Sclerostin expression in trabecular bone is downregulated by osteoclasts.

Bone tissues have trabecular bone with a high bone turnover and cortical bone with a low turnover. The mechanisms by which the turnover rate of these bone tissues is determined remain unclear. Osteocytes secrete sclerostin, a Wnt/ß-catenin signaling antagonist, and inhibit bone formation. We found that sclerostin expression in cortical bone is more marked than in trabecular bone in Sost reporter mice. Leukemia inhibitory factor (LIF) secreted from osteoclasts reportedly suppressed sclerostin expression and promoted bone formation. Here, we report that osteoclasts downregulate sclerostin expression in trabecular bone and promote bone turnover. Treatment of C57BL/6 mice with an anti-RANKL antibody eliminated the number of osteoclasts and LIF-positive cells in trabecular bone. The number of sclerostin-positive cells was increased in trabecular bone, while the number of ß-catenin-positive cells and bone formation were decreased in trabecular bone. Besides, Tnfsf11 heterozygous (Rankl+/-) mice exhibited a decreased number of LIF-positive cells and increased number of sclerostin-positive cells in trabecular bone. Rankl+/- mice exhibited a decreased number of ß-catenin-positive cells and reduced bone formation in trabecular bone. Furthermore, in cultured osteoclasts, RANKL stimulation increased Lif mRNA expression, suggesting that RANKL signal increased LIF expression. In conclusion, osteoclasts downregulate sclerostin expression and promote trabecular bone turnover.

The Biological Efficacy of Natural Products against Acute and Chronic Inflammatory Diseases in the Oral Region.

The oral inflammatory diseases are divided into two types: acute and chronic inflammatory diseases. In this review, we summarize the biological efficacy of herbal medicine, natural products, and their active ingredients against acute and chronic inflammatory diseases in the oral region, especially stomatitis and periodontitis. We review the effects of herbal medicines and a biscoclaurin alkaloid preparation, cepharamthin, as a therapy against stomatitis, an acute inflammatory disease. We also summarize the effects of herbal medicines and natural products against periodontitis, a chronic inflammatory disease, and one of its clinical conditions, alveolar bone resorption. Recent studies show that several herbal medicines such as kakkonto and ninjinto reduce LPS-induced PGE 2 production by human gingival fibroblasts. Among herbs constituting these herbal medicines, shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) strongly reduce PGE 2 production. Moreover, anti-osteoclast activity has been observed in some natural products with anti-inflammatory effects used against rheumatoid arthritis such as carotenoids, flavonoids, limonoids, and polyphenols. These herbal medicines and natural products could be useful for treating oral inflammatory diseases.

Heterogeneous and abnormal localization of desmosomal proteins in oral intraepithelial neoplasms.

To study the relationship between the biological and morphological characteristics of oral intraepithelial neoplasms (OINs), we examined the localization of desmosome-related proteins. Twenty-seven cases of OIN3 were tentatively classified as basaloid (14 cases) or differentiated (13 cases), and the latter were further subdivided into verrucous (five cases) and acanthotic (eight cases) subtypes. All samples were stained using antibodies against desmoglein 1 (DSG1), desmocollin 3 (DSC3), junction plakoglobin (JUP) and serine peptidase inhibitor Kazal type 5 (SPINK5) domain. All variants of OIN3 showed significantly high rates of positivity for DSG1 in the basal layer (basaloid 57%; differentiated 85%), DSC3 in the surface layer (basaloid 93%; differentiated 77%) and JUP in the basal and parabasal layers (basaloid 93%; differentiated 62%). Interestingly, even the basaloid type showed areas of alternating DSG1 positivity and negativity, reflecting keratinocyte maturation. Therefore, most cases of OIN appear to have the characteristics of well differentiated squamous epithelium.

Protein kinase A enhances lipopolysaccharide-induced IL-6, IL-8, and PGE2 production by human gingival fibroblasts.

