Immunoglobulin-like domains of the cargo proteins are essential for protein stability during secretion by the type IX secretion system.

The periodontal pathogen Porphyromonas gingivalis secretes many potent virulence factors using the type IX secretion system (T9SS). T9SS cargo proteins that have been structurally determined by X-ray crystallography are composed of a signal peptide, functional domain(s), an immunoglobulin (Ig)-like domain and a C-terminal domain. Role of the Ig-like domains of cargo proteins in the T9SS has not been elucidated. Gingipain proteases, which are cargo proteins of the T9SS, were degraded when their Ig-like domains were lacking or truncated. The degradation was dependent on the activity of a quality control factor, HtrA protease. Another T9SS cargo protein, HBP35, which has a thioredoxin domain as a functional domain, was analyzed by X-ray crystallography, revealing that HBP35 has an Ig-like domain after the thioredoxin domain and that the hydrophobic regions of the thioredoxin domain and the Ig-like domain face each other. HBP35 with substitution of hydrophobic amino acids in the Ig-like domain was degraded depending on HtrA. These results suggest that the Ig-like domain mediates stability of the cargo proteins in the T9SS.

Functional analysis of differences in transcriptional activity conferred by genetic variants in the 5' flanking region of the IL12RB2 gene.

Interleukin 12 receptor ß chain (IL12RB2) is a crucial regulatory factor involved in cell-mediated immune responses, and genetic variants of the gene encoding IL12RB2 are associated with susceptibility to various immune-related diseases. We previously demonstrated that haplotypes with single nucleotide polymorphisms (SNPs) in the 5' flanking region of IL12RB2, including -1035A>G (rs3762315) and -1023A>G (rs3762316), affect the expression of IL12RB2, thereby altering susceptibility to leprosy and periodontal diseases. In the present study, we identified transcription factors associated with the haplotype-specific transcriptional activity of IL12RB2 in T cells and NK cells. The -1023G polymorphism was found to create a consensus binding site for the transcription factor activating protein (AP)-1, and enzyme-linked immunosorbent assay (ELISA)-based binding assays showed that these SNPs enhanced AP-1 binding to this region. In reporter assays, suppression of JunB expression using siRNA eliminated differences in the -1035G/-1023G and -1035A/-1023A regions containing IL12RB2 promoter activity in Jurkat T cells and NK3.3 cells. These results suggested that the -1035/-1023 polymorphisms created differential binding affinities for JunB that could lead to differential IL12RB2 expression. Moreover, the -1035G and -1035A alleles formed binding sites for GATA-3 and myocyte enhancer factor-2 (MEF-2), respectively. Our data indicated that in addition to JunB, the SNP at -1035/-1023 influenced GATA-3 and MEF-2 binding affinity, potentially altering IL12RB2 transcriptional activity. These findings confirm the effects of rs3762315 and rs3762316 on IL12RB2 transcription. These genetic variants may alter cellular activation of T cells and NK cells and modify cell-mediated immune responses.

Fibroblast VEGF-receptor 1 expression as molecular target in periodontitis.

AIM: Degradation of extracellular matrices is an integral part in periodontitis. For antagonizing this pathophysiological mechanism, we aimed at identifying gene expression profiles in disease progression contributing periodontitis-associated fibroblasts (PAFs) versus normal gingival fibroblasts to determine their molecular repertoire, and exploit it for therapeutic intervention. MATERIALS AND METHODS: Applying an exploratory analysis using a small number of microarrays in combination with a three dimensional (3D) in vitro culture model that incorporates some aspects of periodontitis, PAFs were initially characterized by gene-expression analyses, followed by targeted gene down-regulation and pharmacological intervention in vitro. Further, immunohistochemistry was applied for phosphorylation analyses in tissue specimens. RESULTS: PAFs were characterized by 42 genes being commonly up-regulated >1.5-fold, and by five genes that were concordantly down-regulated (<0.7-fold). Expression of vascular endothelial growth factor (VEGF)-receptor 1 (Flt-1) was highly enhanced, and was thus further explored in in vitro culture models of periodontal fibroblasts without accounting for the microbiome. Phosphorylation of the VEGF-receptor 1 was enhanced in PAFs. Receptor inhibition by a specific VEGF-receptor inhibitor or intrinsic down-regulation by RNAi of the VEGF-receptor kinase in 3D gel cultures resulted in significant reduction in collagen degradation associated with increased tissue inhibitor of metalloproteinase expression, suggesting that Flt-1 may contribute to periodontitis. CONCLUSION: Based on the finding that VEGF-receptor kinase inhibition impaired collagen degradation pathways, Flt-1 may represent a candidate for therapeutic approaches in periodontitis.

