Repression of inflammatory responses in the absence of DNA binding by the glucocorticoid receptor.

The glucocorticoid receptor (GR) acts both as a transcription factor itself on genes carrying GR response elements (GREs) and as a modulator of other transcription factors. Using mice with a mutation in the GR, which cannot activate GRE promoters, we examine whether the important anti-inflammatory and immune suppressive functions of glucocorticoids (GCs) can be established in this in vivo animal model. We find that most actions are indeed exerted in the absence of the DNA-binding ability of the GR: inhibition of the inflammatory response of locally irritated skin and of the systemic response to lipopolysaccharides. GCs repress the expression and release of numerous cytokines both in vivo and in isolated primary macrophages, thymocytes and CD4(+) splenocytes. A transgenic reporter gene controlled by NF-kappa B exclusively is also repressed, suggesting that protein- protein interaction with other transcription factors such as NF-kappa B forms the basis of the anti-inflammatory activity of GR. The only defect of immune suppression detected so far concerns the induced apoptosis of thymocytes and T lymphocytes.

CD44-dependent lymphoma cell dissemination: a cell surface CD44 variant, rather than standard CD44, supports in vitro lymphoma cell rolling on hyaluronic acid substrate and its in vivo accumulation in the peripheral lymph nodes.

Cell motility is an essential element of tumor dissemination, allowing organ infiltration by cancer cells. Using mouse LB lymphoma cells transfected with standard CD44 (CD44s) cDNA (LB-TRs cells) or with the alternatively spliced CD44 variant CD44v4-v10 (CD44v) cDNA (LB-TRv cells), we explored their CD44-dependent cell migration. LB-TRv cells, but not LB-TRs or parental LB cells, bound soluble hyaluronic acid (HA) and other glycosaminoglycans (GAGs), and exclusively formed, under physiological shear force, rolling attachments on HA substrate. Furthermore, LB-TRv cells, but not LB-TRs cells or their parental LB cells, displayed accelerated local tumor formation and enhanced accumulation in the peripheral lymph nodes after s.c. inoculation. The aggressive metastatic behavior of i.v.-injected LB-TRV cells, when compared with that of other LB-transfectants, is attributed to more efficient migration to the lymph nodes, rather than to local growth in the lymph node. Injection of anti-CD44 monoclonal antibody or of the enzyme hyaluronidase also prevented tumor growth in lymph nodes of BALB/c mice inoculated with LB-TRv cells. The enhanced in vitro rolling and enhanced in vivo local tumor growth and lymph node invasion disappeared in LB cells transfected with CD44v cDNA bearing a point mutation at the HA binding site, located at the distal end of the molecule constant region. These findings show that the interaction of cell surface CD44v with HA promotes cell migration both in vitro and in vivo, and they contribute to our understanding of the mechanism of cell trafficking, including tumor spread.

Regulation of alternative pre-mRNA splicing by the ERK MAP-kinase pathway.

Differential gene expression through alternative pre-mRNA splicing is crucial to various physiological and pathological conditions. Upon activation of B and T lymphocytes during an immune response, variant isoforms of the cell surface molecule CD44 are generated by alternative pre-mRNA splicing. We show here that in primary mouse T cells as well as in the murine LB-17 T-cell line upregulation of variant CD44 mRNA species upon T-cell activation requires activation of the MEK-ERK pathway. By employing mutant signaling molecules and a novel luciferase-based splice reporter system we demonstrate that the Ras-Raf-MEK-ERK signaling cascade, but not the p38 MAP-kinase pathway, activates a mechanism that retains variant CD44 exon v5 sequence in mature mRNA. The findings demonstrate that a highly conserved pleiotropic signaling pathway links extracellular cues to splice regulation, providing an avenue for tissue-specific, developmental or pathology-associated splicing decisions.

Soluble CD44 inhibits melanoma tumor growth by blocking cell surface CD44 binding to hyaluronic acid.

