Amphiregulin-EGFR Signaling Mediates the Migration of Bone Marrow Mesenchymal Progenitors toward PTH-Stimulated Osteoblasts and Osteocytes

Intermittent administration of parathyroid hormone (PTH) dramatically increases bone mass and currently is one of the most effective treatments for osteoporosis. However, the detailed mechanisms are still largely unknown. Here we demonstrate that conditioned media from PTH-treated osteoblastic and osteocytic cells contain soluble chemotactic factors for bone marrow mesenchymal progenitors, which express a low amount of PTH receptor (PTH1R) and do not respond to PTH stimulation by increasing cAMP production or migrating toward PTH alone. Conditioned media from PTH-treated osteoblasts elevated phosphorylated Akt and p38MAPK amounts in mesenchymal progenitors and inhibition of these pathways blocked the migration of these progenitors toward conditioned media. Our previous and current studies revealed that PTH stimulates the expression of amphiregulin, an epidermal growth factor (EGF)-like ligand that signals through the EGF receptor (EGFR), in both osteoblasts and osteocytes. Interestingly, conditioned media from PTH-treated osteoblasts increased EGFR phosphorylation in mesenchymal progenitors. Using several different approaches, including inhibitor, neutralizing antibody, and siRNA, we demonstrate that PTH increases the release of amphiregulin from osteoblastic cells, which acts on the EGFRs expressed on mesenchymal progenitors to stimulate the Akt and p38MAPK pathways and subsequently promote their migration in vitro. Furthermore, inactivation of EGFR signaling specifically in osteoprogenitors/osteoblasts attenuated the anabolic actions of PTH on bone formation. Taken together, these results suggest a novel mechanism for the therapeutic effect of PTH on osteoporosis and an important role of EGFR signaling in mediating PTH's anabolic actions on bone

The critical role of the epidermal growth factor receptor in endochondral ossification

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification. © 2011 American Society for Bone and Mineral Research

Tumor-expressed adrenomedullin accelerates breast cancer bone metastasis

INTRODUCTION: Adrenomedullin (AM) is secreted by breast cancer cells and increased by hypoxia. It is a multifunctional peptide that stimulates angiogenesis and proliferation. The peptide is also a potent paracrine stimulator of osteoblasts and bone formation, suggesting a role in skeletal metastases-a major site of treatment-refractory tumor growth in patients with advanced disease. METHODS: The role of adrenomedullin in bone metastases was tested by stable overexpression in MDA-MB-231 breast cancer cells, which cause osteolytic bone metastases in a standard animal model. Cells with fivefold increased expression of AM were characterized in vitro, inoculated into immunodeficient mice and compared for their ability to form bone metastases versus control subclones. Bone destruction was monitored by X-ray, and tumor burden and osteoclast numbers were determined by quantitative histomorphometry. The effects of AM overexpression on tumor growth and angiogenesis in the mammary fat pad were determined. The effects of AM peptide on osteoclast-like multinucleated cell formation were tested in vitro. A small-molecule AM antagonist was tested for its effects on AM-stimulated ex vivo bone cell cultures and co-cultures with tumor cells, where responses of tumor and bone were distinguished by species-specific real-time PCR. RESULTS: Overexpression of AM mRNA did not alter cell proliferation in vitro, expression of tumor-secreted factors or cell cycle progression. AM-overexpressing cells caused osteolytic bone metastases to develop more rapidly, which was accompanied by decreased survival. In the mammary fat pad, tumors grew more rapidly with unchanged blood vessel formation. Tumor growth in the bone was also more rapid, and osteoclasts were increased. AM peptide potently stimulated bone cultures ex vivo; responses that were blocked by small-molecule adrenomedullin antagonists in the absence of cellular toxicity. Antagonist treatment dramatically suppressed tumor growth in bone and decreased markers of osteoclast activity. CONCLUSIONS: The results identify AM as a target for therapeutic intervention against bone metastases. Adrenomedullin potentiates osteolytic responses in bone to metastatic breast cancer cells. Small-molecule antagonists can effectively block bone-mediated responses to tumor-secreted adrenomedullin, and such agents warrant development for testing in vivo

Targeting the osteosarcoma cancer stem cell

<p>Abstract</p> <p>Osteosarcoma is the most common type of solid bone cancer and the second leading cause of cancer-related death in pediatric patients. Many patients are not cured by the current osteosarcoma therapy consisting of combination chemotherapy along with surgery and thus new treatments are urgently needed. In the last decade, cancer stem cells have been identified in many tumors such as leukemia, brain, breast, head and neck, colon, skin, pancreatic, and prostate cancers and these cells are proposed to play major roles in drug resistance, tumor recurrence, and metastasis. Recent studies have shown evidence that osteosarcoma also possesses cancer stem cells. This review summarizes the current knowledge about the osteosarcoma cancer stem cell including the methods used for its isolation, its properties, and its potential as a new target for osteosarcoma treatment.</p

Conditioned media from PTH-treated osteoblastic cells contain chemotactic factors for bone marrow mesenchymal progenitors.