Periodontal and peri-implant wound healing following laser therapy.

Laser irradiation has numerous favorable characteristics, such as ablation or vaporization, hemostasis, biostimulation (photobiomodulation) and microbial inhibition and destruction, which induce various beneficial therapeutic effects and biological responses. Therefore, the use of lasers is considered effective and suitable for treating a variety of inflammatory and infectious oral conditions. The CO2 , neodymium-doped yttrium-aluminium-garnet (Nd:YAG) and diode lasers have mainly been used for periodontal soft-tissue management. With development of the erbium-doped yttrium-aluminium-garnet (Er:YAG) and erbium, chromium-doped yttrium-scandium-gallium-garnet (Er,Cr:YSGG) lasers, which can be applied not only on soft tissues but also on dental hard tissues, the application of lasers dramatically expanded from periodontal soft-tissue management to hard-tissue treatment. Currently, various periodontal tissues (such as gingiva, tooth roots and bone tissue), as well as titanium implant surfaces, can be treated with lasers, and a variety of dental laser systems are being employed for the management of periodontal and peri-implant diseases. In periodontics, mechanical therapy has conventionally been the mainstream of treatment; however, complete bacterial eradication and/or optimal wound healing may not be necessarily achieved with conventional mechanical therapy alone. Consequently, in addition to chemotherapy consisting of antibiotics and anti-inflammatory agents, phototherapy using lasers and light-emitting diodes has been gradually integrated with mechanical therapy to enhance subsequent wound healing by achieving thorough debridement, decontamination and tissue stimulation. With increasing evidence of benefits, therapies with low- and high-level lasers play an important role in wound healing/tissue regeneration in the treatment of periodontal and peri-implant diseases. This article discusses the outcomes of laser therapy in soft-tissue management, periodontal nonsurgical and surgical treatment, osseous surgery and peri-implant treatment, focusing on postoperative wound healing of periodontal and peri-implant tissues, based on scientific evidence from currently available basic and clinical studies, as well as on case reports.

The inflammation-lipocalin 2 axis may contribute to the development of chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is an important risk factor for coronary heart disease, and previous studies indicated the involvement of low-grade inflammation in the pathogenesis of CKD. METHODS: The study was designed to (i) identify and confirm genes and their products upregulated in mesangial cells cocultured with endotoxin-stimulated macrophages and (ii) determine the clinical relevance of genes and proteins upregulated in mesangial cells under inflammatory conditions by an epidemiological approach. RESULTS: DNA microarray analysis revealed upregulated expression of many genes and their products including several cytokines and chemokines, as well as the inflammatory marker, lipocalin 2 gene. The gene expression and protein upregulation of lipocalin 2 were synergistically affected by endotoxin and tumor necrosis factor (TNF)-α stimulation. In human studies, lipocalin 2 level was significantly associated with creatinine (r = 0.419, P < 0.001) and negatively associated with eGFR (r = -0.365, P < 0.001). Multiple logistic regression analysis revealed a significant association between lipocalin 2 and soluble tumor necrosis factor receptor 2 (sTNF-R2), eGFR and uric acid in general subjects attending regular annual medical check-up (n = 420). When subjects with diabetes were excluded from the analysis, lipocalin 2 remained associated with sTNF-R2, eGFR and uric acid. CONCLUSIONS: Since an activated TNF system, as demonstrated by elevated sTNF-R2, and elevated uric acid were recently implicated in an elevated CKD risk, we conclude that inflammation could play an important role in the pathogenesis of CKD, and that lipocalin 2 is a potential universal marker for impaired kidney function. Furthermore, the results obtained by the current microarray analysis could improve the understanding of gene profiles associated with the pathophysiology of CKD under inflammatory conditions.