Proteolytic cleavage of the extracellular domain of CD44 from the surface of cells has been observed recently in different cell types. In cell culture supernatants of human melanoma cell lines a 70 kDa soluble CD44 protein (solCD44) was detected at concentrations of 250-300 ng/ml. Protease inhibitor studies revealed that serine proteases and metalloproteases are involved in the cleavage of CD44 from the surface of melanoma cells. To analyse a possible function of soluble CD44 a human malignant melanoma cell line was stably transfected with cDNAs encoding either wild type soluble CD44s or mutated forms with defective HA binding properties (CD44sR41A and CD44sR150A/R154A). Soluble CD44s almost completely inhibited hyaluronic acid binding by melanoma cells, whereas soluble CD44 mutated in the HA binding domain had no effect. When cultivated on hyaluronic acid, melanoma cell proliferation was induced by 30% for both the parental and the control transfected cells. This increase in proliferation was blocked completely in solCD44s-secreting transfectants, whereas solCD44sR41A and solCD44sR150A/R154A-secreting cells again showed hyaluronic acid-induced cell proliferation. These cell lines were subcutaneously injected into MF1 nu/nu mice to compare their growth as tumors in vivo. Compared to tumors derived from parental and control transfected cells, we observed a dramatic reduction of primary tumor growth with solCD44s expressing MM cells. Transfectants expressing solCD44s mutated in the HA binding domain in contrast developed fast-growing primary tumors. These results provide strong evidence that direct solCD44 interactions with hyaluronic acid interfere competitively with processes induced by hyaluronic acid binding to surface CD44. Autocrine, or drug-induced secretion of solCD44 by human melanoma cells may thus exert potent antitumoral effects in vivo.

Cross-talk between glucocorticoid receptor and AP-1.

Cross-talk between different transcription factors, notably between the glucocorticoid receptor and AP-1, has been discovered more than 10 years ago: a bona fide transcription factor, without apparent need for its own direct DNA contact, influences the activity of another transcription factor. Recent experiments have added interesting aspects: in addition to major insights into the mechanism of cross-talk, it is now clear that the cross-talk ability of glucocorticoid receptor is essential for mouse development, while the activation of target promoters carrying a glucocorticoid response element (GRE), is surprisingly, dispensable for survival under animal house conditions. Interestingly, the cross-talk function is responsible for almost all regulatory actions of cortisol in the immune system. It is possible that the two functions of the glucocorticoid receptor can be activated separately by specific ligands. Future goals will be to define whether adverse effects of long-term corticosteroid treatment, e.g. osteoporosis, joint necroses, metabolic effects, can be ascribed to GRE-target gene activation and thus be dissociated from the desirable actions in the treatment e.g. of autoimmune disease.

The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44.

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.

Gene expression patterns associated with the metastatic phenotype in rodent and human tumors.

Using subtractive technology, we have generated metastasis-associated gene expression profiles for rat mammary and pancreatic adenocarcinomas. Several genes whose expression is thought to be related to tumor progression such as c-Met, urokinase-type plasminogen activator receptor, ezrin, HMG-1, oncomodulin, cathepsin, and caveolin were thereby isolated. Half of the metastasis-associated clones showed no significant homology to genes with known function. Notably, several of the metastasis-associated clones were also expressed in metastatic lines but not in nonmetastatic lines of other tumor models. Furthermore, in situ hybridization using selected clones documents the relevance of these results for human cancer because strong expression in tumor cells including metastases was detected in human colorectal cancer samples and, to a lesser extent, in mammary cancer samples. These data support the concept that tumors express a "metastatic program" of genes.

CD44 is the principal mediator of hyaluronic-acid-induced melanoma cell proliferation.

