Monoamine Oxidase-B Inhibitor Reduction in Pro-Inflammatory Cytokines Mediated by Inhibition of cAMP-PKA/EPAC Signaling.

Mucosal epithelial cell integrity is an important component of innate immunity and it protects the host from an environment rich in microorganisms. Virulence factors from Gram-negative bacteria [e.g. lipopolysaccharide (LPS)] induce significant pro-inflammatory cytokine expression. Monoamine oxidase (MAO) inhibitors reduce cytokine expression in a variety of inflammatory models and may therefore have therapeutic potential for a number of inflammatory diseases. We tested the anti-inflammatory therapeutic potential of a recently developed reversible MAO-B inhibitor (RG0216) with reduced transport across the blood-brain barrier. In an epithelial cell culture model, RG0216 significantly decreased LPS-induced interleukin (IL)-6 and IL-1ß gene and protein expression and was as effective as equimolar concentrations of deprenyl (an existing irreversible MAO-B inhibitor). Hydrogen peroxide and modulating dopamine receptor signaling had no effect on cytokine expression. We showed that LPS-induced expression of IL-6 and IL-1ß was cAMP dependent, that IL-6 and IL-1ß expression were induced by direct cAMP activation (forskolin) and that RG0216 and deprenyl effectively reduced cAMP-mediated cytokine expression. Targeted protein kinase A (PKA) and Exchange Protein Activated by cAMP (EPAC) activation regulated IL-6 and IL-1ß expression, albeit in different ways, but both cytokines were effectively decreased with RG0216. RG0216 reduction of LPS-induced cytokine expression occurred by acting downstream of the cAMP-PKA/EPAC signaling cascade. This represents a novel mechanism by which MAO-B selective inhibitors regulate LPS-induced IL-6 and IL-1ß expression.

Monoamine Oxidase Inhibitors: A Review of Their Anti-Inflammatory Therapeutic Potential and Mechanisms of Action.

Chronic inflammatory diseases are debilitating, affect patients' quality of life, and are a significant financial burden on health care. Inflammation is regulated by pro-inflammatory cytokines and chemokines that are expressed by immune and non-immune cells, and their expression is highly controlled, both spatially and temporally. Their dysregulation is a hallmark of chronic inflammatory and autoimmune diseases. Significant evidence supports that monoamine oxidase (MAO) inhibitor drugs have anti-inflammatory effects. MAO inhibitors are principally prescribed for the management of a variety of central nervous system (CNS)-associated diseases such as depression, Alzheimer's, and Parkinson's; however, they also have anti-inflammatory effects in the CNS and a variety of non-CNS tissues. To bolster support for their development as anti-inflammatories, it is critical to elucidate their mechanism(s) of action. MAO inhibitors decrease the generation of end products such as hydrogen peroxide, aldehyde, and ammonium. They also inhibit biogenic amine degradation, and this increases cellular and pericellular catecholamines in a variety of immune and some non-immune cells. This decrease in end product metabolites and increase in catecholamines can play a significant role in the anti-inflammatory effects of MAO inhibitors. This review examines MAO inhibitor effects on inflammation in a variety of in vitro and in vivo CNS and non-CNS disease models, as well as their anti-inflammatory mechanism(s) of action.

Development of Novel Monoamine Oxidase-B (MAO-B) Inhibitors with Reduced Blood-Brain Barrier Permeability for the Potential Management of Noncentral Nervous System (CNS) Diseases.

