CBAP promotes thymocyte negative selection by facilitating T-cell receptor proximal signaling.

T-cell receptor (TCR)-transduced signaling is critical to thymocyte development at the CD4/CD8 double-positive stage, but the molecules involved in this process are not yet fully characterized. We previously demonstrated that GM-CSF/IL-3/IL-5 receptor common ß-chain-associated protein (CBAP) modulates ZAP70-mediated T-cell migration and adhesion. On the basis of the high expression of CBAP during thymocyte development, we investigated the function of CBAP in thymocyte development using a CBAP knockout mouse. CBAP-deficient mice showed normal early thymocyte development and positive selection. In contrast, several negative selection models (including TCR transgene, superantigen staphylococcal enterotoxin B, and anti-CD3 antibody treatment) revealed an attenuation of TCR-induced thymocyte deletion in CBAP knockout mice. This phenotype correlated with a reduced accumulation of BIM upon TCR crosslinking in CBAP-deficient thymocytes. Loss of CBAP led to reduced TCR-induced phosphorylation of proteins involved in both proximal and distal signaling events, including ZAP70, LAT, PLCγ1, and JNK1/2. Moreover, TCR-induced association of LAT signalosome components was reduced in CBAP-deficient thymocytes. Our data demonstrate that CBAP is a novel component in the TCR signaling pathway and modulates thymocyte apoptosis during negative selection.

CBAP interacts with the un-liganded common beta-subunit of the GM-CSF/IL-3/IL-5 receptor and induces apoptosis via mitochondrial dysfunction.

The cytoplasmic domain of the common beta-chain (betac) of the granulocyte-macrophage-colony-stimulating factor (GM-CSF)/interleukin-3 (IL-3)/IL-5 receptor contains a membrane proximal region that is sufficient to mediate ligand-dependent mitogenic activity. Within this region two motifs, designated as box 1 and box 2, are highly conserved among members of the cytokine receptor superfamily. Whereas box 1 is required for the recruitment and phosphorylation of Janus kinase-2, the function of box 2 remains largely unknown. Here, we report the identification of a novel transmembrane protein (common beta-chain associated protein (CBAP)) which directly associated with betac via the box 2 motif. Interestingly, such an association only occurred in the absence of GM-CSF in vivo. Ectopic overexpression of CBAP triggered apoptosis of factor-dependent cells via mitochondrial dysfunction, which could be inhibited by Bcl-2 overexpression. Reduced expression of endogenous CBAP by small interfering RNA did not interfere GM-CSF-activated signaling molecules, but such treatment significantly inhibited apoptosis induced by GM-CSF deprivation, but not other death stimuli. Domain mapping studies indicated that one apoptogenic domain of CBAP correlated with its ability to interact with betac. Taken together, these results suggest that CBAP modulates GM-CSF-deprivation-induced apoptosis possibly via a novel mechanism involving interaction with an un-liganded betac molecule.

The osteopontin-CD44 survival signal involves activation of the phosphatidylinositol 3-kinase/Akt signaling pathway.

We have recently demonstrated that the gene encoding the osteopontin (OPN) protein is activated both by interleukin-3 and granulocyte-macrophage colony-stimulating factor signaling pathways and that, through binding to the cell surface receptor CD44, OPN contributes to the survival activities of interleukin (IL)-3 and GM-CSF (Lin, Y.-H., Huang, C.-J., Chao, J.-R., Chen, S.-T., Lee, S.-F., Yen, J. J.-Y., and Yang-Yen, H.-F. (2000) Mol. Cell. Biol. 20, 2734-2742). In this report, we demonstrate that the CD44-binding domain of OPN involves a region containing amino acid residues from 121 to 140 and that both threonine and serine at positions 137 and 147, respectively, are essential for the survival stimulatory effect of OPN. Substitution of either residue with alanine results into a dominant negative mutant that overrides the survival effect of IL-3. Upon binding to the CD44 receptor, the wild-type OPN but not the inactive mutant induces activation of phosphatidylinositol 3-kinase and Akt. Last, we demonstrate that two waves of Akt activation are detected in IL-3-treated cells and that the survival promoting effect of OPN is mediated predominantly through the phosphatidylinositol 3-kinase/Akt signaling pathway. Together, our results suggest that a positive autoregulatory loop is involved in the survival pathway of IL-3.

