Sirt6 Activation Ameliorates Inflammatory Bone Loss in Ligature-Induced Periodontitis in Mice.

Periodontitis is an inflammatory disease caused by microorganisms that induce the destruction of periodontal tissue. Inflamed and damaged tissue produces various inflammatory cytokines, which activate osteoclasts and induce alveolar bone loss and, eventually, tooth loss. Sirt6 expression suppresses inflammation and bone resorption; however, its role in periodontitis remains unclear. We hypothesized that Sirt6 has a protective role in periodontitis. To understand the role of Sirt6 in periodontitis, we compared periodontitis with ligature placement around the maxillary left second molar in 8-week-old control (C57BL/6J) male mice to Sirt6-overexpressing Tg (Sirt6Tg) mice, and we observed the resulting phenotypes using micro-CT. MDL801, a Sirt6 activator, was used as a therapy for periodontitis through oral gavage. Pro-inflammatory cytokines and increased osteoclast numbers were observed in alveolar bone tissue under periodontitis surgery. In the same condition, interestingly, protein levels from Sirt6 were the most downregulated among sirtuins in alveolar bone tissue. Based on micro-CT and CEJ-ABC distance, Sirt6Tg was observed to resist bone loss against ligature-induced periodontitis. Furthermore, the number of osteoclasts was significantly reduced in Sirt6Tg-ligated mice compared with control-ligated mice, although systemic inflammatory cytokines did not change. Consistent with this observation, we confirmed that bone loss was significantly reduced when MDL801, a Sirt6 activator, was included in the ligation mouse model. Our findings demonstrate that Sirt6 activation prevents bone loss against ligature-induced periodontitis. Thus, a Sirt6 activator may provide a new therapeutic approach for periodontitis.

Overexpression of COMP-Angiopoietin-1 in K14-Expressing Cells Impairs Hematopoiesis and Disturbs Erythrocyte Maturation.

Numerous studies highlight the potential benefits potentials of supplemental cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) through improved angiogenic effects. However, our recent findings show that excessive overexpression of COMP-Ang1 induces an impaired bone marrow (BM) microenvironment and senescence of hematopoietic stem cells (HSCs). Here, we investigated the underlying mechanisms of how excessive COMP-Ang1 affects the function of BM-conserved stem cells and hematopoiesis using K14-Cre;inducible-COMP-Ang1-transgenic mice. Excessive COMP-Ang1 induced peripheral egression and senescence of BM HSCs and mesenchymal stem cells (MSCs). Excessive COMP-Ang1 also caused abnormal hematopoiesis along with skewed differentiation of HSCs toward myeloid lineage rather than lymphoid lineage. Especially, excessive COMP-Ang1 disturbed late-stage erythroblast maturation, followed by decreased expression of stromal cell-derived factor 1 (SDF-1) and globin transcription factor 1 (GATA-1) and increased levels of superoxide anion and p-p38 kinase. However, transplantation with the mutant-derived BM cells or treatment with rhCOMP-Ang1 protein did not alter the frequency or GATA-1 expression of erythroblasts in recipient mice or in cultured BM cells. Together, our findings suggest that excessive COMP-Ang1 impairs the functions of BM HSCs and MSCs and hematopoietic processes, eventually leading to abnormal erythropoiesis via imbalanced SDF-1/CXCR4 axis and GATA-1 expression rather than Ang1/Tie2 signaling axis alterations.

Genetic overexpression of COMP-Ang1 impairs BM microenvironment and induces senescence of BM HSCs.

Supplemental Angiopoietin 1 (Ang1) exerts its therapeutic potential on microvascular regression-associated diseases, and this potential is linked with the function of hematopoietic stem cells (HSCs). However, the underlying mechanisms of the effect of enhanced angiogenesis on the modulation of HSCs are not yet defined. Here, we generated transgenic mice expressing Cartilage Oligomeric Matrix Protein (COMP)-Ang1 in keratin 14-expressing cells. The mutant animals expressed excessive angiogenic characteristics in the skin and bone marrow (BM) along with redder skin with more numerous and branched vessels compared with their wild-type (WT) littermates. The mutants displayed reduced long bone formation and osteoclast activity than did WT littermates and had fewer CD150+CD48-Lineage-Sca-1+c-Kit+ (LSK) cells in the BM. The mutants also exhibited greater senescence-associated (SA) ß-gal activity, p16INK4a protein expression, and superoxide anion levels in CD150+CD48-LSK cells in the BM. Furthermore, transplantation assay revealed that the mutant-derived LSK cells were inferior to the cells derived from WT littermate in inducing competitive repopulating capacity in the recipients. Collectively, our results demonstrate that persistent and prolonged administration of COMP-Ang1 by inducible transgenic expression mediates excessive angiogenesis in the body and impairs BM microenvironment, eventually leading to senescence of BM HSCs.

Constitutive Activation of Smoothened in the Renal Collecting Ducts Leads to Renal Hypoplasia, Hydronephrosis, and Hydroureter.

Sonic Hedgehog (Shh) signaling plays a major role in and is essential for regulation, patterning, and proliferation during renal development. Smoothened (Smo) plays a pivot role in transducing the Shh-glioma-associated oncogene Kruppel family member. However, the cellular and molecular mechanism underlying the role of sustained Smo activation in postnatal kidney development is still not clearly understood. Using a conditional knockin mouse model that expresses a constitutively activated form of Smo (SmoM2) upon Homeobox-B7-mediated recombination (Hoxb7-Cre), the effects of Shh signaling were determined in postnatal kidney development. SmoM2;Hoxb7-Cre mutant mice showed growth retardation with a reduction of body weight. Constitutive activation of Smo in the renal collecting ducts caused renal hypoplasia, hydronephrosis, and hydroureter. The parenchymal area and glomerular numbers were reduced, but the glomerular density was increased in SmoM2;Hoxb7-Cre mutant mice. The expression of Patched 1, the receptor of Shh and a downstream target gene of the Shh signaling pathway, was highly restricted and it was upregulated in the inner medullary collecting ducts of the kidney. The proliferative cells in the mesenchyme and collecting ducts were decreased in SmoM2;Hoxb7-Cre mutant mice. This study showed for the first time that sustained Smo inhibits postnatal kidney development by suppressing the proliferation of the mesenchyme and medullary collecting ducts in mice.

COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways.

Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

Constitutive activation of smoothened leads to impaired developments of postnatal bone in mice.

Sonic hedgehog (Shh) signaling regulates patterning, proliferation, and stem cell self-renewal in many organs. Smoothened (Smo) plays a key role in transducing Shh signaling into the nucleus by activating a glioma family of transcription factors; however, the cellular and molecular mechanisms underlying the role of sustained Smo activation in postnatal development are still unclear. In this study, we explored the effects of Shh signaling on bone development using a conditional knock-in mouse model that expresses a constitutively activated form of Smo (SmoM2) upon osteocalcin (OCN)-Cre-mediated recombination (SmoM2; OCN-Cre mice). We also evaluated the expression pattern of bone formation-related factors in primary calvarial cultures of mutant and control mice. The SmoM2; OCN-Cre mutant showed growth retardation and reduction of bone mineral density compared to control mice. Constitutively activated SmoM2 also repressed mRNA expression of Runx2, osterix, type I collagen, and osteocalcin. Further, sustained SmoM2 induction suppressed mineralization in calvarial primary osteoblasts cultures, whereas such induction did not affect cell proliferation in the mutant cultures as compared with SmoM2 only control cultures. These results suggest that sustained Smo activation inhibits postnatal development of bone by suppressing gene expression of bone formation regulatory factors in mice.