CCN6 enhances ICAM-1 expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. CCN6 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, and Nov) family of matricellular proteins. However, the effects of CCN6 on human chondrosarcoma cells are largely unknown. In this study, we found that CCN6 increased the migration and the expression of intercellular adhesion molecule-1 (ICAM-1) in human chondrosarcoma cells. αvβ3 and αvβ5 integrin monoclonal antibody and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the CCN6-induced increase of the migration and ICAM-1 up-regulation of chondrosarcoma cells. CCN6 stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK, and extracellular signal-regulated kinase (ERK). In addition, activator protein-1 (AP-1) inhibitors suppressed the cell migration and ICAM-1 expression enhanced by CCN6. Moreover, CCN6 increased AP-1 luciferase activity and binding of c-Jun to the AP-1 element on the ICAM-1 promoter. Taken together, our results indicate that CCN6 enhances the migration of chondrosarcoma cells by increasing ICAM-1 expression through the αvβ3 and αvβ5 integrin receptor, FAK, MEK, ERK, c-Jun, and AP-1 signal transduction pathway.     

The Increased Expression of HLA-DR and ICAM-1 Molecules by Human Bronchial Epithelial Cells, Induced by Activated Mononuclear Cells, is Downregulated by Nedocromil Sodium

To test the hypothesis that mononuclear cell products could increase the expression of HLA-DR and ICAM-1 molecules in bronchial epithelial cells (BECs), subconfluent cultures of human BECs, obtained from surgically resected bronchi, were incubated with PHA-activated blood mononuclear cell conditioned media (BCM-CM) or recombinant IFN-gamma. The presence of HLA-DR and ICAM-1 molecules on BECs was then evaluated by specific antibody staining and flow-cytometry analysis. The addition to BEC cultures of different concentrations of PHA-stimulated BMC-CM, or of IFN-gamma induced a dosedependent increase of HIA-DR and ICAM-1 expression, while no effect was observed with unstimulated BMC-CM. The ability of nedocromil sodium and, as control, of dexamethasone, to prevent the upregulation of HLA-DR and ICAM-1 expression on BECs was then tested. Increasing concentrations (10(-7) to 10(-4) M) of nedocromil significandy inhibited HLA-DR and ICAM-1 expression by BECs in a dose-dependent fashion. A similarly dose-dependent inhibitory effect was also observed with dexamethasone, which, however, was less active than nedocromil on HL-ADR expression and more active on ICAM-1 expression. Finally, nedocromil and dexamethasone showed a significant synergistic effect on the expression of both cell surface molecules at the lowest concentrations tested.     

Immunohistochemical evidence for loss of ICAM-1 by alveolar epithelial cells in pulmonary fibrosis

ICAM-1 is an intercellular adhesion molecule of the immunoglobulin supergene family involved in adherence of leukocytes to the endothelium and in leukocytic accumulation in pulmonary injury. In the current study, the antigen retrieval technique was used to detect ICAM-1 immunohistochemically in paraffin sections of lungs from human, mouse and rat as well as in bleomycin- or radiation-induced fibrotic lungs from rat and human. In normal lung tissue, the expression of ICAM-1 on alveolar type I epithelial cells is stronger than on alveolar macrophages and on endothelial cells. Preembedding immuno-electron microscopy of normal rat, mouse and human lung samples revealed sclective ICAM-1 expression on the surface of type I alveolar epithelial cells and, to a lesser extent, on the pulmonary capillary endothelium and on alveolar macrophages. In fibrotic specimens, both focal lack and strengthening of immunostaining on the surface of type I cells was found. Alveolar macrophages were found focally lacking ICAM-1 immunoreactivity. In some cases, rat type II pneumocytes exhibited positive immunoreactions for ICAM-1. Immunoelectron microscopy with preembedded rat lungs (bleomycin-exposed cases) confirmed the altered ICAM-1 distribution at the alveolar epithelial surface. In the alveolar fluid of fibrotic rat lungs, in contrast to that from untreated controls, soluble ICAM-1 was detected by western blot analysis.     

