, but not G-CSF or leptin. Deletion of gp130 in Dmp1Cre:Socs3f/f mice suppressed STAT3 phosphorylation in osteocytes and osteoclastic resorption within cortical bone, leading to rescue of the corticalisation defect, and restoration of compromised bone strength. We conclude that cortical bone development includes both pore closure and accumulation of high density bone, and that these processes require suppression of gp130-STAT3 signalling in osteocytes.

Osteoclasts Provide Coupling Signals to Osteoblast Lineage Cells Through Multiple Mechanisms.

Bone remodeling is essential for the repair and replacement of damaged and old bone. The major principle underlying this process is that osteoclast-mediated resorption of a quantum of bone is followed by osteoblast precursor recruitment; these cells differentiate to matrix-producing osteoblasts, which form new bone to replace what was resorbed. Evidence from osteopetrotic syndromes indicate that osteoclasts not only resorb bone, but also provide signals to promote bone formation. Osteoclasts act upon osteoblast lineage cells throughout their differentiation by facilitating growth factor release from resorbed matrix, producing secreted proteins and microvesicles, and expressing membrane-bound factors. These multiple mechanisms mediate the coupling of bone formation to resorption in remodeling. Additional interactions of osteoclasts with osteoblast lineage cells, including interactions with canopy and reversal cells, are required to achieve coordination between bone formation and resorption during bone remodeling.

Author Correction: Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Brief exposure to full length parathyroid hormone-related protein (PTHrP) causes persistent generation of cyclic AMP through an endocytosis-dependent mechanism.

Parathyroid hormone (PTH)-related protein (PTHrP) (gene name Pthlh) was discovered as the factor responsible for the humoral hypercalcemia of malignancy. It shares such sequence similarity with PTH in the amino-terminal region that the two are equally able to act through a single G protein-coupled receptor, PTH1R. A number of biological activities are ascribed to domains of PTHrP beyond the amino-terminal domain. PTH functions as a circulating hormone, but PTHrP is generated locally in many tissues including bone, where it acts as a paracrine factor on osteoblasts and osteocytes. The present study compares how PTH and PTHrP influence cyclic AMP (cAMP) formation through adenylyl cyclase, the first event in cell activation through PTH1R. Brief exposure to full length PTHrP(1-141) in several osteoblastic cell culture systems was followed by sustained adenylyl cyclase activity for more than an hour after ligand washout. This effect was dose-dependent and was not found with shorter PTHrP or PTH peptides even though they were fully able to activate adenylyl cyclase with acute treatment. The persistent activation response to PTHrP(1-141) was seen also with later events in the cAMP/PKA pathway, including persistent activation of CRE-luciferase and sustained regulation of several CREB-responsive mRNAs, up to 24 h after the initial exposure. Pharmacologic blockade of endocytosis prevented the persistent activation of cAMP and gene responses. We conclude that full length PTHrP, the likely local physiological effector in bone, differs in intracellular action to PTH by undergoing endosomal translocation to induce a prolonged adenylyl cyclase activation in its target cells.

Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone.

Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.

IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis.

Interleukin 6 (IL-6) supports development of bone-resorbing osteoclasts by acting early in the osteoblast lineage via membrane-bound (cis) or soluble (trans) receptors. Here, we investigated how IL-6 signals and modifies gene expression in differentiated osteoblasts and osteocytes and determined whether these activities can promote bone formation or support osteoclastogenesis. Moreover, we used a genetically altered mouse with circulating levels of the pharmacological IL-6 trans-signaling inhibitor sgp130-Fc to determine whether IL-6 trans-signaling is required for normal bone growth and remodeling. We found that IL-6 increases suppressor of cytokine signaling 3 (Socs3) and CCAAT enhancer-binding protein δ (Cebpd) mRNA levels and promotes signal transducer and activator of transcription 3 (STAT3) phosphorylation by both cis- and trans-signaling in cultured osteocytes. In contrast, RANKL (Tnfsf11) mRNA levels were elevated only by trans-signaling. Furthermore, we observed soluble IL-6 receptor release and ADAM metallopeptidase domain 17 (ADAM17) sheddase expression by osteocytes. Despite the observation that IL-6 cis-signaling occurs, IL-6 stimulated bone formation in vivo only via trans-signaling. Although IL-6 stimulated RANKL (Tnfsf11) mRNA in osteocytes, these cells did not support osteoclast formation in response to IL-6 alone; binucleated TRAP+ cells formed, and only in response to trans-signaling. Finally, pharmacological, sgp130-Fc-mediated inhibition of IL-6 trans-signaling did not impair bone growth or remodeling unless mice had circulating sgp130-Fc levels > 10 µg/ml. At those levels, osteopenia and impaired bone growth occurred, reducing bone strength. We conclude that high sgp130-Fc levels may have detrimental off-target effects on the skeleton.

Tolerance to sustained activation of the cAMP/Creb pathway activity in osteoblastic cells is enabled by loss of p53.

The loss of p53 function is a central event in the genesis of osteosarcoma (OS). How mutation of p53 enables OS development from osteoblastic lineage cells is poorly understood. We and others have reported a key role for elevated and persistent activation of the cAMP/PKA/Creb1 pathway in maintenance of OS. In view of the osteoblast lineage being the cell of origin of OS, we sought to determine how these pathways interact within the context of the normal osteoblast. Normal osteoblasts (p53 WT) rapidly underwent apoptosis in response to acute elevation of cAMP levels or activity, whereas p53-deficient osteoblasts tolerated this aberrant cAMP/Creb level and activity. Using the p53 activating small-molecule Nutlin-3a and cAMP/Creb1 activator forskolin, we addressed the question of how p53 responds to the activation of cAMP. We observed that p53 acts dominantly to protect cells from excessive cAMP accumulation. We identify a Creb1-Cbp complex that functions together with and interacts with p53. Finally, translating these results we find that a selective small-molecule inhibitor of the Creb1-Cbp interaction demonstrates selective toxicity to OS cells where this pathway is constitutively active. This highlights the cAMP/Creb axis as a potentially actionable therapeutic vulnerability in p53-deficient tumors such as OS. These results define a mechanism through which p53 protects normal osteoblasts from excessive or abnormal cAMP accumulation, which becomes fundamentally compromised in OS.

Retinoic Acid Receptor γ Activity in Mesenchymal Stem Cells Regulates Endochondral Bone, Angiogenesis, and B Lymphopoiesis.

Retinoic acid receptor (RAR) signaling regulates bone structure and hematopoiesis through intrinsic and extrinsic mechanisms. This study aimed to establish how early in the osteoblast lineage loss of RARγ (Rarg) disrupts the bone marrow microenvironment. Bone structure was analyzed by micro-computed tomography (µCT) in Rarg-/- mice and mice with Rarg conditional deletion in Osterix-Cre-targeted osteoblast progenitors or Prrx1-Cre-targeted mesenchymal stem cells. Rarg-/- tibias exhibited less trabecular and cortical bone and impaired longitudinal and radial growth. The trabecular bone and longitudinal, but not radial, growth defects were recapitulated in Prrx1:RargΔ/Δ mice but not Osx1:RargΔ/Δ mice. Although both male and female Prrx1:RargΔ/Δ mice had low trabecular bone mass, males exhibited increased numbers of trabecular osteoclasts and Prrx1:RargΔ/Δ females had impaired mineral deposition. Both male and female Prrx1:RargΔ/Δ growth plates were narrower than controls and their epiphyses contained hypertrophic chondrocyte islands. Flow cytometry revealed that male Prrx1:RargΔ/Δ bone marrow exhibited elevated pro-B and pre-B lymphocyte numbers, accompanied by increased Cxcl12 expression in bone marrow cells. Prrx1:RargΔ/Δ bone marrow also had elevated megakaryocyte-derived Vegfa expression accompanied by smaller sinusoidal vessels. Thus, RARγ expression by Prrx1-Cre-targeted cells directly regulates endochondral bone formation and indirectly regulates tibial vascularization. Furthermore, RARγ expression by Prrx1-Cre-targeted cells extrinsically regulates osteoclastogenesis and B lymphopoiesis in male mice. © 2018 American Society for Bone and Mineral Research.

Parathyroid Hormone-Related Protein Negatively Regulates Tumor Cell Dormancy Genes in a PTHR1/Cyclic AMP-Independent Manner.

Parathyroid hormone-related protein (PTHrP) expression in breast cancer is enriched in bone metastases compared to primary tumors. Human MCF7 breast cancer cells "home" to the bones of immune deficient mice following intracardiac inoculation, but do not grow well and stain negatively for Ki67, thus serving as a model of breast cancer dormancy in vivo. We have previously shown that PTHrP overexpression in MCF7 cells overcomes this dormant phenotype, causing them to grow as osteolytic deposits, and that PTHrP-overexpressing MCF7 cells showed significantly lower expression of genes associated with dormancy compared to vector controls. Since early work showed a lack of cyclic AMP (cAMP) response to parathyroid hormone (PTH) in MCF7 cells, and cAMP is activated by PTH/PTHrP receptor (PTHR1) signaling, we hypothesized that the effects of PTHrP on dormancy in MCF7 cells occur through non-canonical (i.e., PTHR1/cAMP-independent) signaling. The data presented here demonstrate the lack of cAMP response in MCF7 cells to full length PTHrP(1-141) and PTH(1-34) in a wide range of doses, while maintaining a response to three known activators of adenylyl cyclase: calcitonin, prostaglandin E2 (PGE2), and forskolin. PTHR1 mRNA was detectable in MCF7 cells and was found in eight other human breast and murine mammary carcinoma cell lines. Although PTHrP overexpression in MCF7 cells changed expression levels of many genes, RNAseq analysis revealed that PTHR1 was unaltered, and only 2/32 previous PTHR1/cAMP responsive genes were significantly upregulated. Instead, PTHrP overexpression in MCF7 cells resulted in significant enrichment of the calcium signaling pathway. We conclude that PTHR1 in MCF7 breast cancer cells is not functionally linked to activation of the cAMP pathway. Gene expression responses to PTHrP overexpression must, therefore, result from autocrine or intracrine actions of PTHrP independent of PTHR1, through signals emanating from other domains within the PTHrP molecule.

Autocrine and Paracrine Regulation of the Murine Skeleton by Osteocyte-Derived Parathyroid Hormone-Related Protein.

Parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH) have N-terminal domains that bind a common receptor, PTHR1. N-terminal PTH (teriparatide) and now a modified N-terminal PTHrP (abaloparatide) are US Food and Drug Administration (FDA)-approved therapies for osteoporosis. In physiology, PTHrP does not normally circulate at significant levels, but acts locally, and osteocytes, cells residing within the bone matrix, express both PTHrP and the PTHR1. Because PTHR1 in osteocytes is required for normal bone resorption, we determined how osteocyte-derived PTHrP influences the skeleton. We observed that adult mice with low PTHrP in osteocytes (targeted with the Dmp1(10kb)-Cre) have low trabecular bone volume and osteoblast numbers, but osteoclast numbers were unaffected. In addition, bone size was normal, but cortical bone strength was impaired. Osteocyte-derived PTHrP therefore stimulates bone formation and bone matrix strength, but is not required for normal osteoclastogenesis. PTHrP knockdown and overexpression studies in cultured osteocytes indicate that osteocyte-secreted PTHrP regulates their expression of genes involved in matrix mineralization. We determined that osteocytes secrete full-length PTHrP with no evidence for secretion of lower molecular weight forms containing the N-terminus. We conclude that osteocyte-derived full-length PTHrP acts through both PTHR1 receptor-mediated and receptor-independent actions in a paracrine/autocrine manner to stimulate bone formation and to modify adult cortical bone strength. © 2017 American Society for Bone and Mineral Research.