Macrophage-derived IL-1beta stimulates Wnt signaling and growth of colon cancer cells: a crosstalk interrupted by vitamin D3.

Tumor-associated macrophages mediate the link between inflammation and cancer progression. Here, we showed that macrophage-derived soluble factors induce canonical Wnt signaling in colon cancer cells and promote their growth. Tumor cells induced the release of interleukin (IL)-1beta from macrophages, which induced phosphorylation of GSK3beta, stabilized beta-catenin, enhanced T-cell factor (TCF)-dependent gene activation and induced the expression of Wnt target genes in tumor cells. Neutralization experiments using anti-IL-1beta-specific antibodies, or silencing of IL-1beta in THP1 macrophages, showed that IL-1beta was required for macrophages to induce Wnt signaling and to support the growth of tumor cells. Constitutive activation of signal transducer and activator of transcription (STAT)1 in THP1 macrophages was essential for the induction of IL-1beta and thus for the activation of beta-catenin signaling in tumor cells. Vitamin D3, an effective chemopreventive agent, interrupted this crosstalk by blocking the constitutive activation of STAT1 and the production of IL-1beta in macrophages, and therefore-in a vitamin D receptor-dependent manner-inhibited the ability of macrophages to activate Wnt signaling in colon carcinoma cells. Our data therefore established that vitamin D3 exerts its chemopreventive activity by interrupting a crosstalk between tumor epithelial cells and the tumor microenvironment.

STAT1-independent inhibition of cyclooxygenase-2 expression by IFNgamma; a common pathway of IFNgamma-mediated gene repression but not gene activation.

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of prostaglandins, promotes the development of colorectal cancer, and is a key molecular target of non-steroidal anti-inflammatory drugs, compounds that reduce the relative risk of developing colon cancer. In this study, we showed that interferon gamma (IFNgamma) inhibits the expression of COX-2 protein in intestinal epithelial cells (IECs) through a pathway that requires Janus-activated kinase (JAK) activity. In contrast, we demonstrated that transcriptional inhibition of COX-2 by IFNbeta or IFNgamma occurs in cells with silenced signal transducer and activator of transcription 1 (STAT1) expression and that IFNs retained the ability to inhibit COX-2 transcription in cells with activated RasV12, in which IFNgamma failed to induce STAT1. Thus, unlike the activity of JAK, STAT1 is not required for the inhibition of COX-2 expression by IFNgamma. In contrast to COX-2, the activation of genes in response to IFNgamma, such as interferon regulatory factor-1, was severely impaired by both STAT1 silencing and by constitutive Ras signaling. To determine whether there is a general differential requirement for STAT1 in gene activation and gene repression in response to IFNgamma in intestinal cells, we performed genome-wide analysis of IFNgamma target genes in an IEC line in which STAT1 expression was silenced by small interfering RNA. The results confirmed that the activation of the majority of genes by IFNgamma required STAT1. In contrast, the repression of several genes, as we showed for COX-2 specifically, was largely unaffected in cells with silenced STAT1. Our results therefore demonstrate that in general gene activation by IFNgamma is more sensitive to STAT1 deficiency than gene repression, and suggest that IFNgamma activates and represses gene expression via distinct pathways that can be distinguished, at least in part, by their requirement for STAT1.

GM-CSF rescues TF-1 cells from growth factor withdrawal-induced, but not differentiation-induced apoptosis: the role of BCL-2 and MCL-1.

Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 promote the survival and stimulate the proliferation of haematopoietic cells. Using the GM-CSF-dependent TF-1 myeloid leukaemia cell line, the authors show that the endogenous levels of BCL-2 and MCL-1 are downregulated upon GM-CSF withdrawal, whereas the levels of BCL-x(L)and Bax are unchanged. Re-exposure of growth factor deprived cells to GM-CSF resulted in an early and transient increase in MCL-1 expression, and prolonged induction of BCL-2, which prevented apoptosis. In contrast, the expression of BCL-2 and MCL-1 were not modulated during TPA-induced differentiation of TF-1 cells, which was followed by apoptosis despite the presence of GM-CSF. TF-1 cells overexpressing BCL-2 or MCL-1 underwent delayed apoptosis upon growth factor withdrawal, but displayed no impaired apoptosis in response to TPA. Erythropoietin (Epo) induced the expression of BCL-2 and MCL-1 protein in TF-1 cells, however it did not support their long term proliferation, further demonstrating that upregulation of these anti-apoptotic genes is insufficient for the long term proliferation of TF-1 cells.

