A factor produced by human cells in vitro that changes HeLa cell colonial morphology

Summary Colonies of HeLa cells cultured in media supplemented with human or bovine serum or both can be morphologically described as three types: diffuse, intermediate, and compact, with their modal distribution depending on the serum or sera added to the growth medium. We have observed that for a particular medium or serum system, the percentage of compact colonies remains fairly constant under normal culture conditions, 0.2%, whereas the diffuse and intermediate colonies vary over a much wider range. The presence of certain substances as trypsin, heparin and Darvan in the medium favor the increase of compact colonies at the expense of other types. Furthermore, we have discovered that colonial morphology is influenced by cocultivation of the HeLa cells with human fibroblastlike cells, the compact colonies increasing as the density of the fibroblast element introduced into the mixed cultures is increased. Subsequent investigation revealed that conditioned medium from confluent fibroblast and HeLa cell cultures contained a factor(s), that significantly increased the percentage of compact colonies. The factor is nondialyzable, heat-stable and can be neutralized by serum. Recorded in this presentation are preliminary observations on the kinetics of colony formation and the interaction among the three HeLa cell colony types, the diffuse, the intermediate, and the compact. The factor's effect on HeLa cell colonial morphology is time dependent and rapidly reversed if the factor(s) is removed and fresh medium added.     

Retracted: The promoting effect of MMP13 on mediating the development of HFLS-RA by the target of miR-19a through IL-17 signaling pathway

By investigating the expression profiles of miR-19a and metalloproteinases (MMP13) in human fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) and HFL cells lines, this study intends to confirm the directly target connection between them and reveal the effect of suppressing MMP13 on HLFS-RA migration, invasion and apoptosis. After screening the abnormal expressed messenger RNAs and microRNAs in synovial tissues of patients with RA, the underlying pathway was determined by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The HFLS-RA cell line was transfected for the following experiments with pcDNA3.1(+) served as vector. The directly target association between miR-19a and MMP13 was confirmed by Luciferase reporter assay. Microarray analysis suggested that MMP13 was upregulated while miR-19a was downregulated in HFLS of RA tissues compared with the healthy control group. MMP13 was related to many proteins in protein-protein interaction network, which might be the main influencing factor of RA. KEGG pathway analysis identified that interleukin (IL)-17 pathway was activated in the regulation of MMP13 in the development of RA. Through observing the alteration of luciferase activity, miR-19a could indeed bind to the 3′UTR of the downstream of MMP13, the target association was then confirmed. The proliferation and invasion of HFLS-RA were promoted by overexpressing MMP13 protein. miR-19a could function as a suppressor of MMP13 and thereby retard the severity of RA. The results showed that miR-19a could regulate the expression of MMP13 in HFLS-RA by mediating the proliferation and invasion of HFLS-RA through IL-17 signaling pathway, thereby participating in the degradation of chondrocytes in the progression of RA.     

TL1A increased IL-6 production on fibroblast-like synoviocytes by preferentially activating TNF receptor 2 in rheumatoid arthritis

TNF-like protein 1A (TL1A), a member of tumor necrosis factor family, recognized as a ligand of death receptor 3 (DR3) and decoy receptor 3 (DcR3). The interaction of TL1A and DR3 may participate in the pathogenesis of some autoimmune diseases including rheumatoid arthritis (RA). Our previous results showed that high concentrations of TL1A could be found in synovial and serum in RA patients, and it was correlated with disease severity. In addition, TL1A could promote Th17 differentiation induced by TGF-β and IL-6 and increased the production of IL-17A. In the present study, we found that TL1A could promote the expression of IL-6 on fibroblast-like synoviocytes (FLS) of RA patients via NF-κB and JNK signaling pathway. TL1A-stimulated FLS increased the percentage of Th17 of peripheral blood mononuclear cells (PBMC) in RA via the production of IL-6, a critical cytokine involved in the differentiation of Th17. Moreover, the blocking of tumor necrosis factor receptor 2 (TNFR2) decreased TL1A-stimulated IL-6 production by RA FLS. Our results suggest that TL1A was capable of acting on RA FLS to elevate IL-6 expression, which promoted the production of Th17. More importantly, we showed that TL1A could influence RA FLS through binding to TNFR2 rather than DR3 on FLS, which indicated that the treatment of TNF inhibitors not only blocked the TNF but also suppressed the TL1A in RA patients.     

Autophagy protects against redox-active trace metal-induced cell death in rabbit synovial fibroblasts through Toll-like receptor 4 activation

Reactive oxygen species (ROS) are implicated to play a role in initiating rheumatoid arthritis (RA) pathogenesis. We have investigated the mechanism(s) by which essential redox-active trace metals (RATM) may induce cell proliferation and cell death in rabbit synovial fibroblasts. These fibroblast-like synovial (FLS) cells, which express Toll-like receptor 4 (TLR4), were used as a model system that plays a role in potentially initiating RA through oxidative stress. Potassium peroxychromate (PPC, [Cr⁵⁺]), ferrous chloride (FeCl₂, [Fe²⁺]), and cuprous chloride (CuCl, [Cu⁺]) in the indicated valency states were used as exogenous pro-oxidants that can induce oxidative stress through TLR4 coupled activation that also causes HMGB1 release. We measured the proliferation index (PI) of FLS, and examined the effect of RATM oxidants on apoptosis and autophagy by fluorescence cell-sorting flow cytometry (FC). Cell cycle was analysed by FC and autophagy-related protein expression levels were measured by western blot. Our data showed that as RATM as prooxidants increased intracellular ROS (iROS) that can induce oxidative stress. Whereas iROS increased PI in FLS, these reactive species also protected cells against apoptosis by inducing autophagy. Our results indicate that ROS/TLR4-coupled activation may contribute to the pathogenesis of RA in FLS by induction of autophagy. The signalling pathway by which inflammation and its tissue destructive sequel may occur in RA underlies the need for developing therapeutic agents that can inhibit release of tissue-damaging high mobility group box 1 (HMGB1), cytokines, and possess both trace metal chelating capacity and oxidant scavenging properties in a directed combinatorial therapy for RA.     

