Expression and Role of IL-1β Signaling in Chondrocytes Associated with Retinoid Signaling during Fracture Healing

The process of fracture healing consists of an inflammatory reaction and cartilage and bone tissue reconstruction. The inflammatory cytokine interleukin-1β (IL-1β) signal is an important major factor in fracture healing, whereas its relevance to retinoid receptor (an RAR inverse agonist, which promotes endochondral bone formation) remains unclear. Herein, we investigated the expressions of IL-1β and retinoic acid receptor gamma (RARγ) in a rat fracture model and the effects of IL-1β in the presence of one of several RAR inverse agonists on chondrocytes. An immunohistochemical analysis revealed that IL-1β and RARγ were expressed in chondrocytes at the fracture site in the rat ribs on day 7 post-fracture. In chondrogenic ATDC5 cells, IL-1β decreases the levels of aggrecan and type II collagen but significantly increased the metalloproteinase-13 (Mmp13) mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. An RAR inverse agonist (AGN194310) inhibited IL-1β-stimulated Mmp13 and Ccn2 mRNA in a dose-dependent manner. Phosphorylated extracellular signal regulated-kinases (pERK1/2) and p-p38 mitogen-activated protein kinase (MAPK) were increased time-dependently by IL-1β treatment, and the IL-1β-induced p-p38 MAPK was inhibited by AGN194310. Experimental p38 inhibition led to a drop in the IL-1β-stimulated expressions of Mmp13 and Ccn2 mRNA. MMP13, CCN2, and p-p38 MAPK were expressed in hypertrophic chondrocytes near the invaded vascular endothelial cells. As a whole, these results point to role of the IL-1β via p38 MAPK as important signaling in the regulation of the endochondral bone formation in fracture healing, and to the actions of RAR inverse agonists as potentially relevant modulators of this process

The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction.

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b+ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment

A case of adenoid cystic carcinoma associated with IgG4-related disease

Introduction: Immunoglobulin G4-related disease (IgG4-RD) is an inflammatory condition associated with elevated serum IgG4 levels and tissue infiltration by IgG4-expressing plasma cells. We present a case of adenoid cystic carcinoma (ACC) of the submandibular gland with possible involvement of IgG4-RD. Presentation of case: The patient was a 59-year-old man presenting with a swollen right submandibular gland. Laboratory tests revealed IgG4 levels of 176 mg/dl (reference range: 4.8–105). An initial open biopsy for histological diagnosis showed chronic sialadenitis. The region was monitored on an outpatient basis, and finally the right submandibular was totally resected because malignant tumor could not be excluded. Histological examination of the submandibular gland showed an ACC with lymphocytic infiltration containing many IgG4-positive plasma cells in the tumor stroma. Discussion: We have described a case that indicated a possible involvement of ACC with IgG4-RD. This allows us to speculate that longstanding IgG4-RD may progress to malignancy or infiltration of IgG4-positive plasma cells through the signals of tumor stimuli. Further investigations are required to determine the potential pathogenic mechanism underlying this unique tumor. Conclusion: This case underscores that caution is needed in the diagnosis of masses with high serum IgG4 levels, as the differential diagnosis includes malignancy

Polycomb Group Protein Ezh2 Regulates Hepatic Progenitor Cell Proliferation and Differentiation in Murine Embryonic Liver

<div><p>In embryonic liver, hepatic progenitor cells are actively proliferating and generate a fundamental cellular pool for establishing parenchymal components. However, the molecular basis for the expansion of the progenitors maintaining their immature state remains elusive. Polycomb group proteins regulate gene expression throughout the genome by modulating of chromatin structure and play crucial roles in development. <i>Enhancer of zeste homolog 2</i> (<i>Ezh2</i>), a key component of polycomb group proteins, catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3), which trigger the gene suppression. In the present study, we investigated a role of <i>Ezh2</i> in the regulation of the expanding hepatic progenitor population <i>in vivo</i>. We found that Ezh2 is highly expressed in the actively proliferating cells at the early developmental stage. Using a conditional knockout mouse model, we show that the deletion of the SET domain of <i>Ezh2</i>, which is responsible for catalytic induction of H3K27me3, results in significant reduction of the total liver size, absolute number of liver parenchymal cells, and hepatic progenitor cell population in size. A clonal colony assay in the hepatic progenitor cells directly isolated from <i>in vivo</i> fetal livers revealed that the bi-potent clonogenicity was significantly attenuated by the Ezh2 loss of function. Moreover, a marker expression based analysis and a global gene expression analysis showed that the knockout of Ezh2 inhibited differentiation to hepatocyte with reduced expression of a number of liver-function related genes. Taken together, our results indicate that Ezh2 is required for the hepatic progenitor expansion <i>in vivo</i>, which is essential for the functional maturation of embryonic liver, through its activity for catalyzing H3K27me3.</p></div

Ezh2 SET domain depletion caused blockade of the cellular differentiation in embryonic liver.

<p>A: Western blot analysis for Albumin (hepatocyte marker) in the CD45⁻ TER119⁻ non-hematopoietic liver cells from control (Rosa26::CreER(T2)^−/−;Ezh2^F/F mouse) and Ezh2 KO (Ezh2 SET domain depleted mouse; Rosa26::CreER(T2)^+/−;Ezh2^F/F mouse) at ED 18.5 after 3 days TAM injection (TAM; ED 10.5–12.5). B: Expression levels of hepatocytes related genes in the CD45⁻ TER119⁻ non-hematopoietic liver cells of the control and Ezh2 KO at indicated points were measured by qRT-PCR. Data are mean ± SD (n = 3). <i>P</i> values (asterisks) are from the Mann–Whitney <i>U</i>-test. *<i>P</i>− TER119⁻ non-hematopoietic liver cells of the control and Ezh2 KO. D: Significant enrichment of metabolism related liver functional GO terms for decreased genes upon Ezh2KO. Corrected <i>P</i> values of GO terms are shown.</p

Negative regulators of cell cycle were significantly up-regulated in the Ezh2 SET domain depleted fetal livers.

<p>A: The G1_to_S_Cell_Cycle_Control pathway (WP413_41269) from analysis using the WikiPathways platform is shown, in which gene hits with more than 2-fold change up to 8-fold change (<i>P</i> = 0.0018) are highlighted in yellow and hits with more than 8-fold change (<i>P</i> = 0.0046) in red in the Ezh2-KO CD45⁻ TER119⁻ liver cells at ED 13.5 compared with WT control. B: ChIP-PCR analysis with the anti-Ezh2 antibody and the primer sets detecting promoter regions of Cdkn1a, Cdkn2a, and Cdkn2b in the CD45⁻ TER119⁻ liver cells from WT livers at ED 13.5 are shown. C: ChIP-PCR analysis with the anti-H3K27me3 antibody and the primer sets detecting promoter regions of Cdkn1a, Cdkn2a, and Cdkn2b in the CD45⁻ TER119⁻ liver cells from the WT control livers at ED 13.5 are shown. D: Immunohistochemical analysis of Cdkn1a protein in the WT control and Ezh2 KO liver tissues at ED 13.5. Scale bar = 50 µm.</p