Harmine promotes molar root development via SMAD1/5/8 phosphorylation.

Tooth and bone are major tissues involved in physiological calcification in the body, and they use similar molecular pathways for development, homeostasis, and regeneration. Harmine (HMN) is a natural small compound that stimulates osteoblast differentiation in vitro and in vivo. Here we examined the biological effect of HMN on the postnatal development of molar tooth roots and periodontal tissues. HMN supported the formation of tooth roots and periodontal tissues in developing tooth germs. In tooth germ organ culture, HMN promoted the elongation of Hertwig's epithelial root sheath (HERS) and stimulated cell proliferation in HERS and dental follicle-derived tissues, including dental papillae and dental follicles. HMN stimulated cell proliferation and cell movement of HERS-derived cells without mesenchymal cells in vitro and directly induced the phosphorylation of SMAD1/5/8 protein in HERS-derived cells. Our results indicated that HMN was the first natural small compound to stimulate postnatal development of tooth germs.

Excess BMP Signaling in Heterotopic Cartilage Forming in Prg4-null TMJ Discs.

Heterotopic cartilage develops in certain pathologic conditions, including those affecting the human temporomandibular joint (TMJ), but the underlying molecular mechanisms remain obscure. This is in part due to the fact that a reliable animal model of such TMJ diseases is not available. Here, we show that aberrant chondrocyte differentiation and ectopic cartilage formation occur spontaneously in proteoglycan 4 (Prg4) mutant TMJ discs without further invasive procedure. By 2 mo of age, mutant disc cells displayed chondrocyte transdifferentiation, accompanied by strong expression of cartilage master gene Sox9 and matrix genes aggrecan and type II collagen. By 6 mo, heterotopic cartilage had formed in the discs and expressed cartilage hypertrophic markers Runx2 and ColX. The ectopic tissue grew in size over time and exhibited regional mineralization by 12 mo. Bone morphogenetic protein (BMP) signaling was activated with the ectopic chondrogenic cells and chondrocytes, as indicated by phosphorylated Smad 1/5/8 nuclear staining and by elevated expression of Bmp2, Bmpr1b, Bmpr2, and BMP signaling target genes. Likewise, we found that upon treatment with recombinant human BMP 2 in high-density micromass culture, mutant disc cells differentiated into chondrocytes and synthesized cartilage matrix more robustly than control cells. Importantly, a specific kinase inhibitor of BMP receptors drastically attenuated chondrogenesis in recombinant human BMP 2-treated mutant disc cultures. Unexpectedly, we found that Prg4 was expressed at joint-associated sites, including disc/muscle insertion and muscle/bone interface, and all these structures were abnormal in Prg4 mutants. Our data indicate that Prg4 is needed for TMJ disc integrity and function and that its absence leads to ectopic chondrogenesis and cartilage formation in conjunction with abnormal BMP signaling. Our findings imply that the BMP signaling pathway could be a potential therapeutic target for prevention or inhibition of ectopic cartilage formation in TMJ disease.

Effects of glutamine on proliferation, migration, and differentiation of human dental pulp cells.

INTRODUCTION: Although glutamine (Gln) is mitogenic in various cell types, little is known about its role in human dental pulp cells (HDPCs). This study investigated the effects of Gln on proliferation, migration, and odontoblastic differentiation of HDPCs and the underlying signal pathway mechanisms. METHODS: Growth and migration were assessed by cell counting and colorimetric cell migration kits. Differentiation was measured as alkaline phosphatase activity, calcified nodule formation by alizarin red staining, and marker mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Chemokine expression was also evaluated by RT-PCR. Signal transduction pathways were examined by RT-PCR and Western blot analysis. RESULTS: Gln dose-dependently increased proliferation, migration, alkaline phosphatase activity, mineralized nodule formation, and odontoblast-marker mRNA of HDPCs. Gln also up-regulated expression of interleukin-6, interleukin-8, MCP-1, MIP-3α, CCL2, CCL20, and CXCL1. Gln increased BMP-2 and BMP-4 mRNA, phosphorylation of Smad 1/5/8, ß-catenin, and key proteins of the Wnt signaling pathway. Furthermore, Gln resulted in up-regulation of extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. In addition, noggin, DKK1, inhibitors of p38, ERK, and JNK significantly attenuatted Gln-induced growth, migration, and odontoblastic differentiation. CONCLUSIONS: Collectively, this study demonstrated that Gln promoted growth, migration, and differentiation in HDPCs through the BMP-2, Wnt, and MAPK pathways, leading to improved pulp repair and regeneration.