Expression and localization of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in murine and human placentas.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is one of the scavenger receptors that recognizes oxidized low-density lipoprotein as a major ligand. The placenta is a major source of prooxidant during pregnancy, and the level of placental oxidative stress increases rapidly at the end of the first trimester and tapers off later in gestation. In our study, we evaluated placental expression of LOX-1 during different gestational stages in mice and humans. We used immunohistochemistry and ISH to identify LOX-1-expressing cells in murine and human placentas. In both species, higher expression of LOX-1 mRNA during early to midgestational stages compared with late gestation-corresponding to the increased oxidative stress in early pregnancy-was shown by real-time RT-PCR. In murine placenta, we showed that LOX-1-expressing cells were fibroblast-like stromal cells in metrial glands and decidua basalis and that they were glycogen trophoblast cells in the junctional and labyrinth zones. In the human, LOX-1 expression was detected in villous cytotrophoblasts in both first trimester and term placentas. These localization patterns of LOX-1 in murine and human placentas suggest the possible involvement of LOX-1 in high oxidative stress conditions of pregnancy.     

Redox-active trace metal-induced release of high mobility group box 1(HMGB1) and inflammatory cytokines in fibroblast-like synovial cells is Toll-like receptor 4 (TLR4) dependent

Rheumatoid arthritis (RA) is a chronic autoimmune systemic inflammatory disease that is characterized by synovial inflammation and bone erosion. We have investigated the mechanism(s) by which essential trace metals may initiate and propagate inflammatory phenotypes in synovial fibroblasts. We used HIG-82, rabbit fibroblast-like synovial cells (FLS), as a model system for potentially initiating RA through oxidative stress. We used potassium peroxychromate (PPC, Cr⁺⁵), ferrous chloride (FeCl_2, Fe⁺²), and cuprous chloride (CuCl, Cu⁺) trace metal agents as exogenous pro-oxidants. Intracellular ROS was quantified by fluorescence microscopy and confirmed by flow cytometry (FC). Protein expression levels were measured by western blot and FC, while ELISA was used to quantify the levels of cytokines. Trace metal agents in different valence states acted as exogenous pro-oxidants that generate reactive oxygen species (ROS), which signal through TLR4 stimulation. ROS/TLR4- coupled activation resulted in the release of HMGB1, TNF-α, IL-1β, and IL-10 in conjunction with upregulation of myeloid-related protein (MRP8/14) inflammatory markers that may contribute to the RA pathophysiology. Our results indicate that oxidant-induced TLR4 activation can release HMGB1 in combination with other inflammatory cytokines to mediate pro-inflammatory actions that contribute to RA pathogenesis. The pathway by which inflammatory and tissue erosive changes may occur in this model system possibly underlies the need for functioning anti-HMGB1-releasing agents and antioxidants that possess both dual trace metal chelating and oxidant scavenging properties in a directed combinatorial therapy for RA.     

Increased lymphocytic aminopeptidase N/CD13 promoter activity after cell‐cell contact

Aminopeptidase N (APN)/CD13 is a transmembrane ectoenzyme expressed on a wide variety of cells. With respect to haematopoietic cells, APN/CD13 has been considered specific for the myeloid lineage, because granulocytes and monocytes/macrophages, but not lymphocytes of peripheral blood, show a surface expression of CD13 antigen. However, we could recently show that cell‐cell contact of lymphocytes with endothelial cells, monocytes, and fibroblast‐like synoviocytes (SFCs) results in an increase of steady‐state APN/CD13 mRNA and a rapid expression of cell‐surface protein on the lymphocytes. In this study using the Dual‐Luciferase reporter assay, we demonstrate that interaction of the T‐cell line Jurkat with SFCs results in a higher activity of the APN/CD13 myeloid promoter in T cells. An enhancer located between the myeloid and epithelial APN/CD13 promoter increases the response of the promoter to the cell‐cell contact‐induced expression of APN/CD13 in lymphocytes. Adhesion of lymphocytes to extracellular matrix did not result in increased promoter activity. The lymphocytic promoter response induced by direct cell‐cell contact with SFCs is not affected by mutations of a proximal promoter element (nucleotides −48 to −35), which has a possible functional role in the basal APN/CD13 gene expression in lymphocytes. Upregulated peptidase‐promoter activity via cell‐cell contact shown in this study for the first time is discussed as a general mechanism in peptidase induction. J. Cell. Biochem. 80:115–123, 2000. © 2000 Wiley‐Liss, Inc.     

Human platelet lysate enhances the proliferative activity of cultured human fibroblast-like cells from different tissues

Several studies have shown the presence of fibroblast-like cells in the stromal fraction of different tissues with a high proliferative and differentiation potential. Platelet alpha granules contain growth factors released into the environment during activation. The effects of different supplements for culture medium (human serum, bovine serum and platelet lysate) on cultured human fibroblast-like cells from bone marrow, adipose tissue, trabecular bone and dental pulp have been compared. Expression of typical stromal and hematopoietic markers was analyzed and proliferative rates were determined. Flow cytofluorometry showed a homogenous pattern in serial-passaged cells, with a high level of stromal cell-associated markers (CD13, CD90, CD105). The presence of platelet lysate in culture media increased the number of cell generations obtained regardless of cell source. This effect was serum-dependent. Cell-based therapies can benefit by the use of products from human origin for “ex vivo” expansion of multipotent cells.