Sodium salicylate activates caspases and induces apoptosis of myeloid leukemia cell lines.

Nonsteroidal antiinflammatory agents (NSAIA) have been shown to exert potent chemopreventive activity against colon, lung, and breast cancers. In this study, we show that at pharmacological concentrations (1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce programmed cell death in several human myeloid leukemia cell lines, including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells undergo rapid apoptosis on treatment with Na-Sal, as indicated by increased annexin V binding capacity, cpp-32 (caspase-3) activation, and cleavage of poly (ADP-ribose) polymerase (PARP) and gelsolin. In addition, the expression of MCL-1, an antiapoptotic member of the BCL-2 family, is downregulated during Na-Sal-induced cell death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of PARP and gelsolin and rescues cells from Na-Sal-induced apoptosis. In addition, we show that Na-Sal accelerates growth factor withdrawal-induced apoptosis and synergizes with daunorubicin to induce apoptosis in TF-1 cells. Thus, our data provide a potential mechanism for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia.

The AML1/ETO fusion protein activates transcription of BCL-2.

The AML1 gene, located on chromosome 21, is involved in several distinct chromosomal translocations in human leukemia. In t(8;21) acute myelogenous leukemia (AML), the AML1 gene is juxtaposed to the ETO gene located on chromosome 8, generating an AML1/ETO fusion protein. Both AML1/ETO and the AML1 proteins recognize the same consensus DNA-binding motif (TGT/CGGT), which is found in the promoters of several genes involved in hematopoiesis. We found that two myeloid leukemia cell lines with the t(8;21) translocation, Kasumi and SKNO-1, have elevated levels of BCL-2 protein compared with other myeloid cell lines. In addition, we identified a consensus AML1 binding site in the BCL-2 promoter. Thus far, AML1/ETO has been shown to dominantly repress its target genes; however, we found that AML1/ETO activates transcription of the BCL-2 gene in U937 cells. This activation requires the presence of both the runt homology domain (rhd) and the C-terminal portion of AML1/ETO. We demonstrated sequence specific binding of both AML1A and AML1/ETO to the TGTGGT sequence in the BCL-2 promoter and showed that the AML1 binding site is required for responsiveness to AML1/ETO. Interestingly, AML1A and AML1B do not modulate the activity of the BCL-2 promoter. The elevated levels of BCL-2 in cells that express AML1/ETO may prolong their life span and contribute to the development of t(8;21) leukemia.

Promoter structure and transcriptional activation of the murine TSG-14 gene encoding a tumor necrosis factor/interleukin-1-inducible pentraxin protein.

Human TNF-stimulated gene 14 (TSG-14) encodes a secreted 42-kDa glycoprotein that shows significant homology to proteins of the pentraxin family, which includes the acute phase reactants C-reactive protein and serum amyloid P component. Levels of TSG-14 protein (also termed PTX-3) become elevated in the serum of mice and humans after injection with bacterial lipopolysaccharide, but in contrast to conventional acute phase proteins, the bulk of TSG-14 synthesis in the intact organism occurs outside the liver. In the present study we cloned and partially sequenced murine genomic TSG-14 DNA. Analysis of the coding region predicts a high degree of amino acid sequence homology between murine and human TSG-14 (88 and 75% identity in the first and second exons, respectively). The promoter of the TSG-14 gene lacks consensus sequences for either a TATA box or CCAAT box. Primer extension analysis and S1 nuclease protection assay revealed one major transcription start site, situated within a consensus sequence for an initiator element. Sequence analysis of a approximately 1.4-kilobase pair fragment of the 5'-flanking region of the TSG-14 gene revealed the presence of numerous potential enhancer binding elements, including six NF-IL6-like sites, four AP-1, one AP-2, one NF-kB, two Sp1, two interferon-gamma-activated sites (GAS), one Hox-1.3, and five binding sites for Ets family members. Transfection of BALB/c 3T3 cells with promoter DNA fragments linked to the luciferase reporter gene revealed that the 5'-flanking region of the TSG-14 gene comprises elements that can mediate a basal level of transcription and inducibility by TNF.

