Janice E. Berry

Bone Morphogenetic Protein 2 Induces Dental Follicle Cells to Differentiate Toward a Cementoblast/Osteoblast Phenotype†

When triggered appropriately, dental follicle cells are considered to be able to differentiate toward a cementoblast/osteoblast phenotype. However, factors and mechanisms regulating follicle cell differentiation remain undefined. This study focused on determining the ability of bone morphogenetic protein (BMP) 2 to promote the differentiation of follicle cells and periodontal ligament (PDL) cells along a cementoblast/osteoblast pathway. Follicle cells and PDL cells were isolated from the first molar region of CD‐1 mice and immortalized with SV40. Both cell types expressed BMP‐4 and BMP receptors (BMPR) IA and II, but only follicle cells expressed BMP‐2 mRNA. Cells were exposed to recombinant human BMP (rhBMP)‐2 (0–100 ng/ml) and Northern blots were used to determine the expression of mineral‐associated markers. BMP‐2, in a dose‐ and time‐dependent manner, induced cementoblast/osteoblast differentiation of follicle cells, as reflected by enhanced core binding factor α1 (Cbfa1), bone sialoprotein (BSP), and osteocalcin (OCN) mRNA expression and enhanced mineral formation. U0126, a specific inhibitor of MEK‐1/2 members of the MAPK family, abolished BMP‐2‐mediated expression of BSP and OCN. In contrast, exposure of PDL cells to BMP‐2 resulted in modest expression of OCN and minimal promotion of mineralization. These results suggest that BMP‐2 triggers follicle cells to differentiate toward a cementoblast/osteoblast phenotype and that the MAPK pathway is involved.

Recruitment of mesenchymal stem cells into prostate tumours promotes metastasis

Tumors recruit mesenchymal stem cells (MSCs) to facilitate healing, which induces their conversion into cancer-associated fibroblasts that facilitate metastasis. However, this process is poorly understood on the molecular level. Here we show that the CXCR6 ligand CXCL16 facilitates MSC or Very Small Embryonic-Like (VSEL) cells recruitment into prostate tumors. CXCR6 signaling stimulates the conversion of MSCs into cancer-associated fibroblasts, which secrete stromal-derived factor-1, also known as CXCL12. CXCL12 expressed by cancer-associated fibroblasts then binds to CXCR4 on tumor cells and induces an epithelial to mesenchymal transition, which ultimately promotes metastasis to secondary tumor sites. Our results provide the molecular basis for MSC recruitment into tumors and how this process leads to tumor metastasis.

Expression of bone associated markers by tooth root lining cells, in situ and in vitro.

Periodontal disease is marked by inflammation and subsequent loss and/or damage to tooth-supporting tissues including bone, cementum, and periodontal ligament. A key tissue in the initial process of periodontal development as well as regeneration following periodontal disease is cementum. Research efforts aimed toward understanding mechanisms involved in periodontal development and regeneration, and in particular the formation of root cementum, have been hampered by an inability to isolate and culture cells involved in cementum production (i.e., cementoblasts). Much has been learned regarding the processes and mechanisms involved in bone formation and function from experiments using bone cell cultures. Therefore, the purpose of this study was to develop a strategy whereby cementoblasts could be isolated, cultured, and characterized. As a first step, using in situ hybridization, we determined the timed and spatial expression of mineral-associated proteins during first molar root development in CD-1 mice. These proteins included dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), and type I collagen. During root development in mice BSP, OPN, and OCN mRNAs were expressed selectively by cells lining the tooth root surface--cementoblasts--with high levels of expression at day 41. Importantly, at this time point BSP, OPN, and OCN mRNAs were not expressed throughout the periodontal ligament. These findings provided us with markers selective to root-lining cells, or cementoblasts, in situ, and established the time (day 41) for isolating cells for in vitro studies. To isolate cells from tissues adherent to the root surface, enzymatic digestion was used, similar to what are now considered classical techniques for isolation of osteoblasts. To determine whether cells in vitro contained root-lining cells and cementoblasts, cultured cells were analyzed for expression of mineral-associated proteins. Cells within this heterogeneous primary population expressed type I collagen, BSP, OPN, and OCN as determined by in situ hybridization. In contrast, cells within this population did not express dentin sialoprotein, an odontoblast-specific protein. These procedures have provided a means to obtain root-lining cells in vitro that can now be cloned and used for studies directed at determining the properties of root-lining cells, or cementoblasts, in vitro.