Studies indicate that MAO-B is induced in peripheral inflammatory diseases. To target peripheral tissues using MAO-B inhibitors that do not permeate the blood-brain barrier (BBB) the MAO-B-selective inhibitor deprenyl was remodeled by replacing the terminal acetylene with a CO2H function, and incorporating a para-OCH2Ar motif (compounds 10a-s). Further, in compound 32 the C-2 side chain corresponded to CH2CN. In vitro, 10c, 10j, 10k, and 32 were identified as potent reversible MAO-B inhibitors, and all four compounds were more stable than deprenyl in plasma, liver microsomal, and hepatocyte stability assays. In vivo, they demonstrated greater plasma bioavailability. Assessment of in vitro BBB permeability showed that compound 10k is a P-glycoprotein (P-gp) substrate and 10j displayed mild interaction. Importantly, compounds 10c, 10j, 10k, and 32 displayed significantly reduced BBB permeability after intravenous, subcutaneous, and oral administration. These polar MAO-B inhibitors are pertinent leads for evaluation of efficacy in noncentral nervous system (CNS) inflammatory disease models.

Lipopolysaccharide induces a stromal-epithelial signalling axis in a rat model of chronic periodontitis.

AIM: Lipopolysaccharide is a bacterial virulence factor implicated in chronic periodontitis, which may penetrate the junctional epithelial barrier and basement membrane to insult underlying stroma. We sought to identify lipopolysaccharide-induced global gene expression changes responsible for signalling between stroma and epithelium during disease onset. MATERIALS AND METHODS: Using a rat lipopolysaccharide periodontitis model, junctional epithelium and underlying stromal tissue were separately collected from healthy and diseased animals by laser-capture microdissection and subject to gene expression microarray analysis. Key gene products identified were validated in gingival epithelial and fibroblast cell cultures. RESULTS: Global gene expression patterns distinguishing health versus disease were found in and between both tissue types. In stroma, the most significantly altered gene ontology function group (Z ≥ 4.00) was cytokines, containing most significantly (±2-fold; p < 0.05) upregulated genes amphiregulin, IL1-ß and Fas ligand, all positive, diffusible modulators of the epithelial growth factor receptor pathway. In epithelium, the most significant changes were in downregulated FOS-related antigen-1 gene, somatostatin receptor-2 gene and mucin-4 gene, all negative modulators of the epithelial growth factor receptor pathway. CONCLUSION: These results establish a periodontitis model for studying gene product interactions and suggests that the onset of junctional epithelial disease hyperproliferation involves a concerted stromal-epithelial signalling axis.

Craniofacial defect regeneration using engineered bone marrow mesenchymal stromal cells.

Large craniofacial bony defects remain a significant clinical challenge. Bone marrow mesenchymal stromal cells (BM-MSCs) constitute a multipotent population. Previously, we developed a novel approach for BM-MSC expansion on 3D CultiSpher-S gelatin microcarrier beads in spin culture with preservation of their multipotentiality, reduction of apoptosis, and enhancement of bone formation in vivo. Here, we hypothesized that such cultured BM-MSCs without exogenous growth factors would respond to the orthopedic microenvironment, thus promoting craniofacial defect regeneration. BM-MSCs isolated from green fluorescent protein (GFP) transgenic rats were ex vivo expanded and transplanted into critical-sized (5-mm diameter) rat calvaria defects. Gelatin beads or defect alone served as controls. By 28 and 42 days, rats were sacrificed for microcomputed tomography (microCT), histologic, and immunohistochemistry examination. MicroCT results demonstrated that BM-MSCs were a statistically significant factor contributing to new bone volume regeneration. Histologic assessment showed that the BM-MSCs group produced more and higher quality new bone compared with beads or defect-alone groups in both osteoinductive and osteoconductive manners. Specifically, immunohistochemical staining identified GFP(+) cells residing in new bone lacunae in conjunction with non-GFP(+) cells. Therefore, ex vivo expanded BM-MSCs at least in part regenerated critical-sized calvaria defects by osteogenic differentiation in vivo.

Periodontal regeneration using engineered bone marrow mesenchymal stromal cells.