Lysophosphatidic acid promotes phorbol-ester-induced apoptosis in TF-1 cells by interfering with adhesion.

When exposed to PMA, the erythroblastic cell line TF-1 and its cytokine-independent variant D2 cells can be induced to undergo differentiation and apoptosis. In this study we investigated the mechanism responsible for the differential responses to PMA induction and show that serum present in the medium has a major role in promoting PMA-induced apoptosis in TF-1 and D2 cells. Interestingly, lysophosphatidic acid (LPA) could replace serum to co-operate with PMA in inducing apoptosis via the Rho-dependent pathway. The expression of a constitutively active form of RhoA also increased PMA-induced apoptosis. However, by inhibiting adhesion, most cells underwent PMA-induced apoptosis even in the absence of LPA or serum, indicating that adhesion is required for PMA-induced differentiation. Given that LPA could prevent adhesion for cells maintained in the serum-free medium, here we propose that RhoA has a switching role in determining whether TF-1 and D2 cells undergo differentiation or apoptosis in response to PMA, by modulating cell adhesion.

CREB is one component of the binding complex of the Ces-2/E2A-HLF binding element and is an integral part of the interleukin-3 survival signal.

The Ces-2/E2A-HLF binding element (CBE) is recognized by Caenorhabditis elegans death specification gene product Ces-2 and human acute lymphocytic leukemia oncoprotein E2A-HLF. In an attempt to identify a cellular CBE-binding protein(s) that may be involved in apoptosis regulation in mammals, multiple nuclear binding complexes of CBE were identified in various mammalian cell lines and tissues by electrophoretic mobility shift assay. Cyclic AMP (cAMP)-responsive element (CRE)-binding protein (CREB) was present in one major CBE complex of Ba/F3 and TF-1 cells, and both in vitro-translated and Escherichia coli-synthesized CREB bound to CBE. Activation of CREB by cAMP-elevating chemicals or the catalytic subunit of protein kinase A (PKAc) resulted in induction of the CBE-driven reporter gene. Stimulation of Ba/F3 cells with interleukin-3 (IL-3) promptly induced phosphorylation of CREB at serine(133) partially via a PKA-dependent pathway. Consistently, Ba/F3 cell survival in the absence of IL-3 was prolonged by activation of PKA. Conversely, treatment of cells with a PKA inhibitor or expression of the dominant negative forms of the regulatory subunit type I of PKA and CREB overrode the survival activity of IL-3. Last, the bcl-2 gene was demonstrated to be one candidate cellular target of the CREB-containing CBE complex, as mutations in the CRE and CBE sites significantly reduced the IL-3 inducibility of the bcl-2 promoter. Together, our results suggest that CREB is one cellular counterpart of Ces-2/E2A-HLF and is part of IL-3 dependent apoptosis regulation in hematopoietic cells.

Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression.

Large granular lymphocyte (LGL) leukemia is characterized by the expansion of antigen-activated cytotoxic T lymphocytes. These leukemic cells are resistant to Fas-mediated apoptosis despite expressing high levels of Fas. We found that leukemic LGL from 19 patients displayed high levels of activated STAT3. Treatment of leukemic LGL with the JAK-selective tyrosine kinase inhibitor AG-490 induced apoptosis with a corresponding decrease in STAT-DNA binding activity. Moreover, using an antisense oligonucleotide approach to diminish STAT3 expression, we found that Fas sensitivity was restored in leukemic LGL. AG-490-induced apoptosis in leukemic LGL was independent of Bcl-xL or Bcl-2 expression. However, we found that the Bcl-2-family protein Mcl-1 was significantly reduced by AG-490 treatment. Activated STAT3 was shown to bind an SIE-related element in the murine mcl-1 promoter. Using a luciferase reporter assay, we demonstrated that v-src overexpression in NIH3T3 induced STAT3-dependent transcriptional activity from the mcl-1 promoter and increased endogenous Mcl-1 protein levels. We conclude that STAT3 activation contributed to accumulation of the leukemic LGL clones. These findings suggest that investigation should focus on novel strategies targeting STAT3 in the treatment of LGL leukemia.

Coupling of osteopontin and its cell surface receptor CD44 to the cell survival response elicited by interleukin-3 or granulocyte-macrophage colony-stimulating factor.

