SummaryOur previous study demonstrated that annexin A2 (ANX2) on cell surface could function as a mediator and stimulate tissue factor (TF) expression of monocytes by anti-b2-glycoprotein I/b2-glycoprotein I complex (anti-b2GPI/ b2GPI). However, ANX2 is not a transmembrane protein and lacks the intracellular signal transduction pathway. Growing evidence suggests that Toll-like receptor 4 (TLR-4) might act as an 'adaptor' for intracellular signal transduction in anti-b2GPI/b2GPI-induced TF expressing cells. In the current study, we investigated the roles of TLR-4 and its related molecules, myeloid differentiation protein 2 (MD-2) and myeloid differentiation factor 88 (MyD88), in anti-b2GPI/b2GPI-induced TF expressing human monocytic-derived THP-1 (human acute monocytic leukaemia) cells. The relationship of TLR-4 and ANX2 in this process was also explored. Along with TF, expression of TLR-4, MD-2 and MyD88 in THP-1 cells increased significantly when treated by anti-b2GPI (10 mg/ml)/b2GPI (100 mg/ml) complex. The addition of paclitaxel, which competes with the MD-2 ligand, could inhibit the effects of anti-b2GPI/b2GPI on TLR-4, MD-2, MyD88 and TF expression. Both ANX2 and TLR-4 in THP-1 cell lysates could bind to b2GPI that had been conjugated to a column (b2GPI-Affi-Gel). Furthermore, TLR-4, MD-2, MyD88 and TF expression was remarkably diminished in THP-1 cells infected with ANX2-specific RNA interference (RNAi) lentivirus (LV-RNAi-ANX2), in spite of treatment with a similar concentration of anti-b2GPI/b2GPI complex. These results indicate that TLR-4 and its signal transduction pathway contribute to anti-b2GPI/b2GPI-induced TF expression in THP-1 cells, and the effects of TLR-4 with ANX2 are tightly co-operative.
Our previous study has demonstrated that TF/FVIIa and protease-activated receptor 2 (PAR2) are closely related to the proliferation and migration of colon cancer cell line SW620. However, the detailed signaling cascades and underlying molecular mechanisms remain unclear. This study has investigated whether extracellular signal-regulated kinase 1 and 2 (ERK1/2) and nuclear factor kappaB (NF-κB) signaling pathways are involved in the events. The results revealed that PAR2-activating peptide (PAR2-AP) or FVIIa elicited time-dependent upregulation of ERK1/2 phosphorylation in SW620 cells, and the effect of FVIIa was significantly attenuated by anti-TF antibody. PAR2-AP or FVIIa also increased NF-κB (p65/RelA) levels among cell nuclear proteins and simultaneously decreased IκB-α levels in the cytoplasmic proteins. Such effects of FVIIa can be inhibited with anti-PAR2 or anti-TF antibodies. While ERK1/2 inhibitor (U0126) intervened with the regulatory effects of PAR2-AP and FVIIa on IκB-α/NF-κB (p65/Rel) expression in the cells, NF-κB inhibitor (PDTC) partially blocked the enhancing effects of PAR2-AP and FVIIa on the proliferating and migratory ability of SW620 cells. Furthermore, the regulatory effects of PAR2-AP and FVIIa on expressions of certain proteins (IL-8, caspase-7, and TF) were also significantly abolished by PDTC. Collectively, the data in this study suggest that the interaction between FVIIa and TF induces PAR2 activation, thereby triggers the ERK1/2 and IκB-α/NF-κB signal transduction pathway to regulate the gene expression of IL-8, TF, and caspase-7, and ultimately promotes SW620 cell proliferation and migration.
Background: Intrauterine adhesion (IUA) is the second leading cause of secondary infertility in women. Research has shown that stem cells can promote endometrial regeneration and that biomaterials are also helpful in tissue regeneration. Therefore, we compared the efficacy of a collagen scaffold combined with either human umbilical cord mesenchymal stem cells (hUCMSC) or estrogen for the treatment of IUA. Methods: The IUA-induced rats were injected with hUCMSCs or estrogen, and with a collagen scaffold. The endometrial glands and amount of fibrosis were assessed using hematoxylin and eosin and Masson staining. The extent of fibrosis and levels of regeneration-related cytokines were examined by real-time quantitative PCR, and the expression levels of the estrogen receptor, KI67 and cytokeratin were analyzed using an immunochemistry assay. In addition, human nuclear antigen (HuNu) and vimentin were examined by immunofluorescence microscopy. Results: The collagen scaffold administered with hUCMSCs markedly increased the number of endometrial glands and reduced the area of fibrosis compared with either the collagen scaffold or hUCMSCs alone. In addition, the collagen scaffold with hUCMSCs significantly regulated the expression levels of fibrosis, estrogen, and differentiation-related genes relative to the collagen scaffold or hUCMSCs alone. Furthermore, the hUCMSCs alone or in combination with the collagen scaffold increased the expression of HuNu and vimentin in the IUA-induced rat model. In addition, protein levels of the p-transcriptional co-activator with PDZ-binding motif, stromal cell-derived factor-1, and C-X-C chemokine receptor type 4 were upregulated in the group that received the collagen scaffold in combination with hUCMSCs. Conclusion: Our results suggest that the combination of the collagen scaffold with hUCMSCs may be an alternative approach for treating IUA.
Our previous study has shown that Toll-like receptor 4 (TLR4) and its signalling pathway contribute to anti-β₂-glycoprotein I/β₂-glycoprotein I (anti-β₂GPI/β₂GPI)-induced tissue factor (TF) expression in human acute monocytic leukaemia cell line THP-1 and annexin A2 (ANX2) is involved in this pathway. However, its downstream molecules have not been well explored. In this study, we have established that interleukin-1 receptor-associated kinases (IRAKs) and tumour necrosis factor receptor-associated factors (TRAFs) are crucial downstream molecules of anti-β₂GPI/β₂GPI-induced TLR4 signaling pathway in THP-1 cells and explored the potential mechanisms of their self-regulation. Treatment of THP-1 cells with anti-β₂GPI/β₂GPI complex induced IRAKs and TRAFs expression and activation. Anti-β₂GPI/β₂GPI complex firstly induced expression of IRAK4 and IRAK1, then IRAK1 phosphorylation and last IRAK3 upregulation. In addition, anti-β₂GPI/β₂GPI complex simultaneously and acutely enhanced mRNA levels of TRAF6, TRAF4 and zinc finger protein A20 (A20), while chronically increased A20 protein level. Moreover, anti-β₂GPI/β₂GPI complex-induced IRAKs and TRAFs expression and activation were attenuated by knockdown of ANX2 by infection with ANX2-specific RNA interference lentiviruses (LV-RNAi-ANX2) or by treatment with paclitaxel, which inhibits TLR4 as an antagonist of myeloid differentiation protein 2 (MD-2) ligand. Furthermore, both IRAK1/4 inhibitor and a specific proteasome inhibitor MG-132 could attenuate TRAFs expression as well as TF expression induced by anti-β₂GPI/β₂GPI complex. In conclusion, our results indicate that IRAKs and TRAFs play important roles in anti-β₂GPI/β₂GPI-stimulated TLR4/TF signaling pathway in THP-1 cells and contribute to the pathological processes of antiphospholipid syndrome (APS).
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