Regeneration of lost periodontium is a challenge in that both hard (alveolar bone, cementum) and soft (periodontal ligament) connective tissues need to be restored to their original architecture. Bone marrow mesenchymal stromal cells (BM-MSCs) appear to be an attractive candidate for connective tissue regeneration. We hypothesized that BM-MSCs are able to sense biological cues from the local microenvironment and organize appropriately to contribute to the regeneration of both soft and hard periodontal connective tissues. To test this hypothesis, we transplanted GFP(+) rat BM-MSCs expanded ex vivo on microcarrier gelatin beads into a surgically created rat periodontal defect. After three weeks, evidence of regeneration of bone, cementum and periodontal ligament was observed in both transplanted and control animals. However, the animals that received BM-MSCs regenerated significantly greater new bone. In addition, the animals that had received the cells and beads transplant had significantly more appropriately orientated periodontal ligament fibers, indicative of functional restoration. Finally, donor-derived BM-MSCs were found integrated in newly formed bone, cementum and periodontal ligament, suggesting that they can directly contribute to the regeneration of cells of these tissues.

Lipopolysaccharide-induced epithelial monoamine oxidase mediates alveolar bone loss in a rat chronic wound model.

Reactive oxygen species (ROS) production is an antimicrobial response to pathogenic challenge that may, in the case of persistent infection, have deleterious effects on the tissue of origin. A rat periodontal disease model was used to study ROS-induced chronic epithelial inflammation and bone loss. Lipopolysaccharide (LPS) was applied for 8 weeks into the gingival sulcus, and histological analysis confirmed the onset of chronic disease. Junctional epithelium was collected from healthy and diseased animals using laser-capture microdissection, and expression microarray analysis was performed. Of 19,730 genes changed in disease, 42 were up-regulated >/=4-fold. Three of the top 10 LPS-induced genes, monoamine oxidase B (MAO/B) and flavin-containing monooxygenase 1 and 2, are implicated in ROS signaling. LPS-associated induction of the ROS mediator H(2)O(2), as well as MAO/B and tumor necrosis factor (TNF)-alpha levels were validated in the rat histological sections and a porcine junctional epithelial cell culture model. Topical MAO inhibitors significantly counteracted LPS-associated elevation of H(2)O(2) production and TNF-alpha expression in vivo and in vitro, inhibited disease-associated apical migration and proliferation of junctional epithelium and inhibited induced systemic H(2)O(2) levels and alveolar bone loss in vivo. These results suggest that LPS induces chronic wounds via elevated MAO/B-mediated increases in H(2)O(2) and TNF-alpha activity by epithelial cells and is further associated with more distant effects on systemic oxidative stress and alveolar bone loss.

An in vitro analysis of mechanical wounding-induced ligand-independent KGFR activation.

BACKGROUND: KGFR (keratinocyte growth factor receptor), exclusively expressed in epithelial cells, plays an important role in wound healing. However, mechanisms of KGFR activation and signaling in wound healing are not clearly understood. OBJECTIVES: We utilized an in vitro mechanical wounding model to examine ligand-independent KGFR activation, its regulation by reactive oxygen species (ROS) and the functional significance of this activation mechanism. METHODS: Confluent HaCaT cell line cultures were mechanically wounded and KGFR internalization and phosphorylation were examined using immunostaining with confocal microscopy and immunoprecipitation with Western blotting. Wounding-induced generation of reactive oxygen species and ligand-independent activation of KGFR were examined. In addition, phosphorylation of its associated molecules FRS2 and c-Src were examined in the presence and absence of the ROS and pathway specific inhibitors. The importance of this activation process on cell migration was also examined in the presence and absence of these inhibitors. RESULTS: Mechanical wounding induced ligand-independent KGFR activation and internalization. KGFR internalization and phosphorylation was associated with ROS generation along the wound edge and scavenging of ROS with NAC inhibited KGFR phosphorylation. Intracellularly, c-Src was phosphorylated by wounding but its inhibitor, PP1, significantly inhibited KGFR activation and associated FRS2 phosphorylation. Mechanical wounding induced wound edge migration, which was significantly reduced by the selective receptor and pathway inhibitors PP1 (82.7%), KGFR inhibitor SU5402 (70%) and MAPK inhibitor PD98059 (57%). CONCLUSION: Mechanical wounding induces significant ROS generation at the wound edge which, in turn, induced ligand-independent KGFR and FRS2 activation via c-Src kinase signaling. Functionally, downstream MAPK signaling induced wound edge cell migration.