Transcriptional regulation of amelotin gene by proinflammatory cytokines in gingival fibroblasts.

Amelotin (AMTN) is a secreted protein expressed during the late stages of enamel formation and in the junctional epithelium. Among many differentially expressed genes, we found significantly increased AMTN mRNA level in inflamed gingiva by DNA microarray. The inductions of AMTN mRNA expressions in inflamed gingiva and human gingival fibroblasts (HGF) were confirmed by real-time polymerase chain reaction. To determine the molecular basis of the expression of AMTN and its regulation by proinflammatory cytokines, we have isolated and characterized the promoter region of mouse AMTN gene. Transient transfection assays were performed using luciferase constructs including mouse AMTN gene promoter. Interleukin-1ß, Interleukin-6 and tumor necrosis factor-α induced AMTN mRNA levels in HGF. These cytokines increased the luciferase activities of the AMTN promoter constructs in HGF. The results suggest that proinflammatory cytokines induce AMTN gene transcription and a role for AMTN in gingival inflammation.

IL-1ß-mediated up-regulation of DEC1 in human gingiva cells via the Akt pathway.

Growing evidence indicates that inflammation is a contributing factor leading to cancer development. However, pathways involved in this progression are not well understood. The involvement of DEC1 in cancer prompted us to examine whether pro-inflammatory cytokine interleukin-1ß (IL-1ß) induces the expression of DEC1 in oral inflammation. We found that IL-1ß up-regulated DEC1 and hypoxia-inducible factor-1α (HIF-1α) protein and elevated the HIF-1α-responsive gene vascular endothelial growth factor (VEGF) expression in human primary gingival cells. HIF-1α and DEC1 immunoreactivity were significantly higher in the cases of gingival inflammation. We demonstrate that IL-1ß up-regulates DEC1 and HIF-1α protein through a classical inflammatory signaling pathway involving Akt. Our data strongly suggest that PI-3K-Akt is an upstream participant in IL-1ß-mediated DEC1 and HIF-1α induction. This is supported by the following data: (1) IL-1ß induces 473 serine phosphorylation of Akt; (2) IL-1ß-mediated Akt activation occurs in a PI-3K-dependent manner, and specific inhibition of PI-3K prevents Akt phosphorylation; and (3) inhibition of Akt prevents IL-1ß-mediated DEC1 and HIF-1α induction. Taken together, these results suggest that DEC1 is one of the important transcription factors in inflammation.

F-spondin regulates the differentiation of human cementoblast-like (HCEM) cells via BMP7 expression.

Cementum plays an important role in the attachment of connective tissue to the root surface. However, the detailed mechanism of cementum formation has not yet been clarified. We previously established human cementoblast-like cell lines (HCEM) and human periodontal ligament cell lines (HPL) by infection of hTERT gene. Using those cell lines, we compared the gene expression of them and identified F-spondin as a cementoblast specific gene. In this study, to clarify the role of F-spondin in the differentiation of periodontal ligament cells to cementoblasts, we compared the gene expression of F-spondin-overexpressed HPL (HPL-spondin) cells with HPL parent cells. We found that several genes expressed higher level in HPL-spondin cells than in HPL cells, such as heparin sulfate 6-sulfotranferase, calcitonin-related polypeptide beta, bone morphogenetic proteins 7 (BMP7), BMP2 and BMP8B. Among those genes, we focused on BMP7 and examined the interaction between F-spondin and BMP7, because BMP7 was reported to enhance cementoblast function. Moreover, we further examined the effect of BMP7 peptide on the expression of mineralization-associated genes in HCEM cells. RT-PCR and real-time PCR analyses showed that HPL-spondin expressed BMP7, but not HPL cells. And BMP7 and phospho-Smad1/5/8 protein production were detected in HPL-spondin by Western blot. siSPON1 inhibited expression of type I collagen, runt-related transcription factor 2 (RUNX2) and bone sialoprotein (BSP) mRNA in HCEM cells. And the mineralization tended to be decreased in siSPON1 treated cells by ALZ staining and the quantification analysis. Moreover, we examined the effect of BMP7 peptide on the gene expressions of HCEM cells by RT-PCR. Increase of the osteopontin and BSP mRNA was observed in BMP7 treated HCEM cells. These findings indicate that F-spondin regulates the differentiation of HCEM cells via BMP7 expression.

