Porphyromonas gingivalis induce apoptosis in human gingival epithelial cells through a gingipain-dependent mechanism

Background  

The oral pathogen Porphyromonas gingivalis has been shown to modulate apoptosis in different cell types, but its effect on epithelial cells remains unclear.     

Blockade of TGF-beta accelerates mucosal destruction through epithelial cell apoptosis

To clarify the protective role of transforming growth factor (TGF)-beta for the intestinal epithelial injury in vivo, the effect of antibodies against TGF-beta on epithelial destruction and apoptosis was assessed in dextran sulfate sodium (DSS)-induced colitis by histological analysis of colonic sections, account of apoptotic epithelial cells. To evaluate the pathways of epithelial apoptosis, we analyzed the activities of caspases, the level of Fas and cellular FLICE-inhibitory protein (cFLIP) expression in epithelial cells. Apoptotic epithelial cells were increased prior to the onset of ulceration in DSS-induced colitis, and the neutralization of TGF-beta exacerbated epithelial apoptosis and histological damage score. The up-regulation of caspase-8 activity and Fas expression and reduced cFLIP expression were observed in intestinal epithelial cells from anti-TGF-beta antibody-treated mice. The present study revealed that suppression of TGF-beta deteriorated epithelial apoptosis, and the increase of apoptotic epithelial cells may amplify the inflammation in gut mucosa.     

Activated ras protein accelerates cell cycle progression to perturb madin-darby canine kidney cystogenesis

In a number of human cancer cells, K-RAS is frequently mutated and activated constitutively, culminating in the induction of continuous cell growth, a hallmark of cancer cells. It is still unclear, however, how the mutated K-RAS induces morphological abnormalities in cancerous tissues. To investigate the mechanism underlying the K-RAS-induced morphological changes, we utilized an auxin-dependent protein expression system, which enabled us to rapidly induce and evaluate constitutively active K-Ras in MDCK (Madin-Darby canine kidney) cysts, a model for polarized epithelial structure. Cells carrying the constitutively active KRasV12 gene were morphologically indistinguishable from normal cells in two-dimensional culture. However, in a gel of extracellular matrix, KRasV12-expressing cells failed to form a spherical cyst. When KRasV12 induction was delayed until after cyst formation, some cells in the cyst wall lost polarity and were extruded into and accumulated in the luminal space. With effector-specific mutants of KRasV12 and inhibitors for MEK and PI3-kinase, we found that both the Raf-MEK-ERK and PI3-kinase axes are necessary and sufficient for this phenotype. Live cell imaging with cell cycle indicators showed that KRasV12 expression promoted cell cycle progression, which was prevented by either MEK or PI3-kinase inhibitors. From these results, we provide a model wherein active-Ras induces cell cycle progression leading to apical cell extrusion through Raf and PI3-kinase in a cooperative manner. The system developed here can be applied to drug screening for various cancers originating from epithelial cells.     

Mechanisms of signalling-memory governing progression through the eukaryotic cell cycle

As cells pass through each replication–division cycle, they must be able to postpone further progression if they detect any threats to genome integrity, such as DNA damage or misaligned chromosomes. Once a ‘decision’ is made to proceed, the cell unequivocally enters into a qualitatively different biochemical state, which makes the transitions from one cell cycle phase to the next switch-like and irreversible. Each transition is governed by a unique signalling network; nonetheless, they share a common characteristic of bistable behaviour, a hallmark of molecular memory devices. Comparing the cell cycle signalling mechanisms acting at the restriction point, G1/S, G2/M and meta-to-anaphase transitions, we deduce a generic network motif of coupled positive and negative feedback loops underlying each transition.     

Calcium oscillations in gingival epithelial cells infected with Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis modulates epithelial cell signal transduction pathways including Ca2+ signaling, and internalizes within the host cell cytoplasm. Since nuclear and cytoplasmic [Ca2+] increases can induce different host cell responses, P. gingivalis-related [Ca2+] changes in these compartments were measured by digital fluorescent imaging microscopy. Non-deconvolved and deconvolved fura-2 images showed that P. gingivalis exposure caused human gingival epithelial cells cultured in physiologic [Ca2+] levels to undergo sustained oscillations of [Ca2+] in nuclear and cytoplasmic spaces. However, P. gingivalis invasion was not tightly correlated with intracellular [Ca2+] oscillations, since invasion could significantly precede, or even occur in the absence of, oscillations. [Ca2+] oscillations required a Ca2+ influx, which was completely inhibited by La3+ or 2-APB (2-aminoethoxydiphenyl borate), indicating Ca2+ entry was via a Ca(2+)-permeable channel. Ca2+ entry was likely not via a store-operated channel, since Ca2+ release from intracellular stores was not observed during cellular uptake of P. gingivalis. Hence, uptake of P. gingivalis in gingival epithelial cells induces oscillations in nuclear and cytoplasmic spaces by activating a Ca2+ influx through Ca2+ channels.     