Regulation of Cementoblast Gene Expression by Inorganic Phosphate In Vitro

Examination of mutant and knockout phenotypes with altered phosphate/pyrophosphate distribution has demonstrated that cementum, the mineralized tissue that sheathes the tooth root, is very sensitive to local levels of phosphate and pyrophosphate. The aim of this study was to examine the potential regulation of cementoblast cell behavior by inorganic phosphate (Pi). Immortalized murine cementoblasts were treated with Piin vitro, and effects on gene expression (by quantitative real-time reverse-transcriptase polymerase chain reaction [RT-PCR]) and cell proliferation (by hemacytometer count) were observed. Dose-response (0.1–10 mM) and time-course (1–48 hours) assays were performed, as well as studies including the Na-Pi uptake inhibitor phosphonoformic acid. Real-time RT-PCR indicated regulation by phosphate of several genes associated with differentiation/mineralization. A dose of 5 mM Pi upregulated genes including the SIBLING family genes osteopontin (Opn, >300% of control) and dentin matrix protein-1 (Dmp-1, >3,000% of control). Another SIBLING family member, bone sialoprotein (Bsp), was downregulated, as were osteocalcin (Ocn) and type I collagen (Col1). Time-course experiments indicated that these genes responded within 6–24 hours. Time-course experiments also indicated rapid regulation (by 6 hours) of genes concerned with phosphate/pyrophosphate homeostasis, including the mouse progressive ankylosis gene (Ank), plasma cell membrane glycoprotein-1 (Pc-1), tissue nonspecific alkaline phosphatase (Tnap), and the Pit1 Na-Pi cotransporter. Phosphate effects on cementoblasts were further shown to be uptake-dependent and proliferation-independent. These data suggest regulation by phosphate of multiple genes in cementoblasts in vitro. During formation, phosphate and pyrophosphate may be important regulators of cementoblast functions including maturation and regulation of matrix mineralization.

Role of two mineral-associated adhesion molecules, osteopontin and bone sialoprotein, during cementogenesis.

Adhesion molecules and their cell membrane receptors are known to play important regulatory roles in cell differentiation. Consequently, the following experiments were conducted to determine the role of two adhesion molecules, bone sialoprotein (BSP) and osteopontin (OPN) in tooth root formation. Developing murine molar tooth germs at sequential stages of development (developmental days 21-42) were analyzed using immunohistochemical and in situ hybridization techniques. While BSP was localized to alveolar bone and odontoblasts early in development, BSP was distinctly localized to the cemental root surface at latter periods coincident with the initiation of root formation and cementogenesis. Conversely, OPN was distributed in a nonspecific fashion throughout the PDL and the eruption pathway of the forming tooth. In situ hybridization confirmed that cells lining the root surface express BSP. The fact that BSP is specifically localized to the cemental surface suggests that this protein is involved in cementoblast differentiation and/or early mineralization of the cementum matrix. Localization of OPN to non-mineralized tissues further suggests that OPN functions as an inhibitor of mineralization during periodontal ligament formation. These findings collectively suggest that BSP and OPN are intimately involved in the sequence of cellular and molecular events accompanying cementogenesis.

Tumor expressed PTHrP facilitates prostate cancer-induced osteoblastic lesions

Expression of parathyroid hormone‐related protein (PTHrP) correlates with prostate cancer skeletal progression; however, the impact of prostate cancer‐derived PTHrP on the microenvironment and osteoblastic lesions in skeletal metastasis has not been completely elucidated. In this study, PTHrP overexpressing prostate cancer clones were stably established by transfection of full length rat PTHrP cDNA. Expression and secretion of PTHrP were verified by western blotting and IRMA assay. PTHrP overexpressing prostate cancer cells had higher growth rates in vitro, and generated larger tumors when inoculated subcutaneously into athymic mice. The impact of tumor‐derived PTHrP on bone was investigated using a vossicle co‐implant model. Histology revealed increased bone mass adjacent to PTHrP overexpressing tumor foci, with increased osteoblastogenesis, osteoclastogenesis and angiogenesis. In vitro analysis demonstrated pro‐osteoclastic and pro‐osteoblastic effects of PTHrP. PTHrP enhanced proliferation of bone marrow stromal cells and early osteoblast differentiation. PTHrP exerted a pro‐angiogenic effect indirectly, as it increased angiogenesis but only in the presence of bone marrow stromal cells. These data suggest PTHrP plays a role in tumorigenesis in prostate cancer, and that PTHrP is a key mediator for communication and interactions between prostate cancer and the bone microenvironment. Prostate cancer‐derived PTHrP is actively involved in osteoblastic skeletal progression.