Requirement of species-specific interactions for the activation of human gamma delta T cells by pamidronate

Human gammadelta T cells bearing Vgamma2Vdelta2-TCR recognize various kinds of small nonpeptide Ags, and activation of them by a nitrogen-containing bisphosphonate Ag, pamidronate, requires Ag presentation by cells other than gammadelta T cells, including many human tumor cells. Present results demonstrated that tumor cell lines of nonhuman origins pulsed with pamidronate failed to activate human gammadelta T cells without exception, whereas most if not all human tumor cell lines could do so. Gammadelta T cells formed stable conjugates with pamidronate-pulsed human tumor cells and both conjugate formation and gammadelta T cell activation were inhibited significantly by anti-LFA-1 mAb, suggesting the requirement of LFA-1-mediated interaction with APC for efficient gammadelta T cell activation. Consistently, ICAM-1(low) tumor cell lines pulsed with pamidronate induced no or only weak activation of gammadelta T cells, whereas similarly treated ICAM-1(high) cell lines could activate them. One of the two ICAM-1(low) tumor cell lines pulsed with pamidronate induced strong gammadelta T cell activation after ICAM-1 gene transfer. However, another ICAM-1(low) human cell line as well as murine tumor cell lines pulsed with pamidronate remained totally defective in gammadelta T cell activation even after expression of human ICAM-1. These results suggested that activation of human gammadelta T cells by nonpeptide Ags required species-specific interactions in addition to LFA-1/ICAM-1-mediated cell adhesion with APC.     

Regulation of ICAM-1 mRNA stability by cycloheximide: Role of serine/threonine phosphorylation and protein synthesis

Cycloheximide is a protein synthesis inhibitor that superinduces the expression of many genes by preventing the degradation of otherwise labile mRNAs. In some genes this depends on the presence of the AUUUA destabilizing multimers in the 3′UTR. We examined the effect of cycloheximide on the murine intercellular adhesion molecule-1 (ICAM-1; CD54) gene expression in several cell lines including A20 (B cell lymphoma), T28 (T cell hybridoma), P388D1 (monocytic cell), SVEC4-10 (lymphoid endothelial cell), and ICAM-1-transfected murine fibroblast L cells. Cycloheximide was indeed able to dramatically increase the accumulation of ICAM-1 mRNA in all the cell lines examined except T28, and this seemed to be due to the stablization of the ICAM-1 mRNA as indicated by the half-life analysis. To determine whether this effect is dependent on the 3′UTR containing the AUUUA sequences, L cells were transfected with either the full-length ICAM-1 cDNA or a truncated form lacking the AUUUA sequences in the 3′UTR (ICAM-1Δ3). There was no discernible difference in the effect of cycloheximide on ICAM-1 mRNA accumulation or half-life between the two types of transfected cells. The effect of cycloheximide on ICAM-1 mRNA was markedly suppressed by serine/threonine (ser/thr) kinase inhibitors, H-7 and staurosporine, whereas the ser/thr phosphatase inhibitor, okadaic acid, augmented the cycloheximide effect. Inhibitors of protein tyrosine kinases and phosphatases had no effect. Unexpectedly, the level of cell surface ICAM-1 as well as de novo synthesis of ICAM-1 in SVEC4-10 and the ICAM-1-transfected L cells were also upregulated by cycloheximide, whereas the overall protein synthesis in these cells was profoundly inhibited, suggesting that ICAM-1 protein synthesis in these cells escapes the translational inhibition by cycloheximide. These results suggest that the stabilization of ICAM-1 mRNA by cycloheximide is independent of its translational inhibition and that ser/thr phosphorylation of unidentified protein(s) seems to play a crucial role in this effect. © 1995 Wiley-Liss, Inc.     