The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) share a common beta subunit, the distal cytoplasmic domain of which is essential for the promotion of cell survival by these two cytokines. Genes whose expression is specifically induced by signaling through the distal cytoplasmic domain of this receptor beta subunit were screened by a subtraction cloning approach in derivatives of a mouse pro-B-cell line. One gene thus identified was shown to encode a protein highly homologous (with only 7 amino acid substitutions) to murine osteopontin (OPN), a secreted adhesion protein. Conditioned medium from cells expressing wild-type OPN, but not that from cells expressing a deletion mutant lacking residues 79 to 140, increased the viability of a non-OPN-producing cell line in the presence of human GM-CSF. Antibody blocking experiments revealed that OPN produced as a result of IL-3 or GM-CSF signaling was secreted into the medium and, through binding to its cell surface receptor, CD44, contributed to the survival-promoting activities of these two cytokines. Furthermore, coupling of the OPN-CD44 pathway to the survival response to IL-3 was also demonstrated in primary IL-3-dependent mouse bone marrow cells. These results thus show that induction of an extracellular adhesion protein and consequent activation of its cell surface receptor are important for the antiapoptotic activities of IL-3 and GM-CSF.

Cytokine receptor common beta chain as a potential activator of cytokine withdrawal-induced apoptosis.

Growth factors and cytokines play an important role in supporting cellular viability of various tissues during development due to their ability to suppress the default cell death program in each cell type. To date, neither the triggering molecule nor the transduction pathway of these default apoptosis programs is understood. In this study, we explored the possibility that cytokine receptors are involved in modulating cytokine withdrawal-induced apoptosis (CWIA) in hematopoietic cells. Expression of the exogenous cytokine receptor common beta chain (betac), but not the alpha chains, accelerated CWIA in multiple cytokine-dependent cell lines. Reduction of the expression level of endogenous betac by antisense transcripts resulted in prolonged survival during cytokine deprivation, suggesting a critical role of betac in modulating CWIA. Fine mapping of the betac subunit revealed that a membrane-proximal cytoplasmic sequence, designated the death enhancement region (DER), was critical to the death acceleration effect of betac. Furthermore, DER accelerated cell death either as a chimeric membrane protein or as a cytosolic protein, suggesting that DER functions independently of the cytokine receptor and membrane anchorage. Cross-linking of the chimeric membrane-bound DER molecules by antibody or of the FK506-binding protein-DER fusion protein by a synthetic dimerizing agent, AP1510, did not abrogate the death acceleration effect. Transient transfection assays further indicated that DER promoted cell death in the absence of serum in the nonhematopoietic 293 cell line. In summary, our data suggest that betac plays an important role in modulating CWIA via an anchorage-independent and aggregation-insensitive mechanism. These findings may facilitate further studies on the signaling pathways of CWIA.

The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB.

mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) signaling pathways and plays an important role in the viability response of these cytokines. In this study, we demonstrated that cytokine stimulation of mcl-1 mRNA and protein expression were attenuated by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-K) inhibitors. Reporter gene assays further showed that the PI3-K/Akt signaling pathway was involved in IL-3 activation of mcl-1 gene transcription. Analysis of the mcl-1 promoter revealed that both promoter elements, SIE at position -87 and CRE-2 at -70, contribute to IL-3 stimulation of mcl-1 gene expression. Although either the SIE site or the CRE-2 site alone was sufficient to confer IL-3 inducibility on a heterologous promoter, only IL-3 activation of the CRE-2 reporter was mediated via the PI3-K/Akt pathway. The SIE binding activity was constitutively high in cells deprived of or stimulated by IL-3. In contrast, the CRE-2 binding activity was low in cytokine-starved cells and was strongly induced within 1 h following cytokine treatment of cells. In addition, cytokine induction of the CRE-2 but not of the SIE binding activity was dependent on activation of the PI3-K/Akt signaling pathway. Lastly, we showed that CREB was one component of the CRE-2 binding complex and played a role in IL-3 activation of the mcl-1 reporter gene. Taken together, our results suggest that both PI3-K/Akt-dependent and -independent pathways contribute to the IL-3 activation of mcl-1 gene expression. Activation of mcl-1 by the PI3-K/Akt-dependent pathway is through a transcription factor complex containing CREB.

Optimal proliferation of a hematopoietic progenitor cell line requires either costimulation with stem cell factor or increase of receptor expression that can be replaced by overexpression of Bcl-2.