OBJECTIVE: Periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Interleukin (IL)-6, IL-8, and the chemical mediator prostaglandin E2 (PGE2) are known to play important roles in inflammatory responses and tissue degradation. Recently, we reported that the protein kinase A (PKA) inhibitor H-89 suppresses lipopolysaccharide (LPS)-induced IL-8 production by human gingival fibroblasts (HGFs). In the present study, the relevance of the PKA activity and two PKA-activating drugs, aminophylline and adrenaline, to LPS-induced inflammatory cytokines (IL-6 and IL-8) and PGE2 by HGFs were examined. METHODS: HGFs were treated with LPS from Porphyromonas gingivalis and H-89, the cAMP analog dibutyryl cyclic AMP (dbcAMP), aminophylline, or adrenaline. After 24 h, IL-6, IL-8, and PGE2 levels were evaluated by ELISA. RESULTS: H-89 did not affect LPS-induced IL-6 production, but suppressed IL-8 and PGE2 production. In contrast, dbcAMP significantly increased LPS-induced IL-6, IL-8, and PGE2 production. Up to 10 µg/ml of aminophylline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at 100 µg/ml. Similarly, 0.01 µg/ml of adrenaline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at concentrations of 0.1 and 1 µg/ml. In the absence of LPS, H-89, dbcAMP, aminophylline, and adrenaline had no relevance to IL-6, IL-8, or PGE2 production. CONCLUSION: These results suggest that the PKA pathway, and also PKA-activating drugs, enhance LPS-induced IL-6, IL-8, and PGE2 production by HGFs. However, aminophylline may not have an effect on the production of these molecules at concentrations used in clinical settings (8 to 20 µg/ml in serum). These results suggest that aminophylline does not affect inflammatory responses in periodontal disease.

Human gingival fibroblasts are critical in sustaining inflammation in periodontal disease.

BACKGROUND AND OBJECTIVE: A major factor in the pathogenesis of periodontal disease, which is one of the biofilm infectious diseases, is thought to be lipopolysaccharide (LPS), owing to its ability to cause inflammation and promote tissue destruction. Moreover, the elimination of pathogens and their component LPSs is essential for the successful treatment of periodontal disease. Lipopolysaccharide tolerance is a mechanism that prevents excessive and prolonged responses of monocytes and macrophages to LPS. Since persistence of inflammation is necessary for inflammatory cytokine production, cells other than monocytes and macrophages are thought to maintain the production of cytokines in the presence of LPS. In this study, we investigated whether human gingival fibroblasts (HGFs), the most abundant structural cell in periodontal tissue, might be able to maintain inflammatory cytokine production in the presence of LPS bynot displaying LPS tolerance. MATERIAL AND METHODS: Human gingival fibroblasts were pretreated with LPS (from Porphyromonas gingivalis and Escherichia coli) and then treated with LPS, and the amounts of interleukin (IL)-6 and IL-8 in the cell culture supernatants were measured. The expression of negative regulators of LPS signalling (suppressor of cytokine signalling-1, interleukin-1 receptor-associated-kinase M and SH2 domain-containing inositol-5-phosphatase-1) was also examined in LPS-treated HGFs. RESULTS: Human gingival fibroblasts did not display LPS tolerance but maintained production of IL-6 and IL-8 when pretreated with LPS, followed by secondary LPS treatment. Lipopolysaccharide-treated HGFs did not express negative regulators. CONCLUSION: These results demonstrate that HGFs do not show LPS tolerance and suggest that this characteristic of HGFs sustains the inflammatory response in the presence of virulence factors.

Preventive effects of a Kampo medicine, Shosaikoto, on inflammatory responses in LPS-treated human gingival fibroblasts.

