Regulatory roles of beta-catenin and AP-1 on osteoprotegerin production in interleukin-1alpha-stimulated periodontal ligament cells.

BACKGROUND: Periodontitis is a chronic inflammatory disease characterized by the enhanced expression of inflammatory mediators leading to alveolar bone resorption. Osteoprotegerin (OPG) plays a suppressive role in cytokine-induced osteoclastogenesis. In osteoblasts, OPG expression is upregulated by beta-catenin but downregulated by the transcription factor activator protein-1 (AP-1; c-fos/c-jun). The purpose of this study was to examine the roles of beta-catenin and AP-1 in interleukin-1alpha (IL-1alpha) -induced OPG production in human gingival fibroblasts (hGFs) and periodontal ligament (PDL) cells. METHODS: Expression of c-fos and c-jun messenger RNA was measured by reverse transcription-polymerase chain reaction and OPG production was analysed by enzyme-linked immunosorbent assay. The nuclear AP-1 activity was quantified using an AP-1 microplate assay. The effect of the Wnt canonical pathway on OPG production was evaluated using small interfering (si) RNA for beta-catenin and the effect of AP-1 on OPG production was evaluated using the AP-1 inhibitor curcumin. RESULTS: Levels of c-fos messenger RNA and nuclear AP-1 activity were higher in PDL cells than in hGFs. When stimulated with IL-1alpha, PDL cells had significantly higher c-fos expression and lower OPG production compared with hGFs. The siRNA for beta-catenin suppressed the IL-1alpha-induced OPG production in both PDL cells and hGFs, whereas the AP-1 inhibitor curcumin augmented the IL-1alpha-induced OPG production in PDL cells, but not in hGFs. CONCLUSION: The present study suggests that beta-catenin enhances IL-1alpha-induced OPG production in both PDL cells and hGFs, whereas AP-1 suppresses IL-1alpha-induced OPG production in PDL cells. Higher expression of c-fos in PDL cells than in hGFs may implicate a role of PDL cells in alveolar bone resorption in periodontitis.

Internal prostaglandin synthesis augments osteoprotegerin production in human gingival fibroblasts stimulated by lipopolysaccharide.

Periodontitis is an inflammatory bone disease caused by Gram-negative anaerobic bacteria. Osteoclast differentiation is regulated by the balance between receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG). The purpose of this study was to examine the mechanism of OPG production in human gingival fibroblasts (HGF) stimulated by lipopolysaccharide (LPS) from periodontopathic bacteria. The expressions of Toll-like receptor 2 (TLR-2) and TLR-4 in HGF were examined using flow-cytometry. HGF were stimulated with whole cell extracts or LPS from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis with or without polymyxin B, a LPS inhibitor. In addition, HGF were stimulated with LPS, prostaglandin E(2) (PGE(2)), various agonists of PGE receptors (EP1, EP2, EP3 and EP4 agonists) with or without indomethacin (IND), a prostaglandin synthesis inhibitor. OPG and PGE(2) production was measured using an enzyme-linked immunosorbent assay (ELISA). HGF expressed both TLR-2 and TLR-4. Both A. actinomycetemcomitans and P. gingivalis LPS augmented OPG expression in HGF. Whole cell extracts from A. actinomycetemcomitans and P. gingivalis augmented OPG production by HGF; the augmentation was suppressed by polymyxin B. IND suppressed OPG production in LPS-stimulated HGF. PGE(2) stimulated HGF to produce OPG. EP1 and EP2 agonists, but not EP3 and EP4 agonists, increased OPG production by HGF. These results suggest that LPS-induced OPG production by HGF is regulated via EP1 and/or EP2 receptors by endogenously generated PGE(2).

Transforming growth factor-beta stimulates interleukin-11 production by human periodontal ligament and gingival fibroblasts.

BACKGROUND: Transforming growth factor (TGF)-beta is a potent multifunctional polypeptide, abundant in the bone matrix. Interleukin (IL)-11 is a pleiotropic cytokine with effects on multiple cell types. The present study was performed to evaluate the regulatory effects of TGF-beta on IL-11 production by human periodontal ligament cells (PDL) and human gingival fibroblasts (HGF). MATERIAL AND METHODS: The expression of TGF-beta receptor in PDL and HGF were observed using flow cytometry. PDL and HGF were stimulated with TGF-beta with or without protein kinase C (PKC) inhibitors and activator. IL-11, bone morphogenetic protein-2 (BMP-2) and TGF-beta mRNA expression was quantified by real-time polymerase chain reaction (PCR). IL-11 production was measured using enzyme-linked immunosorbent assay. RESULTS: PDL and HGF expressed both TGF-beta receptor I and TGF-beta receptor II on the cell surfaces. IL-11 mRNA expression and IL-11 production were augmented by TGF-beta in both PDL and HGF, with higher values in PDL. PKC inhibitors partially suppressed TGF-beta-induced IL-11 production in PDL and HGF, whereas activator enhanced it. TGF-beta mRNA and BMP-2 mRNA expression were up-regulated by TGF-beta in PDL. CONCLUSION: These results suggest that PDL produce IL-11 in response to TGF-beta.

Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts.

Periodontitis, a chronic inflammatory disease, is characterized by increased expression of interleukin (IL)-1 and other inflammatory mediators resulting in extensive osteoclast formation and bone loss. Expression of receptor activator of nuclear factor kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), by osteoblasts is important to regulate osteoclast differentiation. The aim of the present study was to investigate the regulatory effects of IL-1 on RANKL and OPG production by mesenchymal fibroblasts in periodontal tissue. Human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) were stimulated with IL-1alpha with or without protein synthesis inhibitor cycloheximide (CHX), protein kinase A (PKA) inhibitors, protein kinase C (PKC) inhibitors and prostaglandin E(2) (PGE(2)) inhibitor. In some experiments, the cultured cells were directly stimulated with either PKA or PKC activators. In HGF, IL-1alpha-stimulated OPG mRNA expression was high and could be reduced by CHX. PKA inhibitor completely abrogated IL-1alpha-induced OPG mRNA expression and OPG production. Endogenous PGE(2) further enhanced IL-1alpha-induced OPG production in HGF. In PDL, RANKL mRNA expression was greatly augmented by IL-1alpha. IL-1alpha induced OPG mRNA expression and protein production. PKC inhibitor partially reduced IL-1alpha-induced OPG production and PKC activator enhanced OPG production in PDL. The IL-1alpha-stimulated OPG mRNA expression in HGF was greater than PDL. These results provide new evidence for the possible osteoclastogenesis-inhibitory function of HGF through PKA activity pathway. PDL utilized PKC for OPG production. Thus, we emphasize that HGF and PDL have different characteristics of host defence mechanism against inflammatory process.