The human cytomegalovirus UL82 gene product (pp71) accelerates progression through the G1 phase of the cell cycle

As viruses are reliant upon their host cell to serve as proper environments for their replication, many have evolved mechanisms to alter intracellular conditions to suit their own needs. For example, human cytomegalovirus induces quiescent cells to enter the cell cycle and then arrests them in late G(1), before they enter the S phase, a cell cycle compartment that is presumably favorable for viral replication. Here we show that the protein product of the human cytomegalovirus UL82 gene, pp71, can accelerate the movement of cells through the G(1) phase of the cell cycle. This activity would help infected cells reach the late G(1) arrest point sooner and thus may stimulate the infectious cycle. pp71 also induces DNA synthesis in quiescent cells, but a pp71 mutant protein that is unable to induce quiescent cells to enter the cell cycle still retains the ability to accelerate the G(1) phase. Thus, the mechanism through which pp71 accelerates G(1) cell cycle progression appears to be distinct from the one that it employs to induce quiescent cells to exit G(0) and subsequently enter the S phase.     

Fluorescence image analysis of the association between Porphyromonas gingivalis and gingival epithelial cells

We have developed a fluorescence imaging technique using a DNA-binding dye to visualize, over time, the physical interactions between Porphyromonas gingivalis and human gingival epithelial cells in vitro . The results extend previous observations of P. gingivalis invasion of gingival epithelial cells based on indirect measurements. An intracellular location for P. gingivalis was established by optical sectioning of images in the z -plane. Kinetic analysis showed that P. gingivalis invasion of epithelial cells is a rapid and efficient process, reaching completion after 12 min. Imaging of infected monolayers revealed that over 90% of a population of gingival epithelial cells contained bacteria. Furthermore, only vital bacteria were capable of invasion, and intracellular bacteria congregated in the perinuclear region of the epithelial cells. P. gingivalis remained inside the epithelial cells over a 24 h period and induced rearrangement of the actin cytoskeleton along with alteration of the size and shape of the epithelial cells. These findings provide direct evidence that entry rates of P. gingivalis into gingival epithelial cells are high and rapid, and that internalized bacteria initially localize in a specific region of the epithelial cells.     

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis

Abstract Three major polypeptides of 34, 48 and 50 kDa which appear to copurify with 1,3-β-glucan synthase activity were isolated by glycerol gradient centrifugation of Chaps-solubilized proteins from the fungus Saprolegnia monoica . The antiserum produced against the 34-kDa polypeptide revealed by protein immunoblotting that this polypeptide copurified with 1,3-β-glucan synthase during enzyme purification. This antiserum adsorbs the enzymatic activity as well as the 48- and 50-kDa polypeptides. These results indicate that the 34-kDa peptide is a component of the multisubunit protein complex involved in 1,3-β-glucan synthase activity.     

The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle.

The RB gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To test whether RB regulates cell cycle progression, purified RB proteins, either full-length or a truncated form containing the T antigen-binding region, were injected into cells. Injection of either protein early in G1 inhibits progression into S phase. Co-injection of anti-RB antibodies antagonizes this effect. Injection of RB into cells arrested at G1/S or late in G1 has no effect on BrdU incorporation, suggesting that RB does not inhibit DNA synthesis in S phase. These results indicate that RB regulates cell proliferation by restricting cell cycle progression at a specific point in G1 and establish a biological assay for RB activity. Neither co-injection of RB with a T antigen peptide nor injection into cells expressing T antigen prevents cells from progressing into S phase, which supports the hypothesis that T antigen binding has functional consequences for RB.     

The c-Myc protein induces cell cycle progression and apoptosis through dimerization with Max.

The c-Myc protein (Myc) is involved in cellular transformation and mitogenesis, but is also a potent inducer of programmed cell death, or apoptosis. Whether these apparently opposite functions are mediated through common or distinct molecular mechanisms remains unclear. Myc and its partner protein, Max, dimerize and bind DNA in vitro and in vivo through basic/helix-loop-helix/leucine zipper motifs (bHLH-LZ). By using complementary leucine zipper mutants (termed MycEG and MaxEG), which dimerize efficiently with each other but not with their wild-type partners, we demonstrate that both cell cycle progression and apoptosis in nontransformed rodent fibroblasts are induced by Myc-Max dimers. MycEG or MaxEG alone are inactive, but co-expression restores ability to prevent withdrawal from the cell cycle and to induce cell death upon removal of growth factors. Thus, Myc can control two alternative cell fates through dimerization with a single partner, Max.     