Induction by IL 1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1)

ICAM-1 is a cell surface glycoprotein originally defined by a monoclonal antibody (MAb) that inhibits phorbol ester-stimulated leukocyte aggregation. Staining of frozen sections and immunofluorescence flow cytometry showed intercellular adhesion molecule-1 (ICAM-1) is expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal center dendritic cells in tonsils, lymph nodes, and Peyer's patches. ICAM-1 staining on vascular endothelial cells is most intense in T cell areas in lymph nodes and tonsils showing reactive hyperplasia. ICAM-1 is expressed in low amounts on peripheral blood leukocytes. Phorbol ester-stimulated differentiation of myelomonocytic cell lines greatly increases ICAM-1 expression. ICAM-1 expression on dermal fibroblasts is increased threefold to fivefold by either interleukin 1 (IL 1) or interferon-gamma at 10 U/ml over a period of 4 or 10 hr, respectively. The induction is dependent on protein and mRNA synthesis and is reversible. ICAM-1 displays Mr heterogeneity in different cell types with a Mr of 97,000 on fibroblasts, 114,000 on the myelomonocytic cell line U937, and 90,000 on the B lymphoblastoid cell JY. ICAM-1 biosynthesis involves a Mr approximately 73,000 intracellular precursor. The non-N-glycosylated form resulting from tunicamycin treatment has a Mr of 55,000. ICAM-1 isolated from phorbol myristic acetate (PMA) stimulated U937 and from fibroblasts yields an identical major product of Mr = 60,000 after chemical deglycosylation. ICAM-1 MAb interferes with the adhesion of phytohemagglutinin blasts, and the adhesion of the cell line SKW3 to human dermal fibroblast cell layers. Pretreatment of fibroblasts but not lymphocytes with ICAM-1 MAb, and of lymphocytes but not fibroblasts with lymphocyte function-associated antigen 1 MAb inhibits adhesion. Intercellular adhesion is increased by prior exposure of fibroblasts to IL 1, and correlates with induction of ICAM-1.     

siRNA mediated inhibition of MMP-1 reduces invasive potential of a human chondrosarcoma cell line

Matrix metalloproteinases (MMPs) play a crucial role in tumor cell invasion and metastasis. Expression of MMP-1 has been reported as a prognostic predictor of recurrence in human chondrosarcoma, and studies using human chondrosarcoma cell lines indicate that MMP-1 expression levels correlate with in vitro invasiveness. These observations suggest that MMP-1 activity has a central role in cell egress from the primary tumor at an early step in the metastatic cascade. In this study, siRNA was used to investigate whether knock down of the MMP-1 gene could be used to inhibit invasiveness in a human chondrosarcoma cell line. The inhibitory effect of siRNA on endogenous MMP-1 gene expression and protein synthesis was demonstrated via RT-PCR, Northern blotting, Western blotting, collagenase activity assay, and an in vitro cell migration assay. The siRNA inhibited MMP-1 expression specifically, since it did not affect the expression of endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) nor other collagenases. Most importantly, the siRNA mediated reduction in MMP-1 expression correlated with a decreased ability of chondrosarcoma cells to invade a Type I collagen matrix. The reduction of invasive behavior demonstrated by human chondrosarcoma cells transfected with MMP-1 siRNA and the specificity of this inhibition supports the hypothesis that this metalloproteinase molecule is involved in initiation of chondrosarcoma metastasis.     

Stromal cell-derived factor-1 increase alphavbeta3 integrin expression and invasion in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. The stromal cell-derived factor 1 (SDF-1), constitutively secreted by human lung epithelium cells, has been shown to function in a key role for recruitment of neutrophils. Here, we found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of CXCR4 (SDF-1 receptor), which was higher than normal cartilage and human chondrocyte. SDF-1alpha and lung epithelium cells conditioned medium (LECM) induced the invasiveness of chondrosarcoma cells. SDF-1 siRNA inhibited LECM-induced invasion of chondrosarcoma cells and SDF-1alpha also directly induced the cell surface expression of alphavbeta3 but not alpha2beta1 and alpha5beta1 integrin. Activations of ERK and NF-kappaB pathways after SDF-1 treatment was demonstrated, and SDF-1alpha-induced expression of alphavbeta3 integrin and invasion activity was inhibited by the specific inhibitor and mutant of ERK and NF-kappaB cascades. Taken together, our results indicate that lung derived-SDF-1alpha enhances the invasiveness of chondrosarcoma cells by increasing alphavbeta3 integrin expression through the CXCR4/ERK/NF-kappaB signal transduction pathway.     

