Signaling pathways in osteoblast proinflammatory responses to infection by Porphyromonas gingivalis

Abstract

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. 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. 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. 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.