Transition metal ions induce carnosinase activity in PepD-homologous protein from Porphyromonas gingivalis.

Aminoacylhistidine dipeptidase (EC 3.4.13.3; also Xaa-His dipeptidase, carnosinase, or PepD) catalyzes the cleavage and release of an N-terminal amino acid, which is usually a neutral or hydrophobic residue, from an Xaa-His dipeptide or degraded peptide fragment. PepD enzyme is found extensively in prokaryotes and eukaryotes, and belongs to the metallopeptidase family M20, a part of the metallopeptidase H (MH) clan. Carnosine is a naturally occurring dipeptide (ß-alanyl-l-histidine) present in mammalian tissues that has protective functions in addition to anti-oxidant and free-radical scavenging roles. During bacterial infections, degradation of l-carnosine via carnosinase or PepD-like enzymes may enhance the destructive potential of bacteria, resulting in a pathological impact. This process has been proposed to act in an anti-oxidant manner in vivo. In the present study, the recombinant PepD protein encoded by Porphyromonas gingivalis TDC60 pepD was generated and biochemically characterized. In addition, a recombinant dipeptidase enzyme was found to function not only as an alanine-aminopeptidase, but also as a carnosinase. Furthermore, when carnosine was used as substrate for PepD, the transition metals, Mn(2+), Fe(2+), Co(2+), and Ni(2+) stimulated the hydrolyzing activity of rPepD with ß-alanine and l-histidine. Based on its metal ion specificity, we propose that this enzyme should not only be termed l-aminopeptidase, but also a carnosinase.

Basic helix-loop-helix transcription factor DEC1 negatively regulates cyclin D1.

DEC1 (also known as Stra13/Bhlhb2/Sharp2) and DEC2 (also known as Bhlhb3/Sharp1) are two paralogous basic helix-loop-helix (bHLH) transcriptional regulators which exhibit a robust circadian gene expression pattern in the suprachiasmatic nucleus (SCN) and in peripheral organs. DEC1 has been suggested to play key roles in mammalian cell differentiation, the cell cycle and circadian regulation, hypoxia response, and carcinogenesis. Here we show that DEC1 overexpression exhibits delayed wound healing and reduces cell proliferation, migration, and invasion. DEC1 strongly repressed the promoter activity of cyclin D1. We further identify a possible DEC-response element in the cyclin D1 promoter region, and confirmed the direct binding of DEC1 to that element. Forced expression of DEC1 efficiently repressed the cyclin D1 promoter and expression. Our clinical data provide the first evidence that there is a strong inverse correlation between DEC1 and cyclin D1 expression in oral cancer, and DEC1 expression significantly correlated with clinicopathological parameters. We suggest that radiation-induced DEC1 overexpression and Akt phosphorylation in cancer cells are mediated via PI-3K signalling. Overexpression of DEC1 activates the PI-3K/Akt signalling pathway through reactive oxygen species (ROS).

Identification by DNA microarray of genes involved in Candida albicans-treated gingival epithelial cells.

