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Gregory R Mundy

Publications and source records attributed to Gregory R Mundy.

At least 19 recordsLinked to original sources
PubMed2006

Caspase-2 deficiency enhances aging-related traits in mice.

Alteration of apoptotic activity has been observed in a number of tissues in aging mammals, but it remains unclear whether and/or how apoptosis may affect aging. Caspase-2 is a member of the cysteine protease family that plays a critical role in apoptosis. To understand the impact of compromised apoptosis function on mammalian aging, we conducted a comparative study on caspase-2 deficient mice and their wild-type littermates with a specific focus on the aging-related traits at advanced ages. We found that caspase-2 deficiency enhanced a number of traits commonly seen in premature aging animals. Loss of caspase-2 was associated with shortened maximum lifespan, impaired hair growth, increased bone loss, and reduced body fat content. In addition, we found that the livers of caspase-2 deficient mice had higher levels of oxidized proteins than those of age-matched wild-type mice, suggesting that caspase-2 deficiency compromised the animal's ability to clear oxidatively damaged cells. Collectively, these results suggest that caspase-2 deficiency affects aging in the mice. This study thus demonstrates for the first time that disruption of a key apoptotic gene has a significant impact on aging.

Adipose Tissue↗
PubMed2006

Boning up on ephrin signaling.

The activities of osteoclasts, which degrade bone, and osteoblasts, which form bone, are coordinated to maintain bone homeostasis. Zhao et al. (2006) now demonstrate bidirectional signaling between these two cell populations via the transmembrane ligand ephrinB2 expressed by osteoclasts and its receptor EphB4 expressed by osteoblasts. Such bidirectional signaling limits osteoclast activity while stimulating osteoblast differentiation.

Animals↗
PubMed2006

The hedgehog signaling molecule Gli2 induces parathyroid hormone-related peptide expression and osteolysis in metastatic human breast cancer cells.

Parathyroid hormone-related peptide (PTHrP) is a major factor involved in tumor-induced osteolysis caused by breast cancers that have metastasized to bone. However, the molecular mechanisms that mediate PTHrP production by breast cancer cells are not entirely clear. We hypothesized that Gli2, a downstream transcriptional effector of the Hedgehog (Hh) signaling pathway, regulates PTHrP expression in metastatic breast cancer because the Hh pathway regulates physiologic PTHrP expression in the developing growth plate. Here, we show that Gli2 is expressed in several human cancer cell lines that cause osteolytic lesions in vivo and produce PTHrP (MDA-MB-231, RWGT2, and PC-3) but is not expressed in nonosteolytic cancer cell lines that do not secrete PTHrP (MCF-7, ZR-75, and T47D). Transient expression of Gli2 in MDA-MB-231 and MCF-7 breast cancer cells increased PTHrP promoter-luciferase activity dose dependently. Stable expression of Gli2 in MDA-MB-231 cells resulted in an increase in PTHrP protein in the conditioned medium. Alternatively, MDA-MB-231 cells stably transfected with Gli2-EnR, a repressor of Gli2 activity, exhibited a 72% to 93% decrease in PTHrP mRNA by quantitative real-time PCR when compared with control cells. To examine the effects of Gli2 on breast cancer-mediated osteolysis in vivo, athymic nude mice were inoculated with MDA-MB-231 cells stably expressing Gli2 or the empty vector. Following tumor cell inoculation via the left cardiac ventricle, Gli2-expressing tumors caused significantly more osteolysis. Together, these data suggest that PTHrP expression and osteolysis in vivo in human breast cancer cells is driven at least in part by Gli2.

Animals↗
PubMed2006

Inhibition of pulmonary and skeletal metastasis by a transforming growth factor-beta type I receptor kinase inhibitor.

Transforming growth factor-beta (TGF-beta) signaling has been shown to promote invasion and metastasis in various models of human cancers. In this study, we investigated the efficacy of a TGF-beta type I receptor kinase inhibitor (TbetaRI-I) to limit early systemic metastases in an orthotopic xenograft model of lung metastasis and in an intracardiac injection model of experimental bone and lung metastasis using human breast carcinoma MDA-MB-435-F-L cells, a highly metastatic variant of human breast cancer MDA-MB-435 cells, expressing the enhanced green fluorescent protein (EGFP). Treatment of the cells with the TbetaRI-I had no effect on their growth but blocked TGF-beta-stimulated expression of integrin alpha(v)beta(3) and cell migration in vitro. Systemic administration of the TbetaRI-I via i.p. injection effectively reduced the number and size of the lung metastasis in both orthotopic xenograft and experimental metastasis models with no effects on primary tumor growth rate compared with controls. TbetaRI-I treatment also reduced the incidence of widespread early skeletal metastases in the femur, tibia, mandible, and spine detected by whole-body EGFP fluorescence imaging. Tumor burden in femora and tibiae was also reduced after TbetaRI-I treatment as detected by histomorphometry analysis compared with the placebo controls. Our results indicate for the first time that abrogation of TGF-beta signaling by systemic administration of the TbetaRI-I can inhibit both early lung and bone metastasis in animal model systems and suggest antimetastatic therapeutic potential of the TbetaRI-I.

