TGF-BETA INDUCED HYALURONAN SYNTHESIS IN ORBITAL FIBROBLASTS INVOLVES PROTEIN KINASE C BETAII ACTIVATION IN VITRO

Graves' ophthalmopathy is accompanied by hyaluronan (HA) accumulation in the orbital space and infiltration of immunocompetent cells and cytokines, including IFN-g, IL-1b, and TGF-b. We examined the signal transduction pathways by which TGF-b induces HA synthesis in normal orbital fibroblasts , orbital fibroblasts from patients with Graves' ophthalmopathy, and abdominal fibroblasts. Calphostin C inhibited the stimulation of HA synthesis by TGF-b. Phorbol 12-myristate 13-acetate (PMA) activation of PKC stimulated HA production. The effects of TGF-b and PMA were not synergistic. Stimulation by TGF-b and PMA were dependent on protein synthesis and their effects were inhibited by cycloheximide. Since TGF-b- induced HA synthesis was inhibited by BAPTA or by PKC inhibitors, a calcium-dependent PKC was most likely involved. The PKA inhibitor H-89 enhanced TGF-b- and PMA-induced HA synthesis, thus showing that communication between the PKA and PKC pathways was evident. TGF-b stimulated the translocation of PKCbII to the cell membrane. PKCbII, a key enzyme in the regulation of HA synthesis by TGF-b, might be an appropriate target for therapeutic compounds to be used to treat Graves' ophthalmopathy accompanied by inflammation

Matrix metalloproteases and tissue inhibitors of metalloproteinases in medial plica and pannus-like tissue contribute to knee osteoarthritis progression.

Osteoarthritis (OA) is characterized by degradation of the cartilage matrix, leading to pathologic changes in the joints. However, the pathogenic effects of synovial tissue inflammation on OA knees are not clear. To investigate whether the inflammation caused by the medial plica is involved in the pathogenesis of osteoarthritis, we examined the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the medial plica and pannus-like tissue in the knees of patients with medial compartment OA who underwent either arthroscopic medial release (stage II; 15 knee joints from 15 patients) or total knee replacement (stage IV; 18 knee joints from 18 patients). MMP-2, MMP-3, MMP-9, IL-1β, and TNF-α mRNA and protein levels measured, respectively, by quantitative real-time PCR and Quantibody human MMP arrays, were highly expressed in extracts of medial plica and pannus-like tissue from stage IV knee joints. Immunohistochemical staining also demonstrated high expression of MMP-2, MMP-3, and MMP-9 in plica and pannus-like tissue of stage IV OA knees and not in normal cartilage. Some TIMP/MMP ratios decreased significantly in both medial plica and pannus-like tissue as disease progressed from stage II to stage IV. Furthermore, the migration of cells from the pannus-like tissue was enhanced by IL-1β, while plica cell migration was enhanced by TNF-α. The results suggest that medial plica and pannus-like tissue may be involved in the process of cartilage degradation in medial compartment OA of the knee

Interleukin-1β induces CXCR3-mediated chemotaxis to promote umbilical cord mesenchymal stem cell transendothelial migration

Abstract Background Mesenchymal stem cells (MSCs) are known to home to injured and inflamed regions via the bloodstream to assist in tissue regeneration in response to signals of cellular damage. However, the factors and mechanisms that affect their transendothelial migration are still unclear. In this study, the mechanisms involved in interleukin-1β (IL-1β) enhancing the transendothelial migration of MSCs were investigated. Methods Immunofluorescence staining and Western blotting were used to observe IL-1β-induced CXC chemokine receptor 3 (CXCR3) expression on MSCs. Quantitative real-time PCR and ELISA were used to demonstrate IL-1β upregulated both chemokine (C-X-C motif) ligand 9 (CXCL9) mRNA and CXCL9 ligand secretion in human umbilical vein endothelial cells (HUVECs). Monolayer co-cultivation, agarose drop chemotaxis, and transwell assay were conducted to investigate the chemotaxis invasion and transendothelial migration ability of IL-1β-induced MSCs in response to CXCL9. Results In this study, our immunofluorescence staining showed that IL-1β induces CXCR3 expression on MSCs. This result was confirmed by Western blotting. Following pretreatment with protein synthesis inhibitor cycloheximide, we found that IL-1β induced CXCR3 on the surface of MSCs via protein synthesis pathway. Quantitative real-time PCR and ELISA validated that IL-1β upregulated both CXCL9 mRNA and CXCL9 ligand secretion in HUVECs. In response to CXCL9, chemotaxis invasion and transendothelial migration ability were increased in IL-1β-stimulated MSCs. In addition, we pretreated MSCs with CXCR3 antagonist AMG-487 and p38 MAPK inhibitor SB203580 to confirm CXCR3-CXCL9 interaction and the role of CXCR3 in IL-1β-induced chemotaxis invasion and transendothelial migration. Conclusion We found that IL-1β induces the expression of CXCR3 through p38 MAPK signaling and that IL-1β also enhances CXCL9 ligand secretion in HUVECs. These results indicated that IL-1β promotes the transendothelial migration of MSCs through CXCR3-CXCL9 axis. The implication of the finding could enhance the efficacy of MSCs homing to target sites

