Signaling pathways in osteoblast proinflammatory responses to infection by Porphyromonas gingivalis

INTRODUCTION: We recently investigated global gene expression in ST2 mouse stromal cells infected by the periodontal pathogen Porphyromonas gingivalis using microarray technology, and found that the bacterium induces a wide range of proinflammatory gene expression. Here, we reported the signaling pathways involved in those proinflammatory responses. METHODS: ST2 cells and primary calvarial osteoblasts from C3H/HeN, C57BL/6, and MyD88-deficient (MyD88(-/-)) mice were infected with P. gingivalis ATCC33277 and its gingipain-deficient mutant KDP136. Expression of the chemokines CCL5 and CXCL10, and matrix metalloproteinase-9 (MMP9) were quantified by real-time polymerase chain reaction, while phosphorylation of protein kinases and degradation of an inhibitor of nuclear factor-kappaB, IkappaB-alpha, were detected by Western blotting, and activation of transcriptional factors was determined by a luciferase reporter assay. The effects of inhibitors of transcriptional factors and protein kinases were also investigated. RESULTS: Infection by P. gingivalis elicited gene expression of CCL5, CXCL10, and MMP9 in both ST2 cells and osteoblasts. Western blot and reporter assay results revealed activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 transcription factors. The NF-kappaB inhibitor suppressed the expression of CCL5 and MMP9, but not that of CXCL10, whereas P. gingivalis infection induced significant CCL5 expression in MyD88(-/-) osteoblasts. In addition, activation of protease-activated receptors by trypsin elicited significant induction of CXCL10. CONCLUSION: Our results suggest that various proinflammatory responses in P. gingivalis-infected stromal/osteoblast cells are NF-kappaB-dependent, but not always dependent on the Toll-like receptor/MyD88 pathway, while some responses are related to the activation of protease-activated receptors. Thus, P. gingivalis does not fully utilize well-established pathogen recognition molecules such as Toll-like receptors.