Activin Receptors, Type I↗
PubMed2006

Nutritional modulators of bone remodeling during aging.

Bone mass declines progressively with age in both men and women from the age of approximately 30 y. Increased longevity will inevitability be associated with an increase in the incidence of osteoporosis, its associated complications, and incurred health care costs. Current pharmacologic approaches focus on inhibiting bone resorption in those with osteoporosis but do little to improve bone mass. Increased understanding of the cellular events responsible for normal bone formation has led to multiple pathways that can be targeted to positively influence bone mass. Bone morphogenetic proteins (BMPs) have been shown to stimulate bone formation, and the BMP2 gene was recently linked to osteoporosis. BMP-2 therefore represents one potential molecular target to identify new agents to simulate bone formation. Research is accumulating on the positive effects of dietary sources that stimulate the BMP2 promoter and their effects on bone formation. Flavonoids and statins occur naturally in food products and have been shown to promote bone formation. It may be possible to influence peak bone mass by dietary means and to decrease the risk of osteoporosis in later life. To ease the future burden of osteoporosis, focusing on prevention will be key, and this could include dietary interventions to stimulate bone formation.

Aged↗
PubMed2006

The zinc finger transcription factor Gli2 mediates bone morphogenetic protein 2 expression in osteoblasts in response to hedgehog signaling.

Bone morphogenetic protein 2 (BMP-2) plays a critical role in osteoblast function. In Drosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression of dpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. We hypothesize that Gli2 regulates BMP-2 gene transcription and thus osteoblast differentiation. In the present study, we show that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in osteoblast precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal osteoblast differentiation.

Animals↗
PubMed2005

Advances in the management of myeloma bone disease.

Multiple myeloma is the second most common adult haematological malignancy. One of the major clinical features is the development of a unique osteolytic bone disease, characterised by progressive and devastating bone destruction, bone pain, pathological fractures and hypercalcaemia. Bisphosphonates, inhibitors of osteoclastic bone resorption, are the standard therapy for myeloma-induced bone disease. However, as our understanding of the molecular mechanisms involved in the development of myeloma bone disease increases, new molecular targets have been identified for the treatment of this devastating bone disease.

Animals↗
PubMed2004

Surface plasmon resonance (SPR) confirms that MEPE binds to PHEX via the MEPE-ASARM motif: a model for impaired mineralization in X-linked rickets (HYP).

Matrix Extracellular Phospho-glycoprotEin (MEPE) and proteases are elevated and PHEX is defective in HYP. PHEX prevents proteolysis of MEPE and release of a protease-resistant MEPE-ASARM peptide, an inhibitor of mineralization (minhibin). Thus, in HYP, mutated PHEX may contribute to increased ASARM peptide release. Moreover, binding of MEPE by PHEX may regulate this process in normal subjects. The nature of the PHEX-MEPE nonproteolytic interaction(s) (direct or indirect) is/are unknown. Our aims were to determine (1) whether PHEX binds specifically to MEPE, (2) whether the binding involves the ASARM motif region, and (3) whether free ASARM peptide affects mineralization in vivo in mice. Protein interactions between MEPE and recombinant soluble PHEX (secPHEX) were measured using surface plasmon resonance (SPR). Briefly, secPHEX, MEPE, and control protein (IgG) were immobilized on a Biacore CM5 sensor chip, and SPR experiments were performed on a Biacore 3000 high-performance research system. Pure secPHEX was then injected at different concentrations, and interactions with immobilized proteins were measured. To determine MEPE sequences interacting with secPHEX, the inhibitory effects of MEPE-ASARM peptides (phosphorylated and nonphosphorylated), control peptides, and MEPE midregion RGD peptides on secPHEX binding to chip-immobilized MEPE were measured. ASARM peptide and etidronate-mediated mineralization inhibition in vivo and in vitro were determined by quenched calcein fluorescence in hind limbs and calvariae in mice and by histological Sanderson stain. A specific, dose-dependent and Zn-dependent protein interaction between secPHEX and immobilized MEPE occurs (EC50 of 553 nM). Synthetic MEPE PO4-ASARM peptide inhibits the PHEX-MEPE interaction (K(D(app)) = 15 uM and B(max/inhib) = 68%). In contrast, control and MEPE-RGD peptides had no effect. Subcutaneous administration of ASARM peptide resulted in marked quenching of fluorescence in calvariae and hind limbs relative to vehicle controls indicating impaired mineralization. Similar results were obtained with etidronate. Sanderson-stained calvariae also indicated a marked increase in unmineralized osteoid with ASARM peptide and etidronate groups. We conclude that PHEX and MEPE form a nonproteolytic protein interaction via the MEPE carboxy-terminal ASARM motif, and the ASARM peptide inhibits mineralization in vivo. The binding of MEPE and ASARM peptide by PHEX may explain why loss of functional osteoblast-expressed PHEX results in defective mineralization in HYP.