<p>(A) Migration of freshly flushed rat bone marrow cells toward PTH and conditioned media from UMR 106-01 cells. Bone marrow cells flushed from rat long bones were immediately seeded in the upper chamber of transwell plates. The bottom wells were loaded with media alone (αMEM), media containing 10 nM PTH (PTH), or conditioned media (CM) collected from UMR106-01 cells that had been treated with control (CON) or 10 nM PTH (PTH) for 4 hr. The number of cells that migrated to the bottom wells was counted 24 hr later. αMEM containing 5% FBS (FBS) was used as a positive control for cell migration. *: p<0.05; **: p<0.01 vs. αMEM. (B) Conditioned media from various PTH-treated osteoblastic and osteocytic cells stimulated the migration of either human or rat mesenchymal progenitors (MP) in the Boyden chamber assay. The cells seeded in the upper wells are depicted at the top and conditioned media loaded in the lower wells are shown at the bottom. M: αMEM. ***: p<0.001 vs. αMEM; &: p<0.01; #: p<0.001 vs. CON. (C) Microscopic images of the mesenchymal progenitors that migrated to the lower sides of filters in the assay depicted in B. (D) The migration of mesenchymal progenitors stimulated by conditioned media from PTH-treated osteoblasts is not chemokinesis. Mesenchymal progenitors were suspended in conditioned media harvested from either control or PTH-treated UMR 106-01 cells and seeded in the upper chambers. The lower wells were filled with conditioned media, resulting in a total of 4 combination types. **: p<0.01 vs. CON/CON. (E) Time course of the release of chemotactic factor(s) from osteoblasts after PTH treatment. UMR 106-01 cells were treated with PTH and conditioned media were harvested at indicated time points and loaded in the lower wells for chemotaxis assays. ***: p<0.001 vs. time 0 hr. (F) The dosage effects of PTH on chemotactic factor(s) release from osteoblasts. UMR 106-01 cells were treated with different doses of PTH for 4 hr and then conditioned media were harvested for chemotaxis assays. *: p<0.05; **: p = 0.06; ***: p<0.001 vs. 0 nM.</p

PTH stimulates the release of amphiregulin from osteoblasts to promote mesenchymal progenitor migration.

<p>(A) EGF-like ligands are chemotactic factors for mesenchymal progenitors. αMEM containing various amounts of EGF-like ligands was added in the lower wells of chemotaxis assays using rat mesenchymal progenitors. αMEM containing 5% FBS was used as positive control. *: p<0.05; **: p<0.01; ***: p<0.001 vs. CON. (B) Addition of GM6001 (10 µM, GM) in the chemotaxis assay blocked the migration of mesenchymal progenitors toward conditioned media from PTH-treated UMR 106-01 cells. ***: p<0.001 vs. CON DMSO; #: p<0.001 vs. PTH DMSO. (C) qRT-PCR shows that PTH (10 nM) induced the expression of amphiregulin in osteocytic Ocy491 cells at 1 h. (D) qRT-PCR demonstrates the knockdown of amphiregulin mRNA in UMR 106-01 cells after 1 hr of PTH (10 nM) treatment by siRNAs. **: p<0.01 vs. mock1. (E) Chemotaxis assays reveal that PTH did not stimulate the release of chemotactic factor(s) from UMR106-01 cells transfected with siRNAs for amphiregulin. ***: p<0.001 vs. CON CM; #; p<0.001 vs PTH CM mock1. (F) Amphiregulin (AR) stimulated Akt and p38MAPK phosphorylation in mesenchymal progenitors as shown by immunoblotting.</p

A model for PTH-induced mesenchymal progenitor migration in bone.

<p>A model for PTH-induced mesenchymal progenitor migration in bone.</p

PI3K/Akt and p38MAPK pathways are required for the migration of mesenchymal progenitors toward conditioned media from PTH-treated osteoblastic cells.

<p>(A) Chemotaxis assays were performed with mesenchymal progenitors and conditioned media from either control- or PTH-treated UMR 106-01 cells in the presence or absence of pathway-specific inhibitors. D: DMSO; U: U0126 (20 µM); WT: wortmannin (3 µM); SB: SB202190 (20 µM). Inhibitors were added to both upper and bottom chambers. ***: p<0.001 vs. CON CM D; #: p<0.001 vs. PTH CM D. (B) Conditioned media from PTH-treated UMR 106-01 cells stimulated the phosphorylation of Akt and p38MAPK in MSCs. (C) PTH alone did not activate Akt and p38MAPK pathways in mesenchymal progenitors.</p

EGFR signaling mediates the chemotactic effect of PTH on mesenchymal progenitors.

<p>(A) Conditioned media from PTH-treated UMR 106-01 cells increased the phosphorylation of EGFR in mesenchymal progenitors. In this experiment, mesenchymal progenitors were treated with conditioned media for 5 min and then lysed for Western blot. (B) The enhanced phosphorylation of Akt and p38MAPK in mesenchymal progenitors by conditioned media from PTH-treated UMR106-01 cells is dependent on the EGFR pathway. Mesenchymal progenitors were pre-incubated with either DMSO or gefitinib (10 µM, GEF) for 30 min followed by addition of conditioned media to the culture. Cell lysates were collected 5 min later for Western blot analyses. (C) The EGFR inhibitor PD153035 (10 µM, PD) was added to both the upper and lower wells of the chemotaxis assay and partially blocked the PTH-induced chemotactic activity of conditioned media from UMR 106-01 cells. ***: p<0.001 vs. DMSO CON; &: p<0.01 vs. DMSO PTH. (D) An EGFR neutralizing antibody (4 µg/ml) was mixed with mesenchymal progenitors before the chemotaxis assay and suppressed the migration of mesenchymal progenitors towards conditioned media from PTH-treated UMR 106-01 cells. IgG: isotype control. **: p<0.01; ***: p<0.001 vs. CON; $: p<0.05; #: p<0.001 vs. PTH. (E) qRT-PCR demonstrates the knockdown of EGFR mRNA levels in mesenchymal progenitors by siRNAs. ***: p<0.001 vs. MOCK. (F) Immunoblotting reveals that the EGFR protein level was dramatically decreased in mesenchymal progenitors transfected with siRNAs for EGFR. (G) Blocking of EGFR expression in mesenchymal progenitors by siRNAs abolished the chemotactic migration of these cells toward conditioned media from PTH-treated UMR 106-01 cells. ***: p<0.001 vs. mock CON; #: p<0.001 vs. mock PTH.</p