Activation of the TSG-6 gene by NF-IL6 requires two adjacent NF-IL6 binding sites.

Tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) encodes a protein expressed during inflammation. We have previously shown that transcription factors of the NF-IL6 and AP-1 families cooperatively modulate activation of the TSG-6 gene by TNF or interleukin 1 (IL-1) through a promoter region that contains an NF-IL6 site (-106 to -114) and an AP-1 element (-126 to -119). In this study we report the identification of an additional NF-IL6 site (NF-IL6*) located at positions -92 to -83. Footprinting and electrophoretic mobility shift assay suggested that NF-IL6 binds with higher affinity to the newly identified NF-IL6* site than to the earlier identified promoter-distal NF-IL6 site and that the two sites cooperate in binding NF-IL6. TNF and IL-1 stimulate specific binding of nuclear proteins to the NF-IL6* site more efficiently than to the promoter-distal NF-IL6 site. Moreover, a mutation in the NF-IL6* site abolished transactivation of the TSG-6 promoter by NF-IL6 despite the presence of the intact promoter-distal NF-IL6 site. A mutation in the promoter-distal NF-IL6 site also greatly decreased activation of the TSG-6 promoter by NF-IL6. We conclude that the two NF-IL6 sites are functionally interdependent in the activation of the TSG-6 gene.

NF-IL6 and AP-1 cooperatively modulate the activation of the TSG-6 gene by tumor necrosis factor alpha and interleukin-1.

Tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) activate transcription of the TSG-6 gene in normal human fibroblasts through a promoter region (-165 to -58) that encompasses an AP-1 and a NF-IL6 site. We show by deletion analysis and substitution mutagenesis that both sites are necessary for activation by TNF-alpha. Activation by IL-1 requires the NF-IL6 site and is enhanced by the AP-1 site. These results suggest that the NF-IL6 and AP-1 family transcription factors functionally cooperate to mediate TNF-alpha and IL-1 signals. Consistent with this possibility, IL-1 and TNF-alpha markedly increase the binding of Fos and Jun to the AP-1 site, and NF-IL6 activates the native TSG-6 promoter. Activation by NF-IL6 requires an intact NF-IL6 site and is modulated by the ratio of activator to inhibitor NF-IL6 isoforms that are translated from different in-frame AUGs. However, the inhibitor isoform can also bind to the AP-1 site and repress AP-1 site-mediated transcription. The finding that the inhibitor isoform antagonizes activation of the native TSG-6 promoter by IL-1 and TNF-alpha suggests that NF-IL6 has a physiologic role in these cytokine responses. Thus, the functionally distinct NF-IL6 isoforms cooperate with Fos and Jun to positively and negatively regulate the native TSG-6 promoter by TNF-alpha and IL-1.

TSG-6: a TNF-, IL-1-, and LPS-inducible secreted glycoprotein associated with arthritis.