Amino Acid Sequence↗
PubMed2004

Anti-alpha4 integrin antibody suppresses the development of multiple myeloma and associated osteoclastic osteolysis.

Supporting roles of stromal cells in preferential colonization of myeloma cells in bone marrow and development of associated osteoclastic osteolysis through cell-cell interactions have been indicated. Here we examined the effects of a monoclonal antibody to alpha4 integrin (anti-alpha4 Ab) that disrupts myeloma cell-stromal cell interactions mediated via alpha4beta1 integrin and vascular cell adhesion molecule-1 (VCAM-1) on myeloma cell growth in bone marrow and accompanying osteolysis. The anti-alpha4 Ab decreased VCAM-1-stimulated 5TGM1/luc cell growth in culture. The 5TGM1 murine myeloma cells stably transfected with the firefly luciferase (5TGM1/luc) were inoculated from tail vein in bg/xid/nd mice. Preventative administration of the anti-alpha4 Ab suppressed the elevation of serum IgG2b levels, decreased 5TGM1/luc tumor burden with increased apoptosis in bone and spleen, reduced bone destruction with diminished number of osteoclasts, and prolonged survival of 5TGM1/luc-bearing mice. In contrast, therapeutic administration of the antibody failed to show these effects. However, therapeutic administration of the antibody combined with melphalan significantly suppressed serum IgG2b levels and tumor burden in bone. Our results suggest that the interactions with stromal cells via alpha4beta1/VCAM-1 are critical to the development of myeloma and associated osteolysis and that disruption of these interactions using anti-alpha4 Ab is a potential therapeutic approach for myeloma.