BACKGROUND: Oral epithelial cells significantly influence host inflammatory responses against Candida albicans in oropharyngeal candidiasis. Pro-inflammatory cytokines function as an early innate immune system mediator during C. albicans infection in oral epithelial cells. We sought to elucidate the pattern of the molecular mechanisms governing the human gingival epithelial cells (HGECs) to C. albicans infection likely involve multiple converging signal transduction pathways. MATERIALS AND METHODS: Primary HGECs were cultured with C. albicans ATCC90029. Total RNA was extracted after 8 h of infection and monitored mRNA levels using Affymetrix GeneChip (Human Genome U133 plus 2.0 Array, 48 000 genes). GeneChip data was analyzed by GeneSpring software and Ingenuity Pathway Analysis system. Reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR and immunohistochemistry were used to investigate gene expression changes. RESULTS: The differentially expressed genes represented functions as diverse as immune response and inflammatory disease. IL-8, ICAM-1 and Cox-2 showed a greater than two fold change in expression relative to those in control cells. Altered mRNA levels in GeneChip analysis were confirmed by RT-PCR and real-time RT-PCR. Stronger immunoreactivity against ICAM-1 and Cox-2 was also observed in the infection with C. albicans in rat gingival epithelium. We have identified differential gene expression up-regulated or down-regulated with the up-regulation of IL-8 in C. albicans-treated cells. CONCLUSION: These findings indicate that the molecular mechanisms underlying the IL-8 response of HGECs to C. albicans infection likely involve multiple converging signal transduction pathways.

Blue LED inhibits the growth of Porphyromonas gingivalis by suppressing the expression of genes associated with DNA replication and cell division.

BACKGROUND AND OBJECTIVES: Blue light has been employed or investigated in both the medical and dental fields. Many studies have so far been reported a bactericidal effect of blue light emitting diodes (LED). However, it is still unclear whether exposure to blue LED kills or inhibits the growth of bacteria. We therefore investigated the effect of blue LED irradiation on the growth of Porphyromonas gingivalis compared with the effects of red LED. MATERIALS AND METHODS: P. gingivalis cell suspensions were irradiated with blue or red LED (135 J/cm2) anaerobically, incubated for various lengths of time, and then the total RNAs were isolated. The RNA degradation and gene expression levels of stress-related proteins in blue or red LED-irradiated samples were examined using the RNA integrity number (RIN) and RT-PCR, respectively. Quantitative RT-PCR was done to investigate the gene expression profiles associated with chromosome replication and cell division. RESULTS: Exposure to blue LED delayed the growth of P. gingivalis, while red LED did not. The RIN value indicated no RNA degradation in either the blue or red LED-irradiated samples. In addition, the gene expression levels of stress-related molecules remained either constant or increased 15 minutes after the blue LED irradiation compared to that before irradiation, thus suggesting that blue LED may not kill P. gingivalis cells. However, the blue LED irradiation did lead to a remarkably decreased expression of genes associated with chromosomal DNA replication and cell division after 5 minutes; exposure to the red LED did not. CONCLUSION: The inhibition of the growth of P. gingivalis by blue LED may therefore be induced not by a bactericidal effect, but instead due to a bacteriostatic effect mediated by the suppression of the genes associated with chromosomal DNA replication and cell division at the transcriptional level.

Complete genome sequence of the bacterium Porphyromonas gingivalis TDC60, which causes periodontal disease.

Porphyromonas gingivalis is a black-pigmented asaccharolytic anaerobe and a major causative agent of periodontitis. Here, we report the complete genome sequence of P. gingivalis strain TDC60, which was recently isolated from a severe periodontal lesion in a Japanese patient.

Nasal immunization with a fusion protein consisting of the hemagglutinin A antigenic region and the maltose-binding protein elicits CD11c(+) CD8(+) dendritic cells for induced long-term protective immunity.