In vitro proliferation of hematopoietic stem cells requires costimulation by multiple regulatory factors whereas expansion of lineage-committed progenitor cells generated by stem cells usually requires only a single factor. The distinct requirement of factors for proliferation coincides with the differential temporal expression of the subunits of cytokine receptors during early stem cell differentiation. In this study, we explored the underlying mechanism of the requirement of costimulation in a hematopoietic progenitor cell line TF-1. We found that granulocyte-macrophage colony-stimulating factor (GM-CSF) optimally activated proliferation of TF-1 cells regardless of the presence or absence of stem cell factor (SCF). However, interleukin-5 (IL-5) alone sustained survival of TF-1 cells and required costimulation of SCF for optimal proliferation. The synergistic effect of SCF was partly due to its anti-apoptosis activity. Overexpression of the IL-5 receptor alpha subunit (IL5Ralpha) in TF-1 cells by genetic selection or retroviral infection also resumed optimal proliferation due to correction of the defect in apoptosis suppression. Exogenous expression of an oncogenic anti-apoptosis protein, Bcl-2, conferred on TF-1 cells an IL-5-dependent phenotype. In summary, our data suggested SCF costimulation is only necessary when the expression level of IL5Ralpha is low and apoptosis suppression is defective in the signal transduction of IL-5. Expression of Bcl-2 proteins released the growth restriction of the progenitor cells and may be implicated in leukemia formation.

Activation of Stat3 by v-Src is through a Ras-independent pathway.

V-Src induces tyrosine phosphorylation of various cellular proteins and activates a number of signaling molecules including the Jak family of proteins tyrosine kinases and Stat (signal transducers and activators of transcription) proteins. Many cellular effects elicited by v-Src are mediated through Ras, a molecular switch linking growth factor receptors and non-receptor tyrosine kinases to many downstream effectors. In this report, we demonstrated that v-H-Ras and v-Src both induced cellular transformation. However, the activation of Jak1 and Stat3 were only observed in v-Src transformed cells. Using reporter gene assays, we further showed that activation of Stat3 and possibly of Jak1 by v-Src were mediated through a Ras-independent pathway. As Stat3 activation has recently been shown to be required for cellular transformation by v-Src, our results suggest that activation of the Jak-Stat pathway may serve as a modulator in some but not all transformation processes.

mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway and is one component of the GM-CSF viability response.

mcl-1, a bcl-2 family member, was originally identified as an early gene induced during differentiation of ML-1 myeloid leukemia cells. In the present study, we demonstrate that Mcl-1 is tightly regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway. Upon deprivation of survival factor from TF-1 myeloid progenitor cells, Mcl-1 levels quickly dropped prior to visible detection of apoptosis of these cells. Upon restimulation of these deprived cells with GM-CSF, the mcl-1 mRNA was immediately induced and its protein product was accordingly resynthesized. Analysis with Ba/F3 cells expressing various truncation mutants of the GM-CSF receptor revealed that the membrane distal region between amino acids 573 and 755 of the receptor beta chain was required for mcl-1 induction. Transient-transfection assays with luciferase reporter genes driven by various regions of the mcl-1 promoter demonstrated that the upstream sequence between -197 and -69 is responsible for cytokine activation of the mcl-1 gene. Overexpression of mcl-1 delayed but did not completely prevent apoptosis of cells triggered by cytokine withdrawal. Its down regulation by antisense constructs overcame, at least partially, the survival activity of GM-CSF and induced the apoptosis of TF-1 cells. Taken together, these results suggest that mcl-1 is an immediate-early gene activated by the cytokine receptor signaling pathway and is one component of the GM-CSF viability response.

Characterization of factor-independent variants derived from TF-1 hematopoietic progenitor cells: the role of the Raf/MAP kinase pathway in the anti-apoptotic effect of GM-CSF.