Regulation by retinoic acid of ICAM-1 expression on human tumor cell lines.

In a group of four human tumor cell lines comprising one melanoma, one glioma, one teratocarcinoma and one neuroblastoma, the expression of the intercellular adhesion molecule-1 (ICAM-1) was found to be significantly increased following treatment with 10 microM of all-trans retinoic acid. In the melanoma and glioma cell lines HS 294T and HS 683, greater than 90% of the cells reacted with the anti-ICAM-1 monoclonal antibody (mAb) CL203.4 in the absence of treatment. Retinoic acid increased the cell surface expression of the molecule by 2-fold. In the teratocarcinoma and neuroblastoma cell lines, TERA-2 and SK-N-SH, the constitutive expression of ICAM-1 was weak, the percentage of cells stained above the background being less than 25%. Retinoic acid induced ICAM-1 expression in greater than 80% of the cells and increased the levels of expression by 2.5 to 3-fold. Immunoprecipitation studies in biosynthetically labeled cells as well as RNase protection analysis confirmed that retinoic acid treatment increased the amount of ICAM-1 at both the protein and mRNA level. The induction or stimulation occurred within 24 h, was maximal after 4 days and reversible.     

Gastrin-releasing peptide mediates its morphogenic properties in human colon cancer by upregulating intracellular adhesion protein-1 (ICAM-1) via focal adhesion kinase

Gastrin-releasing peptide (GRP) and its receptor (GRPR) act as morphogens when expressed in colorectal cancer (CRC), promoting the assumption of a better differentiated phenotype by regulating cell motility in the context of remodeling and retarding tumor cell metastasis by enhancing cell-matrix attachment. Although we have shown that these processes are mediated by focal adhesion kinase (FAK), the downstream target(s) of GRP-induced FAK activation are not known. Since osteoblast differentiation is mediated by FAK-initiated upregulation of ICAM-1 (Nakayamada S, Okada Y, Saito K, Tamura M, Tanaka Y. J Biol Chem 278: 45368-45374, 2003), we determined whether GRP-induced activation of FAK alters ICAM-1 expression in CRC and, if so, determined the contribution of ICAM-1 to mediating GRP's morphogenic properties. Caco-2 and HT-29 cells variably express GRP/GRPR. These cells only express ICAM-1 when GRPR are present. In human CRC, GRPR and ICAM-1 are only expressed by better differentiated tumor cells, with ICAM-1 located at the basolateral membrane. ICAM-1 expression was only observed subsequent to GRPR signaling via FAK. To study the effect of ICAM-1 expression on tumor cell motility, CRC cells expressing GRP, GRPR, and ICAM-1 were cultured in the presence and absence of GRPR antagonist or monoclonal antibody to ICAM-1. CRC cells engaged in directed motility in the context of remodeling and were highly adherent to the extracellular matrix, only in the absence of antagonist or ICAM-1 antibody. These data indicate that GRP upregulation of ICAM-1 via FAK promotes tumor cell motility and attachment to the extracellular matrix.     

The effect of metronidazole on stimulation of adhesion molecule expression on the surface of vascular endothelial cells by Bacteroides fragilis endotoxins and enterotoxin]

The influence of metronidazole on the level of expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin on the surface of vascular endothelial cells activated with B. fragilis endotoxins and enterotoxin was examined. Three enterotoxigenic (ETBF) strains and one nonenterotoxigenic (NTBF) strain were used for lipopolysaccharide extraction. Enterotoxin was prepared from the culture supernatant of the reference B. fragilis ATCC 43858 strain. Expression of adhesion molecules on vascular endothelial cells (HMEC-1 cell line) was determined after their stimulation with bacterial compounds at the concentration of 10 micrograms/ml in the presence of metronidazole at the concentration of 4 micrograms/ml. Endothelial cells were activated for 4 hours (E-selectin expression) and for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with mouse, monoclonal antibodies against human ICAM-1, VCAM-1 and E-selectin. The results of experiments suggest, that metronidazole enhances the expression of examined adhesion molecules on endothelial cells. This antimicrobial agent causes some changes in the expression of endothelial ICAM-1, VCAM-1 and E-selectin stimulated by B. fragilis endotoxins and enterotoxin.     