We assessed the efficacy of a fusion protein consisting of the 25-kDa antigenic region of Porphyromonas gingivalis hemagglutinin A and the Escherichia coli maltose-binding protein (25k-hagA-MBP) as a nasal vaccine for the prevention of oral infection with P. gingivalis. Nasal immunization with 25k-hagA-MBP induced high levels of 25k-hagA-specific serum IgG, serum IgA, and salivary IgA antibodies in a Toll-like receptor 4 (TLR4)-dependent manner. These antibody responses were maintained for at least 1 year after immunization. Analysis of cytokine responses showed that nasal administration of 25k-hagA-MBP induced antigen-specific CD4(+) T cells producing interleukin 4 (IL-4) and IL-5, but not gamma interferon (IFN-γ), in the spleen and cervical lymph nodes (CLNs). Furthermore, increased numbers of CD11c(+) CD8α(+), but not CD11c(+) CD11b(+) or CD11c(+) B220(+), dendritic cells with upregulated expression of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) molecules were noted in the spleen, CLNs, and nasopharynx-associated lymphoreticular tissues (NALT). Interestingly, when 25k-hagA-MBP or cholera toxin (CT) was given intranasally to enable examination of their presence in neuronal tissues, the amounts of 25k-hagA-MBP were significantly lower than those of CT. Importantly, mice given 25k-hagA-MBP nasally showed a significant reduction in alveolar bone loss caused by oral infection with P. gingivalis, even 1 year after the immunization. These results suggest that 25k-hagA-MBP administered nasally would be an effective and safe mucosal vaccine against P. gingivalis infection and may be an important tool for the prevention of chronic periodontitis in humans.

Low-level laser irradiation treatment reduces CCL2 expression in rat rheumatoid synovia via a chemokine signaling pathway.

Rheumatoid arthritis (RA) is an inflammatory joint disorder whose progression leads to the destruction of cartilage and bone. Although low-level laser irradiation (LLLI) is currently being evaluated for the treatment of RA, the molecular mechanisms underlying its effectiveness remain unclear. To investigate possible LLLI-mediated antiinflammatory effects, we utilized a collagen-induced arthritis (CIA) rat model and analyzed gene expression profiles in the synovial membranes of the knee joint. Total RNA was isolated from the synovial membrane tissue of the joints of untreated CIA rats or CIA rats treated with LLLI (830 nm Ga-Al-As diode), and gene expression profiles were analyzed by DNA microarray (41,000 rat genes), coupled with Ingenuity pathways analysis (IPA). DNA microarray analysis showed that CCL2 gene expression was increased in CIA tissue, and that LLLI treatment significantly decreased CIA-induced CCL2 mRNA levels. IPA revealed that chemokine signal pathways were involved in the activation of CCL2 production. These microarray data were further validated using real-time PCR and reverse transcription PCR. Immunohistochemistry confirmed that CCL2 production was decreased in CIA rats treated with LLLI. These findings suggest that decreased CCL2 expression may be one of the mechanisms involved in LLLI-mediated RA inflammation reduction.

Low-level laser irradiation enhances BMP-induced osteoblast differentiation by stimulating the BMP/Smad signaling pathway.

Low-level laser irradiation (LLLI) has been shown to induce bone formation and osteoblast differentiation both in vivo and in vitro. However, the molecular mechanism by which LLLI stimulates osteoblast differentiation is still unclear. The aim of the present study was to examine whether Ga-Al-As laser irradiation could enhance BMP2-induced alkaline phosphatase (ALP) activity in C2C12 cells. Laser irradiation at 0.5 W for 20 min enhanced BMP2-induced ALP activity. Laser treatment alone did not affect ALP activity. To exclude the effect of pH or temperature changes during irradiation, we shortened the exposure time to 2 min, with various levels of laser power. At 2.5 W, irradiation stimulated BMP2-induced ALP activity but not cell proliferation, whereas 1 or 5 W laser power did not induce any significant effects. Irradiation stimulated BMP2-induced phosphorylation of Smad1/5/8 and BMP2 expression, but had no effect on the expression of inhibitory Smads 6 and 7, BMP4, or insulin-like growth factor 1. Laser irradiation enhanced Smad-induced Id1 reporter activity as well as expression of bone morphogenetic protein (BMP)-induced transcription factors such as Id1, Osterix, and Runx2. Laser irradiation also stimulated BMP-induced expressions of type I collagen, osteonectin, and osteocalcin mRNA, markers of osteoblasts. This enhancement of BMP2-induced ALP activity and Smad phosphorylation by laser irradiation was also observed in primary osteoblasts. These results suggest that LLLI accelerates the differentiation of BMP-induced osteoblasts by stimulating the BMP/Smad signaling pathway.