Animals↗
PubMed2004

Bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. The roles of BMPs in embryonic development and cellular functions in postnatal and adult animals have been extensively studied in recent years. Signal transduction studies have revealed that Smad1, 5 and 8 are the immediate downstream molecules of BMP receptors and play a central role in BMP signal transduction. Studies from transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and related genes have shown that BMP signaling plays critical roles in heart, neural and cartilage development. BMPs also play an important role in postnatal bone formation. BMP activities are regulated at different molecular levels. Preclinical and clinical studies have shown that BMP-2 can be utilized in various therapeutic interventions such as bone defects, non-union fractures, spinal fusion, osteoporosis and root canal surgery. Tissue-specific knockout of a specific BMP ligand, a subtype of BMP receptors or a specific signaling molecule is required to further determine the specific role of a BMP ligand, receptor or signaling molecule in a particular tissue. BMPs are members of the TGFbeta superfamily. The activity of BMPs was first identified in the 1960s (Urist, M.R. (1965) "Bone formation by autoinduction", Science 150, 893-899), but the proteins responsible for bone induction remained unknown until the purification and sequence of bovine BMP-3 (osteogenin) and cloning of human BMP-2 and 4 in the late 1980s (Wozney, J.M. et al. (1988) "Novel regulators of bone formation: molecular clones and activities", Science 242, 1528-1534; Luyten, F.P. et al. (1989) "Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation", J. Biol. Chem. 264, 13377-13380; Wozney, J.M. (1992) "The bone morphogenetic protein family and osteogenesis", Mol. Reprod. Dev. 32, 160-167). To date, around 20 BMP family members have been identified and characterized. BMPs signal through serine/threonine kinase receptors, composed of type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, type IA and IB BMP receptors (BMPR-IA or ALK-3 and BMPR-IB or ALK-6) and type IA activin receptor (ActR-IA or ALK-2) (Koenig, B.B. et al. (1994) "Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells", Mol. Cell. Biol. 14, 5961-5974; ten Dijke, P. et al. (1994) "Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4", J. Biol. Chem. 269, 16985-16988; Macias-Silva, M. et al. (1998) "Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2", J. Biol. Chem. 273, 25628-25636). Three type II receptors for BMPs have also been identified and they are type II BMP receptor (BMPR-II) and type II and IIB activin receptors (ActR-II and ActR-IIB) (Yamashita, H. et al. (1995) "Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects", J. Cell. Biol. 130, 217-226; Rosenzweig, B.L. et al. (1995) "Cloning and characterization of a human type II receptor for bone morphogenetic proteins", Proc. Natl Acad. Sci. USA 92, 7632-7636; Kawabata, M. et al. (1995) "Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming growth factor-beta receptor", J. Biol. Chem. 270, 5625-5630). Whereas BMPR-IA, IB and II are specific to BMPs, ActR-IA, II and IIB are also signaling receptors for activins. These receptors are expressed differentially in various tissues. Type I and II BMP receptors are both indispensable for signal transduction. After ligand binding they form a heterotetrameric-activated receptor complex consisting of two pairs of a type I and II receptor complex (Moustakas, A. and C.H. Heldi (2002) "From mono- to oligo-Smads: the heart of the matter in TGFbeta signal transduction" Genes Dev. 16, 67-871). The type I BMP receptor substrates include a protein family, the Smad proteins, that play a central role in relaying the BMP signal from the receptor to target genes in the nucleus. Smad1, 5 and 8 are phosphorylated by BMP receptors in a ligand-dependent manner (Hoodless, P.A. et al. (1996) "MADR1, a MAD-related protein that functions in BMP2 signaling pathways", Cell 85, 489-500; Chen Y. et al. (1997) "Smad8 mediates the signaling of the receptor serine kinase", Proc. Natl Acad. Sci. USA 94, 12938-12943; Nishimura R. et al. (1998) "Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12", J. Biol. Chem. 273, 1872-1879). After release from the receptor, the phosphorylated Smad proteins associate with the related protein Smad4, which acts as a shared partner. This complex translocates into the nucleus and participates in gene transcription with other transcription factors (). A significant advancement about the understanding of in vivo functions of BMP ligands, receptors and signaling molecules has been achieved in recent years.

Animals↗
PubMed2003

Smurf1 inhibits osteoblast differentiation and bone formation in vitro and in vivo.

Bone morphogenetic proteins (BMPs) are required for normal postnatal bone formation and osteoblast differentiation. There is evidence from recent studies that BMP signaling in osteoblasts is controlled by an ubiquitin-proteasome regulatory mechanism involving a cascade of enzymatic reactions. The specificity of protein ubiquitination is determined by E3 ubiquitin ligases, which play a crucial role in defining substrate specificity and subsequent protein degradation by 26S proteasomes. We have examined the role of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1), a member of the Hect domain family of E3 ubiquitin ligases in osteoblast function. Smurf1 has been found to interact with BMP-activated Smad1 and -5 and to mediate degradation of these Smad proteins. Recently we have found that Smurf1 mediates the protein degradation of the osteoblast-specific transcription factor Runx2/Cbfa1. To determine the role of Smurf1 in osteoblast differentiation, in the present studies we transfected a Smurf1 expression plasmid into 2T3 osteoblast precursor cells and found that Smurf1 overexpression inhibits BMP signaling and osteoblast differentiation. To further investigate the role of Smurf1 in bone formation in vivo, we generated transgenic mice in which expression of the epitope-tagged Smurf1 transgene was targeted to osteoblasts using the murine 2.3-kb osteoblast-specific type I collagen promoter. In these transgenic mice, bone formation was significantly reduced during postnatal life. Our results demonstrate for the first time that Smurf1 plays a specific role in osteoblast differentiation and bone formation in vivo.

Alkaline Phosphatase↗
PubMed2003

C-SRC tyrosine kinase activity is associated with tumor colonization in bone and lung in an animal model of human breast cancer metastasis.