Mechanical induction of an epithelial cell chymase associated with wound edge migration.

Chymase is a chymotrypsin-like serine protease predominantly produced by mast cells. In this study, human cutaneous and gingival keratinocytes, ovary surface epithelia, and a porcine epithelial cell line were assayed by homology-based cloning, and the amplified DNA fragment was identified as a chymase. In vitro, chymase could not be induced by serum or cytokine treatment alone. Chymase was activated 3-fold within 60 min in basal media by scratch wounding cultured monolayers and further potentiated over 10-fold at 18 h by additional serum and cytokine treatment. Chymase activity was cell-associated and found to peak within 24 h of wounding and then steadily decreased as cultures healed, reaching baseline levels before confluence was reestablished. Affinity column purified enzyme effectively degraded fibronectin and was found by Western blot analysis using a human chymase antibody to be of about 30 kDa. Immunostaining revealed chymase activation at the wound edge colocalizing with reactive oxygen species generation. Specifically, chymase activation was attenuated by inhibition of nitric oxide, superoxide, and peroxynitrite. Exogenous peroxynitrite but not hydrogen peroxide also resulted in chymase activation in unwounded monolayers. Disruption of cytoskeletal stress fibers by cytochalasin D attenuated both wound-activated chymase and reactive oxygen species generation. Chymase inhibitor chymostatin reduced the loss of cell-cell contacts and the onset of porcine and human skin epithelial cell migration at the wound edge. This shows that an epithelial chymase is rapidly activated by a ligand-independent mechanism following mechanical stress via cytoskeletal and reactive oxygen species signaling and is associated with the onset of epithelial cell migration.

Absence of alphavbeta6 integrin is linked to initiation and progression of periodontal disease.

Integrin alphavbeta6 is generally not expressed in adult epithelia but is induced in wound healing, cancer, and certain fibrotic disorders. Despite this generalized absence, we observed that alphavbeta6 integrin is constitutively expressed in the healthy junctional epithelium linking the gingiva to tooth enamel. Moreover, expression of alphavbeta6 integrin was down-regulated in human periodontal disease, a common medical condition causing tooth loss and also contributing to the development of cardiovascular diseases by increasing the total systemic inflammatory burden. Remarkably, integrin beta6 knockout mice developed classic signs of spontaneous, chronic periodontal disease with characteristic inflammation, epithelial down-growth, pocket formation, and bone loss around the teeth. Integrin alphavbeta6 acts as a major activator of transforming growth factor-beta1 (TGF-beta1), a key anti-inflammatory regulator in the immune system. Co-expression of TGF-beta1 and alphavbeta6 integrin was observed in the healthy junctional epithelium. Moreover, an antibody that blocks alphavbeta6 integrin-mediated activation of TGF-beta1 initiated inflammatory periodontal disease in a rat model of gingival inflammation. Thus, alphavbeta6 integrin is constitutively expressed in the epithelium sealing the gingiva to the tooth and plays a central role in protection against inflammatory periodontal disease through activation of TGF-beta1.

Ex vivo expansion of rat bone marrow mesenchymal stromal cells on microcarrier beads in spin culture.

Bone marrow mesenchymal stromal cells (BM-MSC) are attractive candidates for connective tissue regeneration. Currently, their use is limited by poor overall cell survival and high apoptosis rates upon transplantation in vivo. We hypothesized that disruption of cell-extracellular matrix contact either during cell expansion or immediately prior to cell transplantation may impair cell viability and facilitate apoptosis. We therefore investigated whether BM-MSC can be expanded on microcarrier beads in spin culture and directly transplanted. This novel approach removes the need for the repeated trypsinizations that are usually required for expansion and transplantation. CultiSpher-S gelatin microcarrier beads supported Fisher and transgenic green fluorescent protein (GFP)(+) Sprague Dawley rat BM-MSC expansion. Bead-expanded BM-MSC could still be differentiated along the chondrogenic, osteogenic and adipogenic lineages. In the short term, direct subcutaneous transplantation of cells expanded on microcarriers was associated with significantly less apoptosis than trypsinized control cells. In the long term, BM-MSC expanded on microcarrier beads induced de novo trabecular bone formation in vivo. This novel approach present several advantages over current expansion-transplantation protocols for mesenchymal tissue regeneration.

