Invasion of P. gingivalis into gingival epithelial cells induces the nucleation of actin filaments to form microspike-like protrusions and long stable microfilaments distributed throughout the cells [15]. Cytoskeletal reorganization may facilitate phagocytic cup formation and subsequent bacterial engulfment. Cytoskeletal remodeling resulting from bacterial internalization can spatially redistribute enzymes such as MAPK family members and their substrates, and thus influence intracellular signaling pathways [16, 17]. P. gingivalis invasion of human gingival epithelial cells causes activation of JNK (c-Jun N-terminal see more kinase) and down-regulation of ERK1/2 (extracellular

signal regulated kinase), whereas Foretinib molecular weight p38 and NF-κB (Nuclear factor-Kappa click here B) are not affected [18]. After invading gingival cells, P. gingivalis ultimately localizes to the perinuclear region [2, 4]. Despite the burden of a large number of intracellular P. gingivalis, both gingival epithelial cells and fibroblasts demonstrate an initially decreased but later increased rate of apoptosis upon bacterial challenge [19–22]. Presumably, this temporal shift from cell survival to apoptosis is utilized by P. gingivalis to reach an initial intracellular concentration

while escaping host immune surveillance, and a later dismantling of host cells to facilitate disease transmission. This paper reports results from experiments using an in vitro model of P. gingivalis−osteoblast interactions. The findings suggest that P. gingivalis uses its major fimbriae to bind to integrin α5β1 on osteoblasts and reorganize actin microfilaments to invade osteoblasts. In addition, infected osteoblasts demonstrate activation of the JNK pathway, as well as an initial

increase in cellular survival with a subsequent increased cellular death, as reported for other periodontal cells. Methods Osteoblast isolation Primary mouse calvarial osteoblasts were isolated from 7-day-old CD-1 mice using the method described by Wong and Cohn [23]. Briefly, calvaria were subjected to four sequential 15-minute digestions in an enzyme mixture containing Autophagy activator 0.05% trypsin and 0.1% collagenase P at 37°C. Cell fractions 2–4 were pooled and resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, then filtered through a 70 μm cell strainer. Cells were plated at a density of 1 × 104 cells/cm2 and the medium was changed 24 h later. All animal-related experiments were approved by the Center for Laboratory Animal Medicine and Care at the University of Texas Health Science Center at Houston (approved animal protocol number HSC-AWC-10–145). Bacteria and culture conditions Porphyromonas gingivalis strain ATCC 33277 was grown anaerobically at 37°C in a Coy anaerobic chamber under an atmosphere of 86% nitrogen, 10% carbon dioxide, 4% hydrogen.