ICAM-1 expression in vaginal cells as a potential biomarker for inflammatory response

This study aimed to elucidate the mechanisms that may lead to an efficient strategy to induce a suitable host response of the vaginal mucosa upon exposure to intravaginally delivered exogenous compounds. It was hypothesized that the upregulation of intercellular adhesion molecule (ICAM)-1 gene expression may reflect the inflammatory response evoked by exogenous compounds. Major emphasis was placed on ethylenediamine tetraacetic acid (EDTA) which was added as a synergistic agent to conventional spermicidal agents or anti-HIV drugs. The levels of ICAM-1 mRNA were examined as a surrogate marker for inflammatory response in human vaginal epithelial cells upon exposure to EDTA or interleukin (IL)-1β (i.e. positive control, 25 mM). The effects of estrogen on EDTA-induced ICAM-1 expression were also evaluated for the estrogen involvement in the inflammatory process of the vaginal mucosa. ICAM-1 expression in human vaginal cells (VK2/E6E7 cells) increased as EDTA concentration added to human vaginal cell lines increased. The effects of estrogen on EDTA-induced ICAM-1 expression in human vaginal epithelial cells were estrogen-concentration dependent; estrogen at lower concentrations (～1–10 nM) did not affect ICAM-1 expression, whereas estrogen at higher concentrations (～100 nM–1 µM) attenuated ICAM-1 expression. The influence of estrogen in ICAM-1 expression suggests the beneficial effects of estrogen on the regulation of vaginal homeostasis. Identification and quantification of specific surrogate markers for the inflammatory response evoked by exogenous compounds and their regulation by estrogen will lead to an efficient strategy against sexually transmitted diseases including AIDS.     

ICAM-1 expression in vaginal cells as a potential biomarker for inflammatory response

This study aimed to elucidate the mechanisms that may lead to an efficient strategy to induce a suitable host response of the vaginal mucosa upon exposure to intravaginally delivered exogenous compounds. It was hypothesized that the upregulation of intercellular adhesion molecule (ICAM)-1 gene expression may reflect the inflammatory response evoked by exogenous compounds. Major emphasis was placed on ethylenediamine tetraacetic acid (EDTA) which was added as a synergistic agent to conventional spermicidal agents or anti-HIV drugs. The levels of ICAM-1 mRNA were examined as a surrogate marker for inflammatory response in human vaginal epithelial cells upon exposure to EDTA or interleukin (IL)-1β (i.e. positive control, 25 mM). The effects of estrogen on EDTA-induced ICAM-1 expression were also evaluated for the estrogen involvement in the inflammatory process of the vaginal mucosa. ICAM-1 expression in human vaginal cells (VK2/E6E7 cells) increased as EDTA concentration added to human vaginal cell lines increased. The effects of estrogen on EDTA-induced ICAM-1 expression in human vaginal epithelial cells were estrogen-concentration dependent; estrogen at lower concentrations (∼1-10 nM) did not affect ICAM-1 expression, whereas estrogen at higher concentrations (∼100 nM-1 µM) attenuated ICAM-1 expression. The influence of estrogen in ICAM-1 expression suggests the beneficial effects of estrogen on the regulation of vaginal homeostasis. Identification and quantification of specific surrogate markers for the inflammatory response evoked by exogenous compounds and their regulation by estrogen will lead to an efficient strategy against sexually transmitted diseases including AIDS.     

p38 kinase and c-Jun N-terminal kinase oppositely regulates tumor necrosis factor alpha-induced vascular cell adhesion molecule-1 expression and cell adhesion in chondrosarcoma cells