Human hematopoietic progenitor cells (TF-1) undergo apoptosis upon deprivation of their dependent cytokine. In this report, we have isolated and characterized some spontaneously derived cytokine-independent variants from TF-1 cells. Analysis of several signaling molecules known to be activated by the GM-CSF pathway revealed that two non-autocrine variants were still responsive to GM-CSF stimulation. However, both variants, without ligand stimulation, already had some activated forms of Raf and MAP kinases. Given current knowledge, the activated Raf/MAP kinase pathway was likely to be responsible for the survival of both variants in the cytokine-free medium. However, the growth of hybrids between wild type and either variant was unexpectedly dependent on GM-CSF. Both variants like the wild type cells were still susceptible to apoptosis induced by other stimuli. These results suggest that either the activated Raf/MAP kinase pathway in both variants is not sufficient to repress the 'two-fold' death signals generated from the hybrids or that there is another mechanism that is responsible for the factor-independent growth of both variants.

Differential regulation of p53, c-Myc, Bcl-2 and Bax protein expression during apoptosis induced by widely divergent stimuli in human hepatoblastoma cells.

Apoptosis of HepG2 cells triggered by various agents is characterized in an attempt to delineate the common apoptosis signaling pathway in human hepatoma cells. Several hallmarks of apoptosis, including DNA laddering, chromatin condensation and fragmentation, and an apoptosis specific cleavage of 28S and 18S ribosomal RNA were observed after treatment with curcumin. Curcumin treatment however did not alter the expression levels of Bcl-2 and Bax proteins. p53 protein accumulated slowly and decreased abruptly after reaching the maximum. Conversely, c-Myc protein decreased initially and subsequently increased preceding the onset of apoptosis. The accumulation of p53 protein is not due to increased levels of p53 mRNA and does not result in growth arrest. Staurosporine, quinacrine, ultraviolet irradiation, hydrogen peroxide, and cyclohexamide are all capable of triggering apoptosis in HepG2 cells. While most of these agents affect the expression levels of p53 and c-Myc similarly, none of them altered the expression levels of the Bcl-2 and Bax proteins. In conclusion, these data suggest that p53 and c-Myc may play a more important role in the apoptosis signaling pathway in HepG2 cells, than the bcl-2 gene family.

Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines.

Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene-transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT-29, human kidney cancer cell 293, and human hepatocellular carcinoma Hep G2 cells, but not in primary culture of mouse embryonic fibroblast C3H 10T1/2, rat embryonic fibroblast, and human foreskin fibroblast cells in a concentration- and time-dependent manner. Many cellular and biochemical effects of curcumin in mouse fibroblast cells have been reported, such as inhibition of protein kinase C (PKC) activity induced by phorbol 12-myristate 13-acetate treatment, inhibition of tyrosine protein kinase activity, and inhibition of arachidonic acid (AA) metabolism. Treatment of NIH 3T3 cells with the PKC inhibitor staurosporine, the tyrosine kinase inhibitor herbimycin A, and the AA metabolism inhibitor quinacrine induces apoptotic cell death. These results suggest that, in some immortalized and transformed cells, blocking the cellular signal transduction might trigger the induction of apoptosis.

Ras transformation results in an elevated level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells.

Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells resulted in cellular transformation and an acceleration of G1 progression of these cells. A shortened G1 phase was found to be associated with an increased level of cyclin D1 but not cyclin E protein. Using an antisense blocking method, reduced synthesis of cyclin D1 in v-H-Ras transformants resulted in a slower G1 progression rate of these cells. Although constitutive overexpression of cyclin D1 in NIH 3T3 cells accelerated G1 progression, cells remained untransformed. Furthermore, inhibition of cyclin D1 synthesis greatly impaired the soft-agar cloning efficiency of v-H-Ras transformants. These results suggest that increased expression of cyclin D1 is necessary but not sufficient for the transforming activity of v-H-Ras. Similar effect on cell cycle progression was also observed in Raf-transformed cells. In addition to cyclin D1, cyclin E protein was found to be elevated in Src transformants. This may account for the further shortening of the G1 phase of these cells. Activation of an additional Ras-independent pathway was suggested to be responsible for the further acceleration of the G1 phase in Src transformants.

Restoring the apoptosis suppression response to IL-5 confers on erythroleukemic cells a phenotype of IL-5-dependent growth.