RNA integrity and in situ RT-PCR in dento-alveolar tissues after microwave accelerated demineralisation.

OBJECTIVE: The structural organization of oral soft tissue and its relationship with highly calcified teeth are difficult to preserve unless tissues are decalcified, paraffin embedded and subsequently sectioned. However, enamel decalcification time and its negative impact on RNA integrity makes it difficult to effectively analyse in situ gene expression. This study examined the impact of microwave-enhanced decalcification on processing time, RNA integrity and detection of in situ mRNA expression in hard and soft tissue for cell type specific markers of Keratinocyte growth factor receptor, Scleraxis and Osteonectin. DESIGN: Maxillas and mandibles were obtained from three male Wistar strain rats. Right side tissues were decalcified using a microwave plus 10% EDTA solution (M+) while left side tissues were decalcified in 10% EDTA solution alone (M-). RESULTS: Microwave use reduced decalcification time by up to 50% and had no significant impact on morphology, RNA quality and in situ detection of gene expression relative to the M-group. CONCLUSIONS: In situ RT-PCR gene expression of microwave decalcified paraffin-embedded oral tissues is an effective technique to localize in situ gene expression while maintaining excellent soft and hard tissue architecture.

Keratinocyte growth factor-1 expression in healthy and diseased human periodontal tissues.

OBJECTIVES: Keratinocyte growth factor-1 (KGF-1) is up-regulated in chronic inflammation and specifically stimulates epithelial cell proliferation by signaling through the epithelial-specific keratinocyte growth factor receptor (KGFR). We examined KGF-1 and KGFR protein and gene expression in healthy and diseased periodontal tissues. METHODS: Tissues were collected from patients with periodontal health or disease, immediately frozen and stained for KGF-1 and KGFR protein expression. Laser capture microdissection of epithelial and connective tissue cells with reverse transcription-polymerase chain reaction (RT-PCR) examined KGF-1 and KGFR gene expression profiles and enzymatic digestion with heparitinase, chondroitinase ABC or pre-treatment with suramin examined epithelial surface molecule interactions with KGF-1. RESULTS: In tissues collected from healthy patients, KGF-1 protein localized to areas of junctional and basal oral epithelial cells and was significantly increased in periodontal pocket epithelium (p<0.01) and in the oral epithelium (p<0.05) of disease-associated tissues. KGFR localized to the junctional and the parabasal cells of oral epithelium, with the relative staining intensity being increased in disease-associated pocket epithelium (p<0.05). Laser capture microdissection with RT-PCR confirmed KGF-1 and KGFR were specifically expressed by connective tissue and epithelium, respectively. KGF-1 localization to epithelial cells was largely eliminated by suramin pre-treatment, indicating interaction with the KGFR. CONCLUSIONS: KGF-1 and KGFR proteins are expressed in healthy periodontal tissues but significantly increased in diseased periodontal tissues. We hypothesize up-regulation of KGF-1 and KGFR protein associated with disease regulates epithelial cell behavior associated with onset and progression of periodontal pocket formation.

Fusobacterium nucleatum increases collagenase 3 production and migration of epithelial cells.