Interactions of the extracellular matrix component hyaluronic acid and its cellular receptors CD44 and RHAMM/IHABP have been linked to tumor progression and metastasis formation. We investigated the expression and hyaluronic-acid-dependent functions of CD44 and RHAMM/IHABP in human melanoma. Immunohistochemistry of tumor specimens at different stages of melanoma progression revealed an increased expression of CD44 and RHAMM/IHABP. High mRNA expression of CD44 was found in three highly tumorigenic melanoma cell lines compared with less tumorigenic melanoma cells or nontransformed melanocytes. RHAMM/IHABP expression was upregulated in all cell lines analyzed but not in melanocytes. In contrast to the cell surface localization of CD44, RHAMM/IHABP was detected exclusively within the cytoplasm of melanoma cells. Binding and adhesion of melanoma cells to hyaluronic acid is mainly CD44 dependent as it was inhibited to 60%--80% by an anti-CD44 monoclonal antibody whereas anti-RHAMM/IHABP sera had no effect. Culture of melanoma cells in the presence of hyaluronic acid resulted in a dose-dependent, CD44-mediated increase of melanoma cell proliferation and enhanced release of basic fibroblast growth factor and transforming growth factor beta 1. We conclude that (i) the expression of CD44 and RHAMM/IHABP is increased during melanoma progression, (ii) CD44 is the principal hyaluronic acid surface receptor on melanoma cells, and (iii) the hyaluronic-acid-induced increase of the proliferative capacity of melanoma cells is mainly dependent on CD44--hyaluronic acid interactions.

Heterogeneous ribonucleoprotein A1 is part of an exon-specific splice-silencing complex controlled by oncogenic signaling pathways.

Regulation of alternative pre-mRNA splicing, recognized as increasingly important in causing human disease, was studied using the CD44 gene, whose splice variants have been implicated in tumor progression. We identified heterogeneous ribonucleoprotein (hnRNP) A1 as a protein interacting in vitro and in vivo with regulatory splice elements in CD44 variant exon v5. Transient overexpression of hnRNP A1 prevented v5 exon inclusion, dependent on the exonic elements. HnRNP A1-dependent repression was exon-specific and could be relieved by coexpression of oncogenic forms of Ras and Cdc42. The results define hnRNP A1 as a decisive part of an oncogene-regulated splice-silencing complex, which can select between multiple alternatively spliced exons.

CD44 acts both as a growth- and invasiveness-promoting molecule and as a tumor-suppressing cofactor.

High-molecular-weight splice variants of the CD44 transmembrane protein family have been implicated in tumorigenesis and metastasis formation. By contrast, in certain tumors--for example, Burkitt's lymphoma, neuroblastomas, and prostate cancer--loss of CD44 expression seems to accompany transformation. Here we describe two modes of action of CD44 proteins. They can bind growth factors and present them to their authentic high-affinity receptors, and thus promote proliferation and invasiveness of cells. Under these conditions the CD44 proteins recruit ERM proteins--for example, ezrin or moesin--to their cytoplasmic tails, thereby producing links to the cytoskeleton. This mode of action could account for the tumor-promoting action of CD44 proteins. The second mode of action of CD44 proteins comes into play when cells reach confluent growth conditions. Under specific conditions, binding of another ligand, the ECM component hyaluronate, leads to the activation and binding to the CD44 cytoplasmic tail of the tumor suppressor protein merlin. The activation of merlin confers growth arrest, so-called contact inhibition. This function of CD44 proteins defines them as tumor suppressors. The type of action of CD44 on a given cell will depend on the isoform pattern of CD44 expressed, on the cellular equipment with ERM protein members, on the nature of the ECM, and on yet-unknown conditions.

UV-Induced stabilization of c-fos and other short-lived mRNAs.

Irradiation of cells with short-wavelength ultraviolet light (UVC) changes the program of gene expression, in part within less than 15 min. As one of the immediate-early genes in response to UV, expression of the oncogene c-fos is upregulated. This immediate induction is regulated at the transcriptional level and is transient in character, due to the autocatalyzed shutoff of transcription and the rapid turnover of c-fos mRNA. In an experiment analyzing the kinetics of c-fos mRNA expression in murine fibroblasts irradiated with UVC, we found that, in addition to the initial transient induction, c-fos mRNA accumulