Mark Dallas

TGF-β signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development

Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-beta (TGF-beta), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-beta inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-beta responses, any role of TGF-beta in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-beta promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant-negative mutant (TbetaRIIDeltacyt) of the TGF-beta type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-beta. In a murine model of bone metastases, expression of TbetaRIIDeltacyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant-negative signaling blockade by expression of a constitutively active TGF-beta type I receptor in the breast cancer cells increased tumor production of parathyroid hormone-related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant-negative TbetaRIIDeltacyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-beta in the development of breast cancer metastasis to bone, via the TGF-beta receptor-mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.

Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis.

Breast cancer almost invariably metastasizes to bone in patients with advanced disease and causes local osteolysis. Much of the morbidity of advanced breast cancer is a consequence of this process. Despite the importance of the problem, little is known of the pathophysiology of local osteolysis in the skeleton or its prevention and treatment. Observations in patients with bone metastases suggest that breast cancer cells in bone express parathyroid hormone-related protein (PTHrP) more frequently than in soft tissue sites of metastasis or in the primary tumor. Thus, the role of PTHrP in the causation of breast cancer metastases in bone was examined using human breast cancer cell lines. Four of eight established human breast cancer cell lines expressed PTHrP and one of these cell lines, MDA-MB-231, was studied in detail using an in vivo model of osteolytic metastases. Mice inoculated with MDA-MB-231 cells developed osteolytic bone metastasis without hypercalcemia or increased plasma PTHrP concentrations. PTHrP concentrations in bone marrow plasma from femurs affected with osteolytic lesions were increased 2.5-fold over corresponding plasma PTHrP concentrations. In a separate experiment, mice were treated with either a monoclonal antibody directed against PTHrP(1-34), control IgG, or nothing before tumor inoculation with MDA-MB-231 and twice per week for 26 d. Total area of osteolytic lesions was significantly lower in mice treated with PTHrP antibodies compared with mice receiving control IgG or no treatment. Histomorphometric analysis of bone revealed decreased osteoclast number per millimeter of tumor/bone interface and increased bone area, as well as decreased tumor area, in tumor-bearing animals treated with PTHrP antibodies compared with respective controls. These results indicate that tumor-produced PTHrP can cause local bone destruction in breast cancer metastatic to bone, even in the absence of hypercalcemia or increased circulating plasma concentrations of PTHrP. Thus, PTHrP may have an important pathogenetic role in the establishment of osteolytic bone lesions in breast cancer. Neutralizing antibodies to PTHrP may reduce the development of destructive bone lesions as well as the growth of tumor cells in bone.

Von Kossa Staining Alone Is Not Sufficient to Confirm that Mineralization In Vitro Represents Bone Formation