In the present study, we investigated the anti-inflammatory effects of a Kampo medicine Shosaikoto (TJ-9) using in vitro periodontal disease model, in which human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) produce IL-6, IL-8 and prostaglandin E2 (PGE2). Treatment with PgLPS (10 ng/ml), TJ-9 (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Moreover, TJ-9 did not alter LPS-induced IL-6 and IL-8 productions. However, TJ-9 significantly suppressed LPS-induced PGE2 production in a dose-dependent manner but TJ-9 alone did not affect basal PGE2 level. Western blotting demonstrated that TJ-9 decreased cyclooxygenase-2 (COX-2) expression in a dose-dependent manner but not phospholipase A2. Moreover, TJ-9 selectively and dose-dependently inhibited COX-2 activity. These results suggest that TJ-9 decreased PGE2 production by inhibition of both COX-2 expression and activity and that TJ-9 may be useful to improve gingival inflammation in periodontal disease.

Adrenomedullin/cyclic AMP pathway induces Notch activation and differentiation of arterial endothelial cells from vascular progenitors.

OBJECTIVE: The acquisition of arterial or venous identity is highlighted in vascular development. Previously, we have reported an embryonic stem (ES) cell differentiation system that exhibits early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors. In this study, we constructively induced differentiation of arterial and venous endothelial cells (ECs) in vitro to elucidate molecular mechanisms of arterial-venous specification. METHODS AND RESULTS: ECs were induced from VEGFR2+ progenitor cells with various conditions. VEGF was essential to induce ECs. Addition of 8bromo-cAMP or adrenomedullin (AM), an endogenous ligand-elevating cAMP, enhanced VEGF-induced EC differentiation. Whereas VEGF alone mainly induced venous ECs, 8bromo-cAMP (or AM) with VEGF supported substantial induction of arterial ECs. Stimulation of cAMP pathway induced Notch signal activation in ECs. The arterializing effect of VEGF and cAMP was abolished in recombination recognition sequence binding protein at the Jkappa site deficient ES cells lacking Notch signal activation or in ES cells treated with gamma-secretase inhibitor. Nevertheless, forced Notch activation by the constitutively active Notch1 alone did not induce arterial ECs. CONCLUSIONS: Adrenomedullin/cAMP is a novel signaling pathway to activate Notch signaling in differentiating ECs. Coordinated signaling of VEGF, Notch, and cAMP is required to induce arterial ECs from vascular progenitors.

The role of CXCL12 in the organ-specific process of artery formation.

CXC chemokine ligand 12 (CXCL12; stromal cell-derived factor-1 [SDF-1]/pre-B-cell growth-stimulating factor [PBSF]) and its receptor CXCR4 are essential for vascularization in the gastrointestinal tract as well as B lymphopoiesis and colonization of bone marrow by hematopoietic cells. However, the mechanism by which CXCL12/CXCR4 functions in blood vessel formation remains elusive. Here, we have found a novel mode of organ vascularization and determined the roles of CXCL12 in these processes. In the developing small intestine, many short interconnecting vessels form between larger superior mesenteric artery (SMA) and the neighboring primary capillary plexus surrounding the primitive gut, and they elongate and become the arteries supplying the small intestine. Mice lacking CXCL12 or CXCR4 lack the interconnecting vessels but have normal venous networks. The mutants lack filopodial extension and intussusception from endothelial cells of SMAs seen in wild-type embryos. CXCR4 is specifically expressed in arteries in the developing mesenteries and its expression is severely reduced in CXCL12-/- embryos. Mice in which CXCR4 is specifically deleted in the endothelium reveal vascular defects identical to those observed in the conventional CXCR4-/- embryos. Together, CXCL12 acts on arterial endothelial cells of SMA to up-regulate CXCR4 and mediate the connection between the larger artery and neighboring capillary plexus in an organ-specific manner.

Long-term hematopoietic stem cells require stromal cell-derived factor-1 for colonizing bone marrow during ontogeny.

The physiological role of SDF-1 on hematopoietic stem cells (HSCs) remains elusive. We show that colonization of bone marrow by HSCs in addition to myeloid cells is severely impaired in SDF-1(-/-) embryos by a long-term repopulation assay. Colonization of spleen by HSCs was also affected, but to a lesser extent. Enforced expression of SDF-1 under the control of vascular-specific Tie-2 regulatory sequences could completely rescue the reduction of HSCs but not myeloid cells in SDF-1(-/-) bone marrow. SDF-1 was detected in the vicinity of the vascular endothelial cells in fetal bone marrow. SDF-1 plays a critical role in colonization of bone marrow by HSCs and myeloid cells during ontogeny, and the mechanisms by which SDF-1 functions are distinct between HSCs and myeloid cells.