IL-1β stimulated human umbilical cord mesenchymal stem cells ameliorate rheumatoid arthritis via inducing apoptosis of fibroblast-like synoviocytes

Abstract Rheumatoid arthritis (RA) is characterized by synovial proliferation and lymphocyte accumulation leading to progressive damage of the periarticular bone and the articular cartilage. The hyperplasia of the synovial intima lining mainly consists of fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) which exhibit apoptosis-resistance, hyper-proliferation, and high invasiveness. The therapeutic efficacy of mesenchymal stem cells (MSCs) treatment in RA has been shown to be due to its immuno-regulatory ability. However, the exact factors and mechanisms involved in MSCs treatment in RA remain unclear. In this study, TRAIL receptor-Death receptor 4 (DR4), DR5, and LFA-1 ligand-intercellular adhesion molecule-1 (ICAM-1) were upregulated in IL-1β-stimulated HFLS-RA. We demonstrated that the total cell number of IL-1β-stimulated hUCMSCs adhering to IL-1β-stimulated HFLA-RA increased via LFA-1/ICAM-1 interaction. Direct co-culture of IL-1β-stimulated hUCMSCs with IL-1β-stimulated HFLS-RA increased the apoptosis of HFLS-RA. RA symptoms in the CIA mouse model improved after administration of IL-1β-stimulated hUCMSCs. In conclusion, IL-1β-stimulated hUCMSCs adhering to HFLS-RA occurred via LFA-1/ICAM-1 interaction, apoptosis of HFLS-RA was induced via TRAIL/DR4, DR5 contact, and RA symptoms and inflammation were significantly improved in a CIA mouse model. The results of this study suggest that IL-1β-stimulated hUCMSCs have therapeutic potential in RA treatment

Lipopolysaccharide Induces Degradation of Connexin43 in Rat Astrocytes via the Ubiquitin-Proteasome Proteolytic Pathway

<div><p>The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation<b>.</b> Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway.</p></div

Migration assay of cells from medial plica and pannus-like tissue after treatment with IL-1β or TNF-α with or without a cytokine inhibitor.

<p>The medial plica (left panels) and pannus (right panels) cells were treated for 24 h with (A) medium or 50 ng/ml of IL-1β either alone or together with 100 ng/ml of IL-1β antagonist (RA) or (B) medium or 100 ng/ml of TNF-α either alone or together with 100 ng/ml of Enbrel^®, then the cells were fixed and stained with crystal violet and the OD₅₉₅ measured. Each bar represents the mean ± standard deviation for 6 separate experiments (* p < 0.05 versus control, #p < 0.05 versus cytokine alone).</p

MMP and TIMP levels in lysates of medial plica and pannus-like tissue from OA patients.

<p>(A) Measurement of human MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1, TIMP-2, and TIMP-4 using a Quantibody^® human MMP ELISA array. The data are the mean ± standard deviation (SD) (N = 15), *P < 0.05. (B) TIMP/MMP ratios in lysates of stage II and stage IV plica (upper panels) and pannus (lower panels) calculated from the Quantibody® human MMP array results. The data are the mean ± SD (N = 15), * P < 0.05.</p

IL-1β and TNF-α mRNA levels in the medial plica and pannus-like tissue in stage IV OA knees and normal cartilage.

<p>Real-time polymerase chain reaction analysis of IL-1β (left panel) and TNF-α (right panel) mRNA levels in the medial plica and pannus-like tissue in stage IV osteoarthritis knees and normal cartilage expressed relative to levels in normal cartilage. Each bar represents the mean ± standard deviation for 15 samples. * P < 0.05. </p

Immunohistochemical staining for MMP-2, MMP-3, or MMP-9 in the medial plica and pannus tissue of stage IV OA patients and normal cartilage.

<p>The panels on the left are negative controls using normal rabbit IgG instead of primary antibody. The bars represent 10 μm. The results are typical of those seen for all 18 OA knees.</p

IL-1β-Induced Matrix Metalloprotease-1 Promotes Mesenchymal Stem Cell Migration via PAR1 and G-Protein-Coupled Signaling Pathway

Mesenchymal stem cells (MSCs) are known for homing to sites of injury in response to signals of cellular damage. However, the mechanisms of how cytokines recruit stem cells to target tissue are still unclear. In this study, we found that the proinflammation cytokine interleukin-1β (IL-1β) promotes mesenchymal stem cell migration. The cDNA microarray data show that IL-1β induces matrix metalloproteinase-1 (MMP-1) expression. We then used quantitative real-time PCR and MMP-1 ELISA to verify the results. MMP-1 siRNA transfected MSCs, and MSC pretreatment with IL-1β inhibitor interleukin-1 receptor antagonist (IL-1RA), MMP tissue inhibitor of metalloproteinase 1 (TIMP1), tissue inhibitor of metalloproteinase 2 (TIMP2), MMP-1 inhibitor GM6001, and protease-activated receptor 1 (PAR1) inhibitor SCH79797 confirms that PAR1 protein signaling pathway leads to IL-1β-induced cell migration. In conclusion, IL-1β promotes the secretion of MMP-1, which then activates the PAR1 and G-protein-coupled signal pathways to promote mesenchymal stem cell migration