TSG-6 (TNF-stimulated gene 6) was originally discovered by differential screening of a cDNA library prepared from TNF-stimulated human diploid FS-4 fibroblasts. We show that the 35-kDa protein encoded by TSG-6 was undetectable in the medium of untreated FS-4 cultures, whereas its production reached approximately 1400 and 700 ng/10(6) cells after 24-h treatment with IL-1 or TNF, respectively. Stimulation of TSG-6 protein and mRNA levels was also demonstrated in normal human mononuclear cells by treatment with TNF and, especially, by LPS. In view of the inducibility of TSG-6 by inflammatory cytokines and its earlier demonstrated affinity for hyaluronan, we examined the presence of TSG-6 protein in the synovial fluids from patients with various forms of arthritis. TSG-6 protein was undetectable in the joint fluids of persons with no known history of arthritis, but high levels of TSG-6 oere demonstrated in the synovial fluids of a majority of arthritis patients. TSG-6 protein was also detected in the sera of some of the arthritis patients, albeit at concentrations that were less than in the joint fluids. To investigate the source of TSG-6 in the synovial fluids, we examined the production of TSG-6 protein in cultures of synovial cells. Synoviocytes from rheumatoid arthritis patients produced TSG-6 protein constitutively, and this production was increased by treatment with TNF or IL-1, but not with TGF-beta. Steady-state levels of TSG-6 mRNA were also increased in synoviocytes after treatment with TNF or IL-1. The presence of high levels of TSG-6 protein in the synovial fluids of arthritis patients and its inducibility by inflammatory cytokines in fibroblasts, mononuclear cells, synoviocytes, and chondrocytes suggest a role for TSG-6 in arthritis and inflammation.

Transcriptional regulation of TSG6, a tumor necrosis factor- and interleukin-1-inducible primary response gene coding for a secreted hyaluronan-binding protein.

TSG6 was originally identified as a tumor necrosis factor (TNF)-inducible gene in human fibroblasts. Earlier we showed that the secretory TSG6 protein is a member of a family of hyaluronan-binding proteins that includes cartilage link protein, proteoglycan core protein, and the adhesion receptor CD44. In the present study we have used Southern blot analysis to demonstrate that TSG6 is a single-copy gene in the human and murine species. With the aid of a somatic cell hybrid mapping panel, TSG6 was assigned to human chromosome 2. Nuclear run-on analysis revealed that TNF produced a rapid, primary transcriptional activation of the TSG6 gene in normal human FS-4 fibroblasts. In order to learn more about the regulation of TSG6 gene expression, we cloned the TSG6 gene from a genomic library of human white blood cells. Sequencing of a 1.3-kilobase fragment of the 5'-flanking region of the TSG6 gene identified TATA-like and CAAT sequences near the transcription start site. In addition, potential binding sites for NF-IL-6, AP-1, interferon regulatory factors (IRF)-1 and -2, and glucocorticoid response elements were identified in the 5'-flanking region. A single transcription start site was identified by primer extension. Deletion analysis of the 5'-flanking region of the TSG6 DNA linked to the chloramphenicol acetyltransferase reporter gene revealed that a construct containing TSG6 DNA from positions -165 to +78 could be transcriptionally activated by interleukin(IL)-1, and to a lesser extent by TNF, upon transfection into FS-4 fibroblasts. The region that imparts inducibility by IL-1 or TNF (positions -165 to -58) contains potential binding sites for IRF-1 and -2, AP-1, and NF-IL-6. A region mediating transcriptional silencing was localized further upstream (between positions -332 and -165). The results suggest that TSG6 gene expression is regulated by an interplay of positively and negatively acting transactivating factors.

Apyrogenic synthetic desmuramyldipeptide, LK-409, with immunomodulatory properties.

The synthesis and some immunological characteristics of a new desmuramyl dipeptide 7-oxooctanoyl-L-alanyl-D-isoglutamine (LK-409) are presented. The effects of this compound were compared with those of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP). The influence of LK-409 on the number of B and T cells in spleen and the number of peritoneal macrophages was studied; Jerne's plaque forming cells assay was performed to monitor the effect of B cell differentiation. The blast transformation of T cells stimulated with concanavalin A was used to detect the influences on T lymphocytes. The activation of macrophages was studied as well. In contrast to MDP, LK-409 was apyrogenic in the doses applied but had similar immunomodulatory properties. Tested immunological properties and the absence of pyrogenicity and low toxicity make LK-409 a candidate for an immunomodulatory drug and a model molecule suitable for studying and understanding the dual activity of the MDP and its analogues.