Enamel matrix derivative: protein components and osteoinductive properties.

BACKGROUND: Although enamel matrix derivative (EMD) has demonstrated the ability to promote angiogenesis and osteogenesis both in vitro and in vivo, the specific elements within the EMD compound responsible for these effects remain unknown. METHODS: Nine different protein pools from a commercially produced EMD were collected based on molecular weight. Six of these pools, along with the complete EMD unfractionated compound and positive and negative controls, were tested for their ability to induce bone formation in a calvarial induction assay. Immunocytochemistry of phosphorylated SMAD1/5/8 (phospho-SMAD), osterix, and vascular endothelial growth factor A (VEGF-A) was carried out at selected time points. Finally, proteomic analysis was completed to determine the specific protein-peptide content of the various osteoinductive pools. RESULTS: One of the lower-molecular-weight pools tested, pool 7, showed bone induction responses significantly greater than those of the other pools and the complete EMD compound and was concentration dependent. Dynamic bone formation rate analysis demonstrated that pool 7 was optimally active at the 5- to 10-µg concentration. It was demonstrated that EMD and pool 7 induced phospho-SMAD, osterix, and VEGF-A, which is indicative of increased bone morphogenetic protein (BMP) signaling. Proteomic composition analysis demonstrated that pool 7 had the highest concentration of the biologically active amelogenin-leucine-rich amelogenin peptide and ameloblastin 17-kDa peptides. CONCLUSIONS: These studies demonstrate that the low-molecular-weight protein pools (7 to 17 kDa) within EMD have greater osteoinductive potential than the commercially available complete EMD compound and that the mechanism of action, in part, is through increased BMP signaling and increased osterix and VEGF-A. With this information, selected components of EMD can now be formulated for optimal osteo- and angio-genesis.

TGF-ß1, Smad-2/-3, Smad-1/-5/-8, and Smad-4 signaling factors are expressed in ameloblastomas, adenomatoid odontogenic tumors, and calcifying cystic odontogenic tumors: an immunohistochemical study.

OBJECTIVES: The TGF-ß/Smad signaling pathway regulates diverse cellular functions, including tooth development, and is involved in numerous pathological processes such as tumorigenesis. The aim of this study was to investigate the immunoexpression of the TGF-ß/Smad signaling pathway members in ameloblastoma (AM), calcifying cystic odontogenic tumor (CCOT), and adenomatoid odontogenic tumor (AOT). MATERIALS AND METHODS: This retrospective cross-sectional study included 65 tissue specimens: 34 AMs, 13 CCOTs, and 18 AOTs. Serial sections were immunohistochemically stained with TGF-ß1, Smad-4, Smad-1/-5/-8, and Smad-2/-3 antibodies, and a semiquantitative measurement of the positive cells was carried out by two oral pathologists using a 0-3 scale (0: no immunoreactivity, 1: <20% positive cells, 2: 20-50% positive cells, 3: >50% positive cells). RESULTS: All biomarkers studied were found significantly decreased in AM compared to CCOT and AOT. AOT and CCOT expressed Smad-1/-5/-8 more strongly compared to AM (OR = 11.66, P < 0.001 and OR = 5.34, P = 0.013, respectively), and Smad-2/-3 immunostaining was found significantly increased in CCOT (OR = 10.42, P = 0.001) and AOT (OR = 5.16, P < 0.004) compared to AM. Similarly, Smad-4 was expressed more strongly in AOT and CCOT compared to AM (P = 0.001), while AOT demonstrated a fivefold higher chance to express TGF-ß1 compared to AM (P = 0.011). CONCLUSION: TGF-ß/Smad signaling pathway is activated in AM, AOT, and CCOT. The statistically significant reduced TGF-ß1/Smad immunoexpression in AM compared to AOT/CCOT could be associated with the more aggressive biological behavior of AM including increased cell proliferation and reduced apoptosis and differentiation. Thus, the biomarkers TGF-ß, Smad-4, Smad-1/-5/-8, and Smad-2/-3 could serve as supplementary diagnostic indices between odontogenic tumors of high and low neoplastic dynamics.