Overexpression of the Linker histone-binding protein tNASP affects progression through the cell cycle

NASP is an H1 histone-binding protein that is cell cycle-regulated and occurs in two major forms: tNASP, found in gametes, embryonic cells, and transformed cells; and sNASP, found in all rapidly dividing somatic cells (Richardson, R. T., Batova, I. N., Widgren, E. E., Zheng, L. X., Whitfield, M., Marzluff, W. F., and O'Rand, M. G. (2000) J. Biol. Chem. 275, 30378-30386). When full-length tNASP fused to green fluorescent protein (GFP) is transiently transfected into HeLa cells, it is efficiently transported into the nucleus within 2 h after translation in the cytoplasm, whereas the NASP nuclear localization signal (NLS) deletion mutant (NASP-DeltaNLS-GFP) is retained in the cytoplasm. In HeLa cells synchronized by a double thymidine block and transiently transfected to overexpress full-length tNASP or NASP-DeltaNLS, progression through the G(1)/S border is delayed. Cells transiently transfected to overexpress the histone-binding site (HBS) deletion mutant (NASP-DeltaHBS) or sNASP were not delayed in progression through the G(1)/S border. By using a DNA supercoiling assay, in vitro binding data demonstrate that H1 histone-tNASP complexes can transfer H1 histones to DNA, whereas NASP-DeltaHBS cannot. Measurement of NASP mobility in the nucleus by fluorescence recovery after photobleaching indicates that NASP mobility is virtually identical to that reported for H1 histones. These data suggest that NASP-H1 complexes exist in the nucleus and that tNASP can influence cell cycle progression through the G(1)/S border through mediation of DNA-H1 histone binding.     

Haspin inhibition delays cell cycle progression through interphase in cancer cells

Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or the S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby, indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis.     

Heparin prevents vascular smooth muscle cell progression through the G1 phase of the cell cycle

To gain insight into the mechanism of the antiproliferative effects of heparin on vascular smooth muscle cells (SMC), the influence of this glycosaminoglycan on cell cycle progression and the expression of the c-fos, c-myc, and c-myb proto-oncogenes and two other growth-regulated genes was examined. SMC, synchronized by a serum-deprivation protocol, enter S phase 12-16 h after serum stimulation. Pretreatment with heparin for 48 h blocked the induction of histone H3 RNA, an S phase-expressed product, and prevented cell replication. Thus, heparin prevents entry of cells into S phase. Conversely, heparin had essentially no effect on changes in expression of the c-fos and c-myc proto-oncogenes during the G0 to G1 transition. Normal increases in c-fos and c-myc RNA were observed 30 min and 2 h following serum addition, respectively. However, the increase in expression of the mRNA of the c-myb proto-oncogene and the mitochondrial ATP/ADP carrier protein, 2F1, which begins to occur 8 h following serum addition to SMC, was completely inhibited by heparin. Two-dimensional polyacrylamide gel electrophoresis of the products of a rabbit reticulocyte cell-free translation of RNA isolated at various times confirmed this temporal assessment of the effects of heparin. These results suggest that heparin does not inhibit cell proliferation by blocking the G0 to G1 transition. Rather, heparin may affect a critical event in the mid-G1 phase of the cell cycle which is necessary for subsequent DNA synthesis.     

Porphyromonas gingivalis lipopolysaccharide induces shedding of syndecan-1 expressed by gingival epithelial cells

Syndecans are constitutively shed from growing epithelial cells as the part of normal cell surface turnover. However, increased serum levels of the soluble syndecan ectodomain have been reported to occur during bacterial infections. The aim of this study was to evaluate the potential of lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis to induce the shedding of syndecan-1 expressed by human gingival epithelial cells. We showed that the syndecan-1 ectodomain is constitutively shed from the cell surface of human gingival epithelial cells. This constitutive shedding corresponding to the basal level of soluble syndecan-1 ectodomain was significantly increased when cells were stimulated with P. gingivalis LPS and reached a level comparable to that caused by phorbol myristic acid (PMA), an activator of protein kinase C (PKC) which is well known as a shedding agonist. The syndecan-1 shedding was paralleled by pro-inflammatory cytokine interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) release. Indeed, secretion of IL-1beta and TNF-alpha increased following stimulation by P. gingivalis LPS and PMA, respectively. When recombinant forms of these proteins were added to the cell culture, they induced a concentration-dependent increase in syndecan-1 ectodomain shedding. A treatment with IL-1beta converting enzyme (ICE) specific inhibitor prevented IL-1beta secretion by epithelial cells stimulated by P. gingivalis LPS and decreased the levels of shed syndecan-1 ectodomain. We also observed that PMA and TNF-alpha stimulated matrix metalloproteinase-9 secretion, whereas IL-1beta and P. gingivalis LPS did not. Our results demonstrated that P. gingivalis LPS stimulated syndecan-1 shedding, a phenomenon that may be mediated in part by IL-1beta, leading to an activation of intracellular signaling pathways different from those involved in PMA stimulation.