Fusobacterium nucleatum is closely associated with human periodontal diseases and may also be a causative agent in other infections, such as pericarditis, septic arthritis, and abscesses of tonsils and liver. Initiation and outcome of infective diseases depend critically on the host cell signaling system altered by the microbe. Production of proteinases by infected cells is an important factor in pericellular tissue destruction and cell migration. We studied binding of F. nucleatum to human epithelial cells (HaCaT keratinocyte line) and subsequent cell signaling related to collagenase 3 expression, cell motility, and cell survival, using a scratch wound cell culture model. F. nucleatum increased levels of 12 protein kinases involved in cell migration, proliferation, and cell survival signaling, as assessed by the Kinetworks immunoblotting system. Epithelial cells of the artificial wound margins were clearly preferential targets of F. nucleatum. The bacterium colocalized with lysosomal structures and stimulated migration of these cells. Of the 13 anaerobic oral bacterial species, F. nucleatum and Fusobacterium necrophorum were among the best inducers of collagenase 3 mRNA levels, a powerful matrix metalloproteinase. Production of collagenase 3 was detected in fusobacterium-infected cells and cell culture medium by immunocytochemistry, immunoblotting, and zymography. The proteinase production involved activation of p38 mitogen-activated protein kinase in the infected cells. The study suggests that F. nucleatum may be involved in the pathogenesis of periodontal diseases (and other infections) by activating multiple cell signaling systems that lead to stimulation of collagenase 3 expression and increased migration and survival of the infected epithelial cells.

Keratinocyte growth factor 1 inhibits wound edge epithelial cell apoptosis in vitro.

The ability of keratinocyte growth factor 1 to modulate apoptosis in the absence of proliferation was studied in vitro. A HaCaT scrape wound model was developed in which dense monolayers prior to wounding were cultured to quiescence in defined media with hydroxyurea at concentrations that blocked proliferation without loss of cell viability. Scrape wounding was then found to induce apoptosis, originating at the wound edge, but subsequently radiating away over a 24 h period to encompass areas not originally damaged. Keratinocyte growth factor 1 inhibited this radial progression of apoptosis in a concentration-dependent manner up to 20 ng per mL with induced migration present at the wound edge. The extent of this rescue was modulated by the concentration of Ca2+ prior to wounding. In control wound cultures apoptotic bodies were found in cells adjacent to the wound interface but were greatly reduced in keratinocyte-growth-factor-1-treated groups. Keratinocyte growth factor 1 receptor expression was significantly induced within two to three cell widths of the scraped wound edge, at levels far exceeding those found at the leading edge of a nonwounded epithelial sheet. Tumor necrosis factor alpha (1-5 ng per mL) or Escherichia coli lipopolysaccharide (10-50 ng per mL) exacerbated scrape-induced early apoptosis (1-4 h), but was largely ameliorated by coculture with keratinocyte growth factor 1. Keratinocyte growth factor 1 protection was associated with a reduction in both caspase-3 activation and cytokeratin-19 loss. Protected wound edges were also associated with the maintenance of e-cadherin expression and induction of beta1 integrin and actin stress fiber organization. These results suggest that keratinocyte growth factor 1 may play a role in limiting mechanically induced apoptotic processes at the epithelial wound edge in a manner that is distinct from its proliferative function.

Clinical and microbiologic study of periodontitis associated with Kindler syndrome.

BACKGROUND: Little is known about the onset and prevalence of periodontal disease in patients with the rare Kindler syndrome, a genodermatological disorder. This study investigated the level of clinical periodontal attachment in relation to age and presence of putative periodontopathogenic bacteria in individuals with Kindler syndrome. METHODS: Eighteen individuals diagnosed with Kindler syndrome and 13 control subjects, aged 4 to 37 years, from rural Panama received a limited clinical periodontal examination. Subgingival samples were collected for identification of putative periodontal pathogens by polymerase chain reaction. RESULTS: Mild to severe gingivitis was a common finding in all adults of the study population. Seventy-two percent (13/18) of the Kindler patients and 46% (6/13) of the control subjects showed mild to severe periodontal disease (P = 0.001, chi-square test). The onset of periodontitis was earlier and the progression occurred at a faster rate in the Kindler group. There was a strong correlation (r = 0.83) between the level of attachment loss and age in the Kindler group and a weaker correlation (r = 0.66) in the control group. The appearance of gingival tissues suggested atypical periodontitis with spontaneous bleeding and fragile, often desquamative, gingiva. In periodontitis patients, Porphyromonas gingivallis and Diallster pneumosintes tended to occur more frequently in control individuals compared to those with Kindler syndrome. CONCLUSIONS: In the Kindler group, periodontitis had an onset in early teenage years and progressed more rapidly compared to non-Kindler individuals of the same geographic and ethnic group. Clinical and microbiological findings suggest atypical periodontitis in Kindler patients. We propose to include Kindler syndrome in the category of medical disorders predisposing to destructive periodontal disease.

