The Association of Notch2 and NF-κB Accelerates RANKL-Induced Osteoclastogenesis▿

Notch signaling plays a key role in various cell differentiation processes including bone homeostasis. However, the specific involvement of Notch in regulating osteoclastogenesis is still controversial. In the present study, we show that RANKL induces expression of Jagged1 and Notch2 in bone marrow macrophages during osteoclast differentiation. Suppression of Notch signaling by a selective γ-secretase inhibitor or Notch2 short hairpin RNA suppresses RANKL-induced osteoclastogenesis. In contrast, induction of Notch signaling by Jagged1 or by ectopic expression of intracellular Notch2 enhances NFATc1 promoter activity and expression and promotes osteoclastogenesis. Finally, we found that Notch2 and p65 interact in the nuclei of RANKL-stimulated cells and that both proteins are recruited to the NFATc1 promoter, driving its expression. Taken together, our results show a new molecular cross talk between Notch and NF-κB pathways that is relevant in osteoclastogenesis

The Association of Notch2 and NF-B Accelerates RANKL-Induced Osteoclastogenesis

Notch signaling plays a key role in various cell differentiation processes including bone homeostasis. However, the specific involvement of Notch in regulating osteoclastogenesis is still controversial. In the present study, we show that RANKL induces expression of Jagged1 and Notch2 in bone marrow macrophages during osteoclast differentiation. Suppression of Notch signaling by a selective gamma-secretase inhibitor or Notch2 short hairpin RNA suppresses RANKL-induced osteoclastogenesis. In contrast, induction of Notch signaling by Jagged1 or by ectopic expression of intracellular Notch2 enhances NFATc1 promoter activity and expression and promotes osteoclastogenesis. Finally, we found that Notch2 and p65 interact in the nuclei of RANKL-stimulated cells and that both proteins are recruited to the NFATc1 promoter, driving its expression. Taken together, our results show a new molecular cross talk between Notch and NF-kappa B pathways that is relevant in osteoclastogenesis

Interleukin-1 Receptor Antagonist Has a Novel Function in the Regulation of Matrix Metalloproteinase-13 Expression.

Interleukin-1 receptor antagonist (IL-1Ra) is an IL-1 family member, which binds to IL-1 receptors but does not induce any intracellular signaling. We addressed whether IL-1Ra has a novel function in regulation of the extracellular matrix or adhesion molecules. Polymerase chain reaction array analysis demonstrated a ~5-fold increase in matrix metalloproteinase 13 (MMP-13) mRNA expression of IL-1Ra siRNA-transfected Ca9-22 human oral squamous epithelial carcinoma cells compared with the control. In fact, MMP-13 mRNA and protein expression as well as its activity in IL-1Ra siRNA-transfected Ca9-22 cell lines were significantly higher than those in the control. IL-1Ra siRNA treatment resulted in strong elevation of MMP-13 expression, whereas addition of rhIL-1Ra (40 ng/ml) suppressed MMP-13 expression, suggesting that IL-1Ra had a specific effect on MMP-13 induction. IL-1Ra siRNA could potently suppress IL-1α. No significant difference was found between the MMP-13 mRNA expression of IL-1Ra siRNA-transfected cells and those treated with anti-IL-1α or anti-IL-1β antibodies. These results suggested that continuous supply of IL-1 had no effect on the induction of MMP-13 by IL-1Ra siRNA. Histopathological investigation of MMP-13 in periodontal tissue showed specific localization in the junctional epithelial cells of IL-1Ra knockout (KO) mice. Furthermore, infection with Aggregatibacter actinomycetemcomitans to establish an experimental periodontitis model resulted in predominant localization of MMP-13 along apical junctional epithelial cells. Laminin-5, which is degraded by MMP-13, was found in the internal basal lamina of wild-type mice, whereas the internal basal lamina of IL-1Ra KO mice did not show obvious laminin-5 localization. In particular, laminin-5 localization almost disappeared in the internal basal lamina of IL-1Ra KO mice infected with A. actinomycetemcomitans, suggesting that the suppression of IL-1Ra resulted in strong induction of MMP-13 that degraded laminin-5. In conclusion, IL-1Ra is associated with MMP-13 expression and has a novel function in such regulation without interference of the IL-1 signaling cascade

Correction: Interleukin-1 Receptor Antagonist Has a Novel Function in the Regulation of Matrix Metalloproteinase-13 Expression.

[This corrects the article DOI: 10.1371/journal.pone.0140942.]

TIMP expression in IL-1Ra siRNA-transfected cells.

<p>(A) TIMP-1 and TIMP-2 mRNA expression in IL-1Ra and control siRNA-transfected cells. The cells were cultured for 1, 3, 6, 12, and 24 hours, and then mRNA levels were determined using real-time PCR. Values represent fold changes. Differences among groups were analyzed using the Student’s <i>t</i>-test. Data are expressed as the mean ± SD (n = 3). *<i>p</i> < 0.05 vs each time point control. (B) Western blot of TIMP-1 (23 kDa) and -2 (21 kDa) in cells transfected with either IL-1Ra or control siRNAs. Data are representative of three independent experiments.</p

Expression and localization of MMP-13 in periodontal tissue of IL-1Ra KO and WT mice.

<p>(A) At 4 weeks after infection, the mouse gingival epithelial layer was collected to examine MMP-13 levels. The levels of MMP-13 were measured by an ELISA. Differences among groups were analyzed by one-factor ANOVA and Bonferroni’s multiple comparison test. Data are expressed as the mean ± SD (n = 4). **<i>p</i> <0.01 vs IL-1Ra KO mice infected with <i>A</i>. <i>actinomycetemcomitans</i>. ^†<i>p</i> <0.05 vs IL-1Ra KO mice treated with PBS. (B) Localization of MMP-13 in periodontal tissue. Buccolingual sections of the second and third mandibular molars from <i>A</i>. <i>actinomycetemcomitans</i>-infected IL-1Ra KO and WT mice were stained immunohistochemically. In contrast to infected WT mice, immunohistochemical findings in periodontal tissues indicated considerable inflammatory reactions in IL-1Ra KO mice infected with <i>A</i>. <i>actinomycetemcomitans</i>.</p