CXCR4 regulates interneuron migration in the developing neocortex.

The chemotactic factors directing interneuron migration during cerebrocortical development are essentially unknown. Here we identify the CXC chemokine receptor 4 (CXCR4) in interneuron precursors migrating from the basal forebrain to the neocortex and demonstrate that stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for isolated striatal precursors. In addition, we show that CXCR4 is present in early generated Cajal-Retzius cells of the cortical marginal zone. In mice with a null mutation in CXCR4 or SDF-1, interneurons were severely underrepresented in the superficial layers and ectopically placed in the deep layers of the neocortex. In contrast, the submeningeal positioning of Cajal-Retzius cells was unaffected. Thus, our findings suggest that SDF-1, which is highly expressed in the embryonic leptomeninx, selectively regulates migration and layer-specific integration of CXCR4-expressing interneurons during neocortical development.

Impaired colonization of the gonads by primordial germ cells in mice lacking a chemokine, stromal cell-derived factor-1 (SDF-1).

Primordial germ cells (PGCs) are the founders of sperm or oocytes. PGCs migrate through the tissues of the embryos and colonize the gonads during development. However, the cytokines essential for colonization of the gonads by PGCs in mammals remain unclear. Stromal cell-derived factor-1 (SDF-1, also called PBSF and CXCL12) is a member of chemokines, a family of structurally related chemoattractive cytokines. SDF-1 and its primary physiologic receptor CXCR4 have multiple essential functions in development including colonization of bone marrow by hematopoietic cells and neuron localization within cerebellum during embryogenesis as well as B lymphopoiesis and cardiovasculogenesis. Here, we have shown that PGCs have cell-surface expression of CXCR4 and that, in SDF-1(-/-) mice, PGCs undergo directed migration through tissues of embryos, but the numbers of PGCs in the gonads are significantly reduced. The proliferation of PGCs within the gonads seems normal in the mutant mice. These findings reveal the essential role for SDF-1 in murine PGC development likely by controlling colonization of the gonads by PGCs.

A role of CXC chemokine ligand 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its receptor CXCR4 in fetal and adult T cell development in vivo.

The functions of a chemokine CXC chemokine ligand (CXCL) 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its physiologic receptor CXCR4 in T cell development are controversial. In this study, we have genetically further characterized their roles in fetal and adult T cell development using mutant and chimeric mice. In CXCL12(-/-) or CXCR4(-/-) embryos on a C57BL/6 background, accumulation of T cell progenitors in the outer mesenchymal layer of the thymus anlage during initial colonization of the fetal thymus was comparable with that seen in wild-type embryos. However, the expansion of CD3(-)CD4(-)CD8(-) triple-negative T cell precursors at the CD44(-)CD25(+) and CD44(-)CD25(-) stages, and CD4(+)CD8(+) double-positive thymocytes was affected during embryogenesis in these mutants. In radiation chimeras competitively repopulated with CXCR4(-/-) fetal liver cells, the reduction in donor-derived thymocytes compared with wild-type chimeras was much more severe than the reduction in donor-derived myeloid lineage cells in bone marrow. Triple negative CD44(+)CD25(+) T cell precursors exhibited survival response to CXCL12 in the presence of stem cell factor as well as migratory response to CXCL12. Thus, it may be that CXCL12 and CXCR4 are involved in the expansion of T cell precursors in both fetal and adult thymus in vivo. Finally, enforced expression of bcl-2 did not rescue impaired T cell development in CXCR4(-/-) embryos or impaired reconstitution of CXCR4(-/-) thymocytes in competitively repopulated mice, suggesting that defects in T cell development caused by CXCR4 mutation are not caused by reduced expression of bcl-2.