The proto-oncogene, c-src, has been implicated in the tumorigenesis in breast cancer. However, the relationship of c-src with distant metastasis is unclear. Moreover, the role of c-src in organ-preferential metastasis of breast cancer is unknown. Because breast cancer has a strong predilection for metastasizing to bone, we examined the role of c-src in bone metastases using an animal model in which inoculation of the MDA-231 human breast cancer cells into the left cardiac ventricle preferentially developed osteolytic bone metastases in female nude mice. A clone of the MDA-231 with the increased capacity of bone metastasis exhibited elevated c-src tyrosine kinase (TK) activity compared with parental cells. MDAsrc527 cells caused significantly increased size of the osteolytic bone metastases with increased number of osteoclasts and mitotic cancer cells compared with MDA-231EV or MDAsrcWT. In contrast, MDAsrc295 cells caused impaired metastases to bone. Of note, mice inoculated with MDAsrc295 cells via tail vein developed reduced lung metastases and prolonged survival compared with mice with MDA-231EV cells, suggesting that c-src TK is unlikely to play a specific role in bone metastases. The growth in vitro and in vivo and production of parathyroid hormone-related protein, a key cytokine in the pathogenesis of osteolytic bone metastases in breast cancer, were promoted in MDAsrc527 and diminished in MDAsrc295. These results suggest that c-src TK is associated with the capacity of breast cancer to metastasize to bone through regulating cell growth and parathyroid hormone-related protein production. Our results together with the fact that c-src is an essential molecule for bone resorption by osteoclasts, which are central players in osteolytic bone metastases, support the notion that c-src TK is a potential target molecule for designing novel therapeutic interventions, especially for bone metastases in breast cancer.

Animals↗
PubMed2003

NF-kappaB specifically activates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chondrocyte cell line in vitro.

Bone morphogenetic protein-2 (BMP-2) regulates growth plate chondrogenesis during development and postnatal bone growth, but the control mechanisms of BMP-2 expression in growth plate chondrocytes are unknown. Here we have used both in vitro and in vivo approaches to demonstrate that transcription factor, NF-kappaB, regulates BMP-2 gene expression in chondrocytes. Two putative NF-kappaB response elements were found in the -2712/+165 region of the BMP-2 gene. Cotransfection of mutant I-kappaBalpha expression plasmids with BMP-2 promoter-luciferase reporters into TMC-23 chondrocyte cell line suppressed BMP-2 transcription. Mutations in NF-kappaB response elements in the BMP-2 gene lead to decreases in BMP-2 promoter activity. Electrophoretic mobility shift assay using nuclear extracts from TMC-23 chondrocytic cells revealed that the NF-kappaB subunits p50 and p65 bound to the NF-kappaB response elements of the BMP-2 gene. Thus, NF-kappaB may positively regulate BMP-2 gene transcription. Consistent with these findings, expression of BMP-2 mRNA was significantly reduced in growth plate chondrocytes in NF-kappaB p50/p52 dKO mice, which associated with decreased numbers of 5-bromo-2'-deoxyuridine (BrdUrd)-positive cells in the proliferating zone of growth plate in these mice. Therefore, in postnatal growth plate chondrocytes, expression of BMP-2 is regulated by NF-kappaB, which may play an important role in chondrogenesis.

Animals↗
PubMed2003

E3 ubiquitin ligase Smurf1 mediates core-binding factor alpha1/Runx2 degradation and plays a specific role in osteoblast differentiation.

Osteoblast differentiation and bone formation is stimulated by bone morphogenetic protein (BMP)-2 and its downstream signaling molecules Smad1 and -5 and the osteoblast-specific transcription factor core-binding factor alpha1 (Cbfa1). Proteolytic degradation of Smad1 and Cbfa1 is proteasome-dependent, and intracellular concentrations of Smad1 and Cbfa1 are enhanced by inhibition of the 26 S proteasome. Smad1 degradation is mediated by the E3 ubiquitin ligase Smurf1 (Smad ubiquitin regulatory factor 1), but the specific E3 ligase responsible for Cbfa1 degradation has not been identified. Because Cbfa1 interacts with Smad1, whose degradation is mediated by Smurf1, we examined the effect of Smurf1 on Cbfa1 degradation in osteoblast precursor cells. Smurf1 interacts directly with Cbfa1 and mediates Cbfa1 degradation in a ubiquitin- and proteasome-dependent manner. Because Smurf1 controls the intracellular concentrations of several key molecules in the bone formation cascade, we examined the effect of a mutant form of Smurf1 in osteoblasts and found that expression of mutant Smurf1 markedly enhanced osteoblast differentiation. Smurf1 therefore appears to be an important regulatory factor in osteoblast differentiation and a potential molecular target for identification of bone anabolic agents.

Animals↗