Numerous techniques are currently used to characterize biological mineralization in intact tissues and cell cultures; the von Kossa staining method, electron microscopic analysis (EM), X-ray diffraction, and Fourier transform infrared spectroscopy (FTIR) are among the most common. In this study, we utilized three of these methods to compare the mineralization of cultured fetal rat calvarial cells (FRC) and the osteoblast cell lines 2T3 and MC3T3-E1 with the in vivo mineral of rat calvarial bone. The cells were cultured with or without ascorbic acid (100 µg/ml) and β-glycerophosphate (2.5, 5, or 10 mM βGP), and harvested between 16 and 21 days (FRC cells and 2T3 cells) or at 30 days of culture (MC3T3-E1 cells). In the FRC cultures, maximal von Kossa staining was observed with 2.5 and 5 mM βGP in the presence of 100 µg/ml ascorbate. FRC cells also showed some von Kossa staining when cultured with βGP alone. In contrast, maximal von Kossa staining for MC3T3-E1 cells was observed with 10 mM βGP. Only the cultures of MC3T3-E1 cells that received both ascorbate and βGP produced von Kossa positive structures. The 2T3 cultures produced von Kossa positive staining only upon treatment with ascorbic acid and βGP, which was greatly accelerated by bone morphogenic protein-2 (BMP-2). FTIR was performed on the mineral and matrix generated in FRC, MC3T3, and 2T3 cultures, and the results were compared with spectra derived from 16-day-old rat calvaria. The mineral-to-matrix ratios calculated from FTIR spectra for rat calvaria ranged from 2.97 to 7.44. FRC cells made a bonelike, poorly crystalline apatite, and, with increasing βGP, there was a statistically significant (P ≤ 0.02) dose-dependent increase in the mineral-to-matrix ratio (0.56 ± 0.16, 1.00 ± 0.32, and 2.46 ± 0.76, for 2.5, 5, and 10 mM βGP, respectively). The mean carbonate-to-phosphate ratios of the FRC cultures were 0.015, 0.012, and 0.008, in order of increasing βGP concentration, compared with rat calvaria values of 0.009–0.017. The 2T3 cells treated with BMP-2 also made bonelike crystals, similar to those observed in FRC cultures. In contrast, the cultures of von Kossa positive MC3T3-E1 cells did not display a significant amount of mineral (maximum mineral-to-matrix ratio was 0.4). Thus, although the von Kossa stainings of FRC, 2T3, and MC3T3-E1 were very similar, FTIR analysis indicated that calcium phosphate mineral was not present in the MC3T3 cultures. By EM, the mineral in FRC cell cultures and 2T3 cultures was generally associated with collagen, whereas rare or sparse dystrophic mineralization of unknown chemical origin was evident in the MC3T3-E1 cultures. These studies demonstrate that von Kossa staining alone is not appropriate for the identification and quantitation of bonelike mineral and, hence, other techniques such as X-ray diffraction, EM, or FTIR should be utilized to verify the presence and quality of calcium phosphate phases.

E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation.

ABSTRACT Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.

Establishment of an osteoid preosteocyte-like cell MLO-A5 that spontaneously mineralizes in culture

The mechanisms controlling the initiation of mineralization of bone matrix are not clear. To examine this process, we established a cell line called MLO‐A5 that mineralizes in sheets, not nodules, within 3 days of culture in the presence of β‐glycerophosphate (β‐GP) and ascorbic acid and within 7 days in the absence of β‐GP and ascorbic acid. The mineral formed in both cases was shown to be bonelike apatite by Fourier transformed infrared (FTIR) spectroscopy. Mineral‐to‐matrix ratios (min/matrix) calculated from the FTIR data, which are related directly to ash weight, were approximately 0.4 in the absence of β‐GP and ascorbic acid and approximately 1.2 in the presence of β‐GP and ascorbic acid. By comparison, these ratios in fetal rat calvarial cells without β‐GP equal 0 and with β‐GP 1.9. This cell line and three others (MLO‐A2, −D1, and −D6) were isolated from the long bones of transgenic mice expressing the large T‐antigen driven by the osteocalcin promoter, the same mice from which the osteocyte‐like cell line MLO‐Y4 was isolated. (1) The cell lines were selected based on a dendritic or stellate morphology. MLO‐A5 cells express high alkaline phosphatase, collagen type 1, parathyroid hormone/parathyroid hormone‐related peptide (PTH/PTHrP) receptor, bone sialoprotein (BSP), and osteocalcin (767 ng/106 cells compared with <1–2.2 ng/106 cell for primary mouse osteoblasts and five osteoblast cell lines). The single unique feature of the MLO‐A5 cells compared with the other three nonmineralizing cell lines is the high expression of messenger RNA (mRNA) for BSP. These cell lines may represent stages of osteocyte differentiation and the MLO‐A5 cells represent the postosteoblast, preosteocyte responsible for triggering mineralization of osteoid.

Mechanism by which MLO-A5 Late Osteoblasts/Early Osteocytes Mineralize in Culture: Similarities with Mineralization of Lamellar Bone

The mechanisms whereby bone mineralizes are unclear. To study this process, we used a cell line, MLO-A5, which has highly elevated expression of markers of the late osteoblast such as alkaline phosphatase, bone sialoprotein, parathyroid hormone type 1 receptor, and osteocalcin and will mineralize in sheets, not nodules. In culture, markers of osteocytes and dendricity increase with time, features of differentiation from a late osteoblast to an early osteocyte. Mineral formation was examined using transmission electron microscopy, scanning electron microscopy with energy-dispersive X-ray analysis, and atomic force microscopy. At 3–4 days of culture, spheres of approximately 20–50 nm containing calcium and phosphorus were observed budding from and associated with developing cellular projections. By 5–6 days, these calcified spheres were associated with collagen fibrils, where over time they continued to enlarge and to engulf the collagen network. Coalescence of these mineralized spheres and collagen-mediated mineralization were responsible for the mineralization of the matrix. Similar calcified spheres were observed in cultured fetal rat calvarial cells and in murine lamellar bone. We propose that osteoid-osteocytes generate spherical structures that calcify during the budding process and are fully mineralized on their developing cellular processes. As the cellular process narrows in diameter, these mineralized structures become associated with and initiate collagen-mediated mineralization.

Parathyroid hormone-related protein (PTHrP)-(1-139) isoform is efficiently secreted in vitro and enhances breast cancer metastasis to bone in vivo

Parathyroid hormone-related peptide (PTHrP) is a mediator of local osteolysis due to breast cancer. Three isoforms of PTHrP, (1-139), (1-141), and (1-173), are products of alternative splicing in humans, but the specific contribution of each of these isoforms to osteolytic metastasis caused by breast cancer has not been evaluated. To determine the role of PTHrP isoforms in breast cancer metastasis to bone, the human breast cancer cell line MDA-MB-231 (MDA-231) was stably transfected with cDNAs for human prepro PTHrP-(1-139), -(1-141), or -(1-173). Stable MDA/PTHrP-(1-139) clones expressed more PTHrP mRNA and secreted more PTHrP protein, compared with MDA/PTHrP-(1-141), -(1-173), or parental MDA-231. Parental MDA-231 cells and clones expressing each isoform had similar growth rates in vitro. In a mouse model of bone metastases, the osteolytic lesion area of radiographs was greatest in mice bearing MDA/PTHrP-(1-139) compared with those bearing MDA/PTHrP-(1-141), -(1-173), or parental MDA-231. Ca(++) and plasma PTHrP concentrations were significantly higher in the MDA/PTHrP-(1-139) compared with the MDA/PTHrP-(1-141), -(1-173), or parental MDA-231 groups. These data demonstrate that the PTHrP-(1-139) isoform was produced to a greater extent than PTHrP-(1-141) or -(1-173), and in vivo enhanced osteolysis with increased plasma PTHrP concentrations and hypercalcemia compared with overexpression of PTHrP-(1-141) or -(1-173).

Conditional deletion of Pkd1 in osteocytes disrupts skeletal mechanosensing in mice

We investigated whether polycystin‐1 is a bone mechanosensor. We conditionally deleted Pkd1 in mature osteoblasts/osteocytes by crossing Dmp1‐Cre with Pkd1flox/m1Bei mice, in which the m1Bei allele is nonfunctional. We assessed in wild‐type and Pkd1‐deficient mice the response to mechanical loading in vivo by ulna loading and ex vivo by measuring the response of isolated osteoblasts to fluid shear stress. We found that conditional Pkd1 heterozygotes (Dmp1‐Cre;Pkd1flox/+) and null mice (Pkd1Dmp1‐cKO) exhibited a ~40 and ~90% decrease, respectively, in functional Pkd1 transcripts in bone. Femoral bone mineral density (12 vs. 27%), trabecular bone volume (32 vs. 48%), and cortical thickness (6 vs. 17%) were reduced proportionate to the reduction of Pkd1 gene dose, as were mineral apposition rate (MAR) and expression of Runx2‐II, Osteocalcin, Dmp1, and Phex. Anabolic load‐induced periosteal lamellar MAR (0.58±0.14; Pkd1Dmp1‐cKO vs. 1.68±0.34 μm/d; control) and increases in Cox‐2, c‐Jun, Wnt10b, Axin2, and Runx2‐II gene expression were significantly attenuated in Pkd1Dmp1‐cKO mice compared with controls. Application of fluid shear stress to immortalized osteoblasts from Pkd1null/null and Pkd1m1Bei/m1Bei‐derived osteoblasts failed to elicit the increments in cytosolic calcium observed in wild‐type controls. These data indicate that polycystin‐1 is essential for the anabolic response to skeletal loading in osteoblasts/osteocytes.—Xiao, Z., Dallas, M., Qiu, N., Nicolella, D., Cao, L., Johnson, M., Bonewald, L., Quarles, L. D. Conditional deletion of Pkd1 in osteocytes disrupts skeletal mechanosensing in mice. FASEB J. 25, 2418–2432 (2011). www.fasebj.org

Deletion of a Single β-Catenin Allele in Osteocytes Abolishes the Bone Anabolic Response to Loading

The Wnt/β‐catenin signaling pathway is essential for bone cell viability and function and for skeletal integrity. To determine if β‐catenin in osteocytes plays a role in the bone anabolic response to mechanical loading, 18‐ to 24‐week‐old osteocyte β‐catenin haploinsufficient mice (Dmp1‐Cre × β‐catenin fl/ + ; HET cKO) were compared with their β‐catenin fl/fl (control) littermates. Trabecular bone volume (BV/TV) was significantly less (58.3%) in HET cKO females versus controls, whereas male HET cKO and control mice were not significantly different. Trabecular number was significantly less in HET cKO mice compared with controls for both genders, and trabecular separation was greater in female HET cKO mice. Osteoclast surface was significantly greater in female HET cKO mice. Cortical bone parameters in males and females showed subtle or no differences between HET cKO and controls. The right ulnas were loaded in vivo at 100 cycles, 2 Hz, 2500 µϵ, 3 days per week for 3 weeks, and the left ulnas served as nonloaded controls. Calcein and alizarin complexone dihydrate were injected 10 days and 3 days before euthanization, respectively. Micro‐computed tomography (µCT) analysis detected an 8.7% and 7.1% increase in cortical thickness in the loaded right ulnas of male and female control mice, respectively, compared with their nonloaded left ulnas. No significant increase in new cortical bone formation was observed in the HET cKO mice. Histomorphometric analysis of control mice showed a significant increase in endocortical and periosteal mineral apposition rate (MAR), bone‐formation rate/bone surface (BFR/BS), BFR/BV, and BFR/TV in response to loading, but no significant increases were detected in the loaded HET cKO mice. These data show that deleting a single copy of β‐catenin in osteocytes abolishes the anabolic response to loading, that trabecular bone in females is more severely affected and suggest that a critical threshold of β‐catenin is required for bone formation in response to mechanical loading.

Immortalization of osteoclast precursors by targeting Bcl -XL and Simian virus 40 large T antigen to the osteoclast lineage in transgenic mice.

Cellular and molecular characterization of osteoclasts (OCL) has been extremely difficult since OCL are rare cells, and are difficult to isolate in large numbers. We used the tartrate-resistant acid phosphatase promoter to target the bcl-XL and/or Simian Virus 40 large T antigen (Tag) genes to cells in the OCL lineage in transgenic mice as a means of immortalizing OCL precursors. Immunocytochemical studies confirmed that we had targeted Bcl-XL and/or Tag to OCL, and transformed and mitotic OCL were readily apparent in bones from both Tag and bcl-XL/Tag mice. OCL formation in primary bone marrow cultures from bcl-XL, Tag, or bcl-XL/Tag mice was twofold greater compared with that of nontransgenic littermates. Bone marrow cells from bcl-XL/Tag mice, but not from singly transgenic bcl-XL or Tag mice, have survived in continuous culture for more than a year. These cells form high numbers of bone-resorbing OCL when cultured using standard conditions for inducing OCL formation, with approximately 50% of the mononuclear cells incorporated into OCL. The OCL that form express calcitonin receptors and contract in response to calcitonin. Studies examining the proliferative capacity and the resistance of OCL precursors from these transgenic mice to apoptosis demonstrated that the increased numbers of OCL precursors in marrow from bcl-XL/Tag mice was due to their increased survival rather than an increased proliferative capacity compared with Tag, bcl-XL, or normal mice. Histomorphometric studies of bones from bcl-XL/Tag mice also confirmed that there were increased numbers of OCL precursors (TRAP + mononuclear cells) present in vivo. These data demonstrate that by targeting both bcl-XL and Tag to cells in the OCL lineage, we have immortalized OCL precursors that form bone-resorbing OCL with an efficiency that is 300-500 times greater than that of normal marrow.