Advanced breast cancers frequently metastasize to bone, resulting in osteolytic lesions, yet the underlying mechanisms are poorly understood. Here we report that nuclear factor-kappaB (NF-kappaB) plays a crucial role in the osteolytic bone metastasis of breast cancer by stimulating osteoclastogenesis. Using an in vivo bone metastasis model, we found that constitutive NF-kappaB activity in breast cancer cells is crucial for the bone resorption characteristic of osteolytic bone metastasis. We identified the gene encoding granulocyte macrophage-colony stimulating factor (GM-CSF) as a key target of NF-kappaB and found that it mediates osteolytic bone metastasis of breast cancer by stimulating osteoclast development. Moreover, we observed that the expression of GM-CSF correlated with NF-kappaB activation in bone-metastatic tumor tissues from individuals with breast cancer. These results uncover a new and specific role of NF-kappaB in osteolytic bone metastasis through GM-CSF induction, suggesting that NF-kappaB is a potential target for the treatment of breast cancer and the prevention of skeletal metastasis.

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Experimental cell or ex vivo gene therapy for localized bone formation typically uses osteoprogenitor cells propagated from periosteum or bone marrow. Both require bone or marrow biopsies to obtain cells. We have demonstrated that implantation of gingival or dermal fibroblasts transduced with BMP ex vivo, using a recombinant adenovirus (AdCMVBMP) attached to porous biodegradable scaffolds, form bone in vivo. Here we show that BMP-7-transduced fibroblasts suspended in injectable thermoset hydrogels form complete ossicles on subcutaneous injection and repair segmental defects in rat femurs. Bone formation was preceded by an intermediate cartilage stage. To determine the fate of the implanted transduced cells, thermoset hydrogel suspensions of ex vivo BMP-7-transduced or nontransduced fibroblasts were placed in diffusion chambers and implanted to allow development in vivo without direct contact with host cells. Only the BMP-transduced fibroblasts formed bone within the diffusion chambers in vivo, revealing that BMP transduction induces osteoblastic conversion of these cells.

Recombinant human BMP-7 (bone morphogenetic protein-7, osteogenic protein-1) is osteogenic, dentinogenic and cementogenic when implanted into the appropriate tissue in vivo. However, most studies characterizing the induction of these tissues have implanted BMP-7 into freshly surgerized, clinically healthy tissues. To determine if BMP-7 is dentinogenic in inflamed dental pulps, we applied BMP-7 to inflamed ferret pulps. A single application of 5 microg of a commercial preparation of lipopolysaccharide (LPS) from Salmonella typhimurium directly to the coronal pulp induced a reversible mixed inflammatory exudate of moderate intensity within 3 d. Treatment with a single application of 2.5, 7.5 or 25 microg recombinant human BMP-7/mg collagen (2 mg total mass/tooth) induced reparative dentinogenesis in controls but not LPS treated dental pulps. These data reveal that a single application of up to 50 microg/tooth of exogenous recombinant BMP-7 is insufficient to induce reparative dentinogenesis in ferret teeth with reversible pulpitis. Given that pulp cells in the inflamed tissues likely retain the capacity to respond to exogenous BMP-7, it is possible that insufficient active recombinant protein is available to induce tissue formation in experimentally inflamed dental pulps.

Autologous tissue grafting for the restoration of oral tissues is limited by several factors, including the availability of sufficient donor tissue. One solution to this problem may be to develop substitute tissue grafts by attaching disaggregated autologous cells propagated in vitro to scaffolds composed of natural or synthetic polymers. We have earlier demonstrated that human dental pulp and gingival fibroblasts (HPF, HGF) adhere to non-woven polyglycolic acid (PGA) scaffolds, proliferate and produce extracellular matrix in vitro. We now report that such HPF and HGF adhered to PGA scaffolds survive when implanted into subcutaneous sites in immuno-compromised mice. The transplanted cells synthesize and secrete type I collagen, cellular fibronectin and may express genes implicated in transducing bone morphogenetic protein (BMP) signals. Messenger RNA for BMP-2, -4, -7 (OP-1), the BMP type I receptors Act RI, BMPR-1A and 1B, the type II receptor BMPR-II, and type I collagen were detected by reverse transcription-polymerase chain reaction (RT-PCR). These data revealed that three adult human dental pulp and gingival cell populations, each from individual donors, attached to PGA scaffolds and cultured for 24 h in vitro, survive implantation and express genes indicative of a capacity to produce extracellular matrix. The implanted cells may also express genes associated with responsiveness to BMP-mediated tissue inductive signals.

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP; phosphophoryn) are two principal dentin-specific non-collagenous proteins. DPP is extremely acidic and is rich in aspartic acid and serine. By virtue of this structure, DPP may bind large amounts of calcium and may facilitate initial mineralization of dentin matrix collagen as well as regulate the size and shape of the crystals. The function of DSP is not known. DSP and DPP are encoded by a single gene in both rat and mouse, and are uniquely expressed in odontoblasts and transiently in pre-ameloblasts. Because DSP and DPP are isolated from dentin as distinct proteins and appear to be present in different amounts, the nascent dentin sialophosphoprotein (DSPP) is likely cleaved to yield DSP and DPP. However, when, where and how the DSPP is cleaved into DSP and DPP is not clear. To further elucidate the structure and function of human DSP and DPP, we have cloned DPP and DSP cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) strategies, and then cloned and initiated characterization of a human dentin sialophosphoprotein gene. The genomic organization of human DSPP is very similar to that of mouse, containing five exons and four introns, suggesting it is a homologue of mouse dentin sialophosphoprotein (DSPP). Exons 1-4 encode for DSP, while exon 5 encodes for the C-terminus of DSP and the whole DPP. A 4.6-kb RNA transcript was detected on Northern blot analyses of total RNA extracted from immature (open root apices) human teeth using either a human DPP or DSP probe.

Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A has been the subject of extensive studies, however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wildtype mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoters to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoters to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development.

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