Endometrial receptivity and implantation require uterine BMP signaling through an ACVR2A-SMAD1/SMAD5 axis.

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a-PRcre and Acvr2b-PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation.

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.

Overexpression of the BMP4/SMAD signaling pathway in skull base chordomas is associated with poor prognosis.

Chordomas are rare, locally invasive tumors with characteristic expression of the T-box transcription factor Brachyury. Little is yet known of the molecular events involved in the development of these tumors. Bone morphogenesis protein 4 (BMP4) signaling, which acts upstream of Brachyury in embryonic development, has been implicated in carcinogenesis in multiple malignancies. To explore the role of the canonical BMP4/SMAD signaling pathway in the pathogenesis of chordoma, we investigated, in 40 skull base chordomas, the expression of three major components of the signaling axis: BMP4, phospho-SMAD5 and SMAD4. Immunostaining revealed positive expression in 70%, 52.5% and 90% of cases, respectively. Eighteen (45%) of patients exhibited concurrent positive expression of these markers, which we defined as "high" expression of the BMP4/SMAD signaling pathway. Interestingly, when we compared the pattern of expression with clinicopathological parameters, we found that high expression of the pathway was more often observed in larger tumors (≥ 4 cm) than smaller ones (P = 0.010), and correlated significantly with dural invasion (P = 0.024). The Kaplan-Meier log-rank test showed that the 5-year overall survival rate for patients with high expression of the pathway was significantly lower than those with low expression (71.4% vs. 90.2%, P = 0.010). In conclusion, our results demonstrate for the first time that overexpression of the BMP4/SMAD signaling pathway could predict poor clinical outcome in skull base chordomas, suggesting activation of this pathway is involved in chordoma pathogenesis.

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.

Fibromodulin and Biglycan Modulate Periodontium through TGFß/BMP Signaling.

A full understanding of the key regulators controlling periodontal development and homeostasis is necessary for the design of improved periodontal regenerative therapies. Small leucine-rich proteoglycans (SLRPs) are extracellular matrix molecules suggested to regulate collagen organization and cell signaling. Mice with double-deficiency of 2 SLRPs, fibromodulin and biglycan (dKO), acquire skeletal abnormalities, but their roles in regulating the periodontium remain undefined and were the focus of our studies. Transmission electron microscopy studies showed abnormal collagen fibrils in the periodontal ligament (PDL) and altered remodeling of alveolar bone in dKO mice. Immunohistochemistry (IHC) revealed increased staining of SLRPs (asporin, lumican, and decorin) and dentin matrix protein-1 (DMP1, a mechanosensory/osteocyte marker), while osteoblast markers, bone sialoprotein and osteopontin, remained unchanged. Disruption of homeostasis was further evidenced by increased expression of receptor-activator of nuclear factor-κB ligand (RANKL) and elevated numbers of osteoclasts, especially noted around the alveolar bone of molars (buccal side) and incisors. Polymerase chain reaction (PCR) array revealed hyperactive transforming growth factors beta/bone morphogenetic protein (TGFß/BMP) signaling in dKO PDL tissues, which was further confirmed by elevated expression of phosphorylated Smad5 (p-Smad5) by IHC in dKO PDL. These studies highlight the importance of SLRPs in maintaining periodontal homeostasis through regulation of TGFß/BMP signaling, matrix turnover, and collagen organization.

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.

Detection, identification and in vivo treatment responsiveness of bone morphogenetic protein (BMP)-activated cell populations in the synovium of patients with rheumatoid arthritis.

OBJECTIVE: To characterise the bone morphogenetic protein (BMP) target cells positive for phosphorylated (P)-SMAD1/5, in rheumatoid arthritis (RA) synovium. METHODS: Synovial biopsies were obtained by needle arthroscopy. Anti-P-SMAD1/5 antibodies were used for Western blot (WB) on protein extracts from RA and normal synovium and for immunostaining of synovial biopsy sections. Positive cells were further identified by double staining for CD3, CD20, CD68, CD138, CD90, alpha smooth muscle actin (SMA), endoglin (CD105) and von Willebrand factor (VWF). In sections from early patients with RA taken before and under antirheumatic treatment, the degree of inflammation and activation of the BMP pathway were quantified. RESULTS: P-SMAD1/5 protein was detected by WB in RA and to a lesser extent in normal synovium. Different P-SMAD1/5 positive cell populations were identified in RA synovium, mainly in perivascular and sublining cells. P-SMAD1/5 positive perivascular cells were alphaSMA positive and located around VWF positive endothelial cells. Some CD90 positive synovial fibroblasts were P-SMAD1/5 positive, as was part of the CD68 positive synovial cells but other cells of the haematopoietic lineage showed no SMAD1/5 phosphorylation. Treatment resulted in an absolute but not relative decrease in BMP activation in the synovium. CONCLUSION: BMP-activated cells belong to distinct stromal compartments in RA synovium and some of them express markers associated with the mesenchymal progenitor cell lineage. Antirheumatic treatment effectively downregulates synovial inflammation, but BMP activation in the synovium does persist albeit reduced.