Induction of keratinocyte growth factor 1 Expression by lipopolysaccharide is regulated by CD-14 and toll-like receptors 2 and 4.

Periodontal disease is a chronic inflammatory condition that is associated with increased concentrations of gram-negative pathogenic bacteria and epithelial cell proliferation. Regulation of this proliferation is poorly understood but is most likely controlled by locally expressed growth factors. Keratinocyte growth factor 1, an epithelium-specific growth factor, is expressed by gingival fibroblasts, and its expression is regulated in a concentration-dependent manner by lipopolysaccharide. In this study, induction of keratinocyte growth factor 1 protein expression was dependent on gingival fibroblast expression of membrane CD14 (mCD14) and Toll-like receptors 2 and 4. Lipopolysaccharides from Escherichia coli and Porphyromonas gingivalis induced membrane expression of CD14 at 1, 3, and 24 h. Specifically, lipopolysaccharide induced low mCD14 expression gingival fibroblasts to express mCD14 at a level consistent with that of high mCD14 expression cells. Functional studies with specific blocking antibodies for CD14 and Toll-like receptors 2 and 4 implicated all of these molecules in signal transduction. The rapid decrease in cell membrane expression of Toll-like receptors 2 and 4 after treatment with lipopolysaccharide was consistent with receptor internalization, and blocking of either of these receptors completely inhibited keratinocyte growth factor 1 protein expression. The transcription factors AP-1 and NF-kappaB were involved in lipopolysaccharide induction of keratinocyte growth factor 1 mRNA and protein expression. These results suggest that lipopolysaccharide may induce proliferation of periodontal epithelial cells by upregulating keratinocyte growth factor 1 expression via the CD14 and Toll-like receptor signaling pathway.

Keratinocyte growth factor (KGF)-1 and -2 protein and gene expression in human gingival fibroblasts.

The onset and progression of periodontal disease is associated with significant changes in the epithelial component of the attachment complex. From the early to the advanced stages of periodontal disease increased epithelial cell proliferation, migration and invasion into the surrounding connective tissue takes place. Concomitantly there is a significant increase in proinflammatory cytokine expression in periodontal tissue and quantitative and qualitative changes in the subgingival microflora, including an increase in gram-negative microorganisms. One of the most significant virulence factors of these bacteria is lipopolysaccharide (LPS) connected to the outer membrane. Two important growth factors controlling epithelial behavior are Keratinocyte Growth Factor-1 (KGF-1) and -2 (KGF-2). Connective tissue cells express these growth factors, but only epithelial cells respond to them. We studied the effect of proinflammatory cytokines and LPS on gingival fibroblast expression of KGF-1 and KGF-2 in vitro. Gingival fibroblasts were found to express KGF-1 and -2 in culture but only KGF-1 protein and gene expression was stimulated by serum, in a concentration-dependent manner by proinflammatory cytokines IL-1alpha, IL-1beta, TNF-alpha and IL-6 and LPS isolated from Porphyromonas gingivalis and Escherichia coli. The local increase in proinflammatory cytokine expression and the accumulation of LPS in disease sites may therefore stimulate gingival fibroblast expression of KGF-1. We hypothesize that this local increase in KGF-1 expression may, via a paracrine mechanism, stimulate local epithelial cell proliferation, migration and invasion during the onset and progression of periodontitis.