We have established a human IL-5 (hIL-5) dependent cell line, JYTF-1, derived from TF-1 parental human erythroleukemic cells by long-term cultivation in the presence of hIL-5. The ED50 values of hIL-5 for both TF-1 and JYTF-1 cell lines remained similar. However, when cells were grown in an optimal concentration of IL-5, some TF-1 cells but not JYTF-1 cells died via apoptosis. Although the rates of DNA synthesis were similar for TF-1 and JYTF-1 cells grown in IL-5, [3-H]thymidine releasing of pulse-labeled DNA analysis indicated that the viable TF-1 cells in IL-5 were more apoptosis-prone than were JYTF-1 cells. Therefore, in the JYTF-1 variant, the ability to suppress apoptosis has apparently been restored. The following findings suggest that overexpression of the hIL-5 receptor alpha-chain may be responsible for restoring the apoptosis suppression ability of IL-5: 1) the growth of JYTF-1 cells remained cytokine-dependent; 2) the proliferation of JYTF-1 cells in IL-5 was not mediated by autocrine secretion; 3) JYTF-1 and TF-1 cells responded similarly to other cytokines such as human erythropoietin; 4) Northern blot analysis revealed that JYTF-1 cells expressed approximately eightfold more IL-5 receptor alpha-chain mRNA than did TF-1. To our knowledge, JYTF-1 represents the first example in which coupling of mitogenesis stimulation and apoptosis suppression from otherwise uncoupled parental cells confers a phenotype of IL-5-dependent growth.

Various modes of gene regulation by nuclear receptors for steroid and thyroid hormones.

AP-1 is a transcriptional activator composed of homo- and heterodimers of Jun and Fos proteins. It is involved in activation of genes, such as collagenase, stromelysin, IL-2 and TGF beta 1, by tumour promoters, growth factors and cytokines. AP-1 activity is also elevated in response to transforming oncogenes and is required for cell proliferation. AP-1 activity is subject to complex regulation both transcriptionally and post-transcriptionally. Transcriptional control of jun and fos gene expression determines the amount and composition of the AP-1 complex. The jun and fos genes are regulated both positively and negatively and are highly inducible in response to extracellular stimuli. Post translational control is also important. Both cJun and cFos are subject to regulated phosphorylation. In the case of cJun, phosphorylation of sites near the DNA-binding domain inhibits DNA-binding, while dephosphorylation reverses this inhibition. Phosphorylation of cJun on sites within the N-terminal activation domain increases its ability to activate transcription. The protein kinase phosphorylating these sites is stimulated by cytokines and growth factors. Another mechanism modulating AP-1 activity is transcriptional interference by members of the nuclear receptor family and is relevant for the pathophysiology of rheumatoid arthritis (RA). In RA, chronic inflammation leads to increased AP-1 activity in T cells,macrophages and synoviocytes as a response to secretion of cytokines such as IL-1 and TNF alpha. While the IL-2 gene plays a major role in T cell activation, another AP-1 target gene encodes an enzyme, collagenase, responsible for destruction of bone and tendon.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonism between retinoic acid receptors and AP-1: implications for tumor promotion and inflammation.

Retinoids such as retinoic acid (RA) are potent anti-arthritic and anti-neoplastic agents. We investigated the mechanism by which RA inhibits induction of collagenase gene transcription by inflammatory mediators, tumor promoters, and proto-oncogenes. We found that the RA receptors (RARs) are potent inhibitors of AP-1 activity generated either by cJun homodimers or cJun/cFos heterodimers. In addition, both cJun and cFos can inhibit RAR activity. In vitro experiments suggested that this inhibition is due to an interaction between RAR and AP-1 proteins that results in mutual loss of DNA-binding activity. The RARs need not bind to the AP-1 site, neither does AP-1 bind to RA response elements. An understanding of this antagonism between the RAR and AP-1 might help to elucidate the anti-neoplastic and anti-arthritic effects of RA as well as its effects on cell differentiation and proliferation.

Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction.

Glucocorticoids are potent inhibitors of collagenase induction by phorbol esters and inflammatory mediators. The target for this negative effect is the AP-1 site within the collagenase promoter, which also mediates its induction. Negative regulation is due to repression of AP-1 activity by the glucocorticoid receptor (GCR). While the GCR is a potent inhibitor of AP-1 activity (Jun/Fos), both c-Jun and c-Fos are potent repressors of GCR activity. In vitro experiments using purified GCR and c-Jun proteins suggest that mutual repression is due to direct interaction between the two. Direct interaction between GCR and either c-Jun or c-Fos is demonstrated by cross-linking and coimmunoprecipitation. These findings reveal a cross talk between two major signal transduction systems used to control gene transcription in response to extracellular stimuli, and a novel mechanism for transcriptional repression.