We investigated signaling pathways leading to tumor necrosis factor (TNF) alpha-induced intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression in chondrosarcoma cells, and determined the functional significance of their expression by examining Jurkat T cell adhesion. TNFalpha induced VCAM-1 and ICAM-1 expression and Jurkat T cell binding. Antibody blocking assay indicated that VCAM-1 mediates TNFalpha-induced Jurkat T cell adhesion. TNFalpha caused activation of mitogen-activated protein (MAP) kinase subtypes, extracellular signal-regulated protein kinase, p38 kinase, and c-jun N-terminal kinase (JNK). ICAM-1 expression was not altered by the inhibition of MAP kinases. However, VCAM-1 expression and Jurkat T cell adhesion was blocked by the inhibition of p38 kinase, whereas inhibition of JNK enhanced VCAM-1 expression and cell adhesion without any modulation of NFkappaB activation. Our results, therefore, indicate that p38 kinase mediates TNFalpha-induced VCAM-1 expression and cell adhesion, whereas JNK suppresses VCAM-1 expression that is independent to NFkappaB activation.     

Mast cells are potent regulators of endothelial cell adhesion molecule ICAM-1 and VCAM-1 expression

To investigate the possible role of mast cells (MC) in regulating leukocyte adhesion to vascular endothelial cells (EC), microvascular and macrovascular EC were exposed to activated MC or MC conditioned medium (MCCM). Expression of intercellular and vascular adhesion molecules (ICAM-1 and VCAM-1) on EC was monitored. Incubation of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) with activated MC or MCCM markedly increased ICAM-1 and VCAM-1 surface expression, noted as éarly as 4 hr. Maximal levels were observed at 16 hr followed by a general decline over 48 hr. A dose-dependent response was noted using incremental dilutions of MCCM or by varying the number of MC in coculture with EC. At a ratio as low as 1:1,000 of MC:EC, increased ICAM-1 was observed. The ICAM-1 upregulation by MCCM was >90% neutralized by antibody to tumor necrosis factor alpha (TNF-α), suggesting that MC release of this cytokine contributes significantly to inducing EC adhesiveness. VCAM-1 expression enhanced by MCCM was partly neutralized (70%) by antibody to TNF-α; thus other substances released by MC may contribute to VCAM-1 expression. Northern blot analysis demonstrated MCCM upregulated ICAM-1 and VCAM-1 mRNA in both HDMEC and HUVEC. To evaluate the function of MCCM-enhanced EC adhesion molecules, T cells isolated from normal human donors were used in a cell adhesion assay. T-cell binding to EC was increased significantly after exposure of EC to MCCM, and inhibited by antibodies to ICAM-1 or VCAM-1. Intradermal injection of allergen in human atopic volunteers known to develop late-phase allergic reactions led to marked expression of both ICAM-1 and VCAM-1 at 6 hr, as demonstrated by immunohistochemistry. These studies indicate that MC play a critical role in regulating the expression of EC adhesion molecules, ICAM-1 and VCAM-1, and thus augment inflammatory responses by upregulating leukocyte binding. © 1995 Wiley-Liss Inc.     

Cytokine-induced enhancement of ICAM-1 expression results in increased vulnerability of tumor cells to monocyte-mediated lysis.

Pretreatment of the human melanoma cell line, A375, and the human colon carcinoma cell line, HT-29, with certain cytokines was found to increase the vulnerability of these cells to monocyte-mediated killing. This activity was found to correlate with increased expression of intercellular adhesion molecule-1 (ICAM-1) on the tumor cells and was blocked by anti-ICAM-1 antibodies. Both IFN-gamma and TNF induced large increases in the ICAM-1 expression on both cell lines and increased the susceptibility of the tumor cells to monocyte-mediated killing. IFN-alpha and IL-1 beta, however, induced only small increases in ICAM-1 expression and enhanced the lysis of the A375 cells but not the HT-29 cells by monocytes. These differences may be the result of a higher basal expression of ICAM-1 found on the A375 cells when compared with the HT-29 cells. These data indicate that regulation of ICAM-1 expression on tumor cells can alter the vulnerability of these cells to lysis by monocytes.