One-step purification of murine IgM and human α2-macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin

A new mannose-specific plant lectin (GNA) isolated from the snowdrop bulb was immobilized on Sepharose 4B and employed for the purification of certain glycoproteins with high-mannose type glycan chains. Murine IgM bound tightly to this column and was eluted with 0.1 M methyl alpha-D-mannoside whereas bovine and murine IgG were not bound. When a murine hybridoma serum containing IgM monoclonal antibody was applied to this column, highly purified IgM antibody was obtained after elution with methyl alpha-D-mannoside. On the contrary, human IgM was not bound by this column despite reports that it contains high-mannose type glycan chains. alpha 2-Macroglobulin was the sole glycoprotein present in human serum which was bound by the immobilized snowdrop lectin column. It appears that only glycoproteins containing multiple Man(alpha 1,3)Man units are bound to the immobilized lectin.

GAS6 Receptor Status Is Associated with Dormancy and Bone Metastatic Tumor Formation

Disseminated tumor cells (DTCs) are believed to lie dormant in the marrow before they can be activated to form metastases. How DTCs become dormant in the marrow and how dormant DTCs escape dormancy remains unclear. Recent work has shown that prostate cancer (PCa) cell lines express the growth-arrest specific 6 (GAS6) receptors Axl, Tyro3, and Mer, and become growth arrested in response to GAS6. We therefore hypothesized that GAS6 signaling regulates the proliferative activity of DTCs in the marrow. To explore this possibility, in vivo studies were performed where it was observed that when Tyro3 expression levels exceed Axl expression, the PCa cells exhibit rapid growth. When when Axl levels predominate, PCa cells remain largely quiescent. These findings suggest that a balance between the expression of Axl and Tyro3 is associated with a molecular switch between a dormant and a proliferative phenotype in PCa metastases.

Cementum: A Phosphate-sensitive Tissue

Ectopic calcification within joints has been reported in humans and rodents exhibiting mutations in genes that regulate the level of extracellular pyrophosphate, e.g., ank and PC-1; however, periodontal effects of these mutations have not previously been examined. These initial studies using ank and PC-1 mutant mice were done to see if such mineral deposition and resulting ankylosis were occurring in the periodontium as well. Surprisingly, results indicated the absence of ankylosis; however, a marked increase in cementum formation on the root surfaces of fully developed teeth of these mutant mice was noted. Examination of ank mutant mice at earlier ages of tooth root formation indicated that this striking observation is apparent from the onset of cementogenesis. These findings suggest that cells within the periodontal region are highly responsive to changes in phosphate metabolism. This information may prove valuable in attempts to design successful therapies for regenerating periodontal tissues.

Bone Morphogenetic Protein-2 Inhibits Differentiation and Mineralization of Cementoblasts in vitro

As an approach for improving the outcome and predictability of periodontal regenerative therapies, we have focused on determining the responses of cells within the local environment to putative regenerative factors. This study examined the effects of bone morphogenetic protein-2 (BMP-2) on murine cementoblasts in vitro. Northern blot analysis indicated that BMP-2 decreased mRNA levels of bone sialoprotein and type I collagen dose-dependently (10–300 ng/mL). At low doses, up to 100 ng/mL, BMP-2 had no effect on transcripts for osteocalcin and osteopontin, whereas at 300 ng/mL, BMP-2 greatly increased expression of these two genes. BMP-2 also inhibited cementoblast-mediated mineral nodule formation in a dose-dependent manner (inhibition was noted at 10 ng/mL). Noggin reversed the effects of BMP-2 on gene expression and on mineralization. These findings reflect the diverse responses of periodontal cells to BMP-2 and highlight the need to consider the complexity of factors involved in designing predictable regenerative therapies.