Gene expression profiling of cholangiocarcinoma-derived fibroblast reveals alterations related to tumor progression and indicates periostin as a poor prognostic marker.

BACKGROUND: Fibroblasts play important roles in several cancers. It was hypothesized that cholangiocarcinoma (CCA)-associated fibroblasts (Cfs) differ from non-tumorigenic liver fibroblasts (Lfs) in their gene expression profiles resulting in the capability to promote cancer. Periostin (PN) is a multi-functional protein and has emerged as a promising marker for tumor progression. The role of PN in CCA, however, has not yet been explored. RESULTS: In this study, the gene expression profile of Cfs in comparison to Lfs was performed using oligonucleotide microarrays. The common- and unique-expressed genes in Cfs and the promising roles in cancer promotion and progression were determined. PN was markedly over-expressed in Cfs confirmed by real time RT-PCR and western blot analysis. Immunohistochemistry examination of a number of patients with intrahepatic CCA showed the expression of PN solely in stromal fibroblasts, but was expressed neither in cancer cells nor immune cells. Low to no expression of PN was observed in tissues of benign liver disease and hepatocellular carcinoma. CCA patients with high levels of PN had significantly shorter survival time than those with low levels (P = 0.026). Multivariate analysis revealed high levels of PN (P = 0.045) and presence of lymph node metastasis (P = 0.002) as independent poor prognostic factors. The in vitro study revealed that recombinant PN induced CCA cell proliferation and invasion. Interestingly, interference RNA against integrin alpha 5 significantly reduced the cellular response to PN-stimulated proliferation and invasion. CONCLUSION: The gene expression profile of fibroblasts in CCA is apparently explored for the first time and has determined the genes involving in induction of this cancer progression. High PN can be used to distinguish CCA from other related liver diseases and is proposed as a prognostic factor of poor survival. Regulation of fibroblast-derived PN in CCA proliferation and invasion may be considered as an alternative therapeutic approach.

Odd-skipped related 2 regulates genes related to proliferation and development.

Cell proliferation is a biological process in which chromosomes replicate in one cell and equally divide into two daughter cells. Our previous findings suggested that Odd-skipped related 2 (Osr2) plays an important role in cellular quiescence and proliferation under epigenetic regulation. However, the mechanism used by Osr2 to establish and maintain proliferation is unknown. To examine the functional role of Osr2 in cell proliferation, we analyzed its downstream target genes using microarray analysis following adenovirus-induced overexpression of Osr2 as well as knockdown with Osr2 siRNA, which showed that Osr2 regulates a multitude of genes involved in proliferation and the cell cycle, as well as development. Additional proliferation assays also indicated that Osr2 likely functions to elicit cell proliferation. Together, these results suggest that Osr2 plays important roles in proliferation and development.

Characterization of hemin-binding protein 35 (HBP35) in Porphyromonas gingivalis: its cellular distribution, thioredoxin activity and role in heme utilization.

BACKGROUND: The periodontal pathogen Porphyromonas gingivalis is an obligate anaerobe that requires heme for growth. To understand its heme acquisition mechanism, we focused on a hemin-binding protein (HBP35 protein), possessing one thioredoxin-like motif and a conserved C-terminal domain, which are proposed to be involved in redox regulation and cell surface attachment, respectively. RESULTS: We observed that the hbp35 gene was transcribed as a 1.1-kb mRNA with subsequent translation resulting in three proteins with molecular masses of 40, 29 and 27 kDa in the cytoplasm, and one modified form of the 40-kDa protein on the cell surface. A recombinant 40-kDa HBP35 exhibited thioredoxin activity in vitro and mutation of the two putative active site cysteine residues abolished this activity. Both recombinant 40- and 27-kDa proteins had the ability to bind hemin, and growth of an hbp35 deletion mutant was substantially retarded under hemin-depleted conditions compared with growth of the wild type under the same conditions. CONCLUSION: P. gingivalis HBP35 exhibits thioredoxin and hemin-binding activities and is essential for growth in hemin-depleted conditions suggesting that the protein plays a significant role in hemin acquisition.