OBJECTIVETo investigate whether signaling through Toll-like receptor-2 (TLR-2) can affect the expression of some cytokines in human gingival fibroblasts.

METHODSThe gingival fibroblasts were isolated and cultured in vivo, divided into blank control group, lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg) group and Escherichia coli (Ec) group. mRNA expression levels were measured by real-time polymerase chain reaction (PCR). The protein expression levels were detected by the enzyme linked immunosorbent assay (ELISA). The data was statistically analyzed by SPSS16.0 software package.

RESULTSLPS from Pg could stimulate the expression of interleukin (IL)-6 and leukemia inhibitory factor (LIF) mRNA and protein, which reached the peak (5.87 ± 0.83) at 10 h, and the expression level increased with the increase of the Pg concentration. IL-11 or oncostatin-M (OSM) mRNA expression was not affected by LPS. After treated with Pg for 48 h, the protein expression of IL-6 and LIF was up-regulated, (962 ± 57) ng/L and (47 ± 18) ng/L respectively.

CONCLUSIONSSignaling through TLR-2 controls the expression of cytokines of IL-6 family in human gingival fibroblasts.

Adolescent ; Adult ; Cells, Cultured ; Child ; Dose-Response Relationship, Drug ; Escherichia coli ; chemistry ; Fibroblasts ; cytology ; metabolism ; Gingiva ; cytology ; Humans ; Interleukin-11 ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Leukemia Inhibitory Factor ; genetics ; metabolism ; Lipopeptides ; pharmacology ; Lipopolysaccharides ; administration & dosage ; isolation & purification ; pharmacology ; Oncostatin M ; genetics ; metabolism ; Porphyromonas gingivalis ; chemistry ; RNA, Messenger ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; agonists ; Young Adult

This study was aimed to investigate the hematopoietic growth factors expressed in human aorta-gonad-mesonephros (AGM)-derived stromal cells in vitro in order to provide the basic data for elucidating the role of AGM -derived-stromal cells in embryo-hematopoiesis and its hematopoietic suppoitive effect. RT-PCR was used to analyze the expression of IL-6, SCF, Flt3-L, oncostatin M (OSM), IL-3, TPO, M-CSF and LIF in human aorta-gonad-mesonephros-derived stromal cells (hAGMS1-S5) at mRNA level. IL-6, SCF and Flt3-L levels were detected in the supernatant of hAGMS1-S5 stromal cells by ELISA assay. Umbilical cord blood CD34(+) cells were cocultured with hAGMS1-S5 feeder cells, and hematopoietic cells were collected at day 14 for colony analysis in methylcellulose semisolid medium. The results showed that human aorta-gonad-mesonephros-derived stromal cells S1-S5 expressed IL-6, SCF, Flt3-L and OSM mRNA, but did not express IL-3, TPO, M-CSF and LIF mRNA. In the supernatant of hAGMS1-S5 cells, IL-6, SCF and Flt3-L could be detected by ELISA assay at different levels, while there was no significant difference between groups of hAGMS1-S5 (P > 0.05). When cocultured with umbilical cord blood CD34(+) cells, hAGMS1-S5 could support the expansion of CFU-GM, BFU-E, and CFU-Mix. The supportive effects of hAGM S1-S5 were significantly different (P < 0.05), hAGM S3 and S4 were better than hAGM S1, S2, and S5. It is concluded that detection of hematopoietic growth factors expressed in human aorta-gonad-mesonephros-derived stromal cells provided a solid foundation to elucidate the mechanism of hematopoiesis and the hematopoietic supportive effect of these stromal cells.
Aorta ; cytology ; embryology ; metabolism ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; metabolism ; Endothelial Cells ; cytology ; metabolism ; Gonads ; cytology ; embryology ; metabolism ; Hematopoiesis ; physiology ; Hematopoietic Stem Cells ; cytology ; Humans ; Interleukin-6 ; biosynthesis ; genetics ; Mesonephros ; cytology ; metabolism ; Oncostatin M ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Stromal Cells ; cytology ; metabolism ; fms-Like Tyrosine Kinase 3 ; biosynthesis ; genetics

PURPOSE: Our previous studies have shown that oncostatin M (OSM) promotes trophoblast invasion activity through increased enzyme activity of matrix metalloproteinase (MMP)-2 and -9. We further investigated OSM-induced intracellular signaling mechanisms associated with these events in the immortalized human trophoblast cell line HTR8/SVneo. MATERIALS AND METHODS: We investigated the effects of OSM on RNA and protein expression of MMP-2 and -9 in the first-trimester extravillous trophoblast cell line (HTR8/SVneo) via Western blot. The selective signal transducer and activator of transcription (STAT)3 inhibitor, stattic, STAT3 siRNA, and extracellular signal-regulated kinase (ERK) siRNA were used to investigate STAT3 and ERK activation by OSM. The effects of STAT3 and ERK inhibitors on OSM-induced enzymatic activities of MMP-2 and -9 and invasion activity were further determined via Western blot and gelatin zymography. RESULTS: OSM-induced MMP-2 and -9 protein expression was significantly suppressed by STAT3 inhibition with stattic and STAT3 siRNA silencing, whereas the ERK1/2 inhibitor (U0126) and ERK silencing significantly suppressed OSM-induced MMP-2 protein expression. OSM-induced MMP-2 and MMP-9 enzymatic activities were significantly decreased by stattic pretreatment. The increased invasion activity induced by OSM was significantly suppressed by STAT3 and ERK1/2 inhibition, though to a greater extent by STAT3 inhibition. CONCLUSION: Both STAT3 and ERK signaling pathways are involved in OSM-induced invasion activity of HTR8/SVneo cells. Activation of STAT3 appears to be critical for the OSM-mediated increase in invasiveness of HTR8/SVneo cells.
Blotting, Western ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Matrix Metalloproteinase 2/genetics/*metabolism ; Matrix Metalloproteinase 9/genetics/*metabolism ; Oncostatin M/genetics/*metabolism ; Phosphorylation/drug effects ; RNA, Messenger/metabolism ; RNA, Small Interfering ; STAT3 Transcription Factor/*metabolism ; Signal Transduction/*drug effects

BACKGROUND/AIMS: SKI306X, a mixed extract of three herbs, Clematis mandshurica (CM), Prunella vulgaris (PV), and Trichosanthes kirilowii (TK), is chondroprotective in animal models of osteoarthritis (OA). The objectives of this study were to investigate its effect on interleukin (IL)-1beta-induced degradation of glycosaminoglycan (GAG) and the basis of its action in human OA cartilage, as well as to screen for the presence of inhibitors of matrix metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 in SKI306X and its component herbs, as well as in fractions from SKI306X. METHODS: Human OA chondrocytes and cartilage explants were obtained during total knee replacements and incubated with IL-1beta +/- oncostatin M with or without SKI306X or its component herb extracts. GAG degradation was assayed in cartilage explants using a commercial kit. Expression of genes involved in cartilage destruction was measured by real-time polymerase chain reaction using chondrocyte RNA. SKI306X was fractionated by preparative liquid chromatography to test for the presence of inhibitors of MMP-13 and ADAMTS-4. RESULTS: SKI306X and PV inhibited IL-1beta-induced GAG release from cartilage explants, and SKI306X, CM, PV, and TK inhibited IL-1beta-induced MMP gene expression. Unexpectedly, SKI306X greatly stimulated IL-1beta + oncostatin M-induced ADAMTS-4 gene expression, probably due to its TK component. Some fractions of SKI306X also inhibited ADAMTS-4 activity. CONCLUSIONS: SKI306X and its herbal components inhibit GAG degradation and catabolic gene expression in human OA chondrocytes and cartilage explants. SKI306X likely also contains one or more ADAMTS-4 inhibitor.
ADAM Proteins/antagonists & inhibitors ; Cartilage, Articular/*drug effects/*metabolism ; Cells, Cultured ; Chondrocytes/drug effects/metabolism ; Down-Regulation/drug effects ; Drugs, Chinese Herbal/*pharmacology ; Glycosaminoglycans/*metabolism ; Humans ; Interleukin-1beta/metabolism ; Matrix Metalloproteinase 13/metabolism ; Matrix Metalloproteinase Inhibitors/pharmacology ; Oncostatin M/metabolism ; Osteoarthritis, Knee/drug therapy/genetics/metabolism ; Procollagen N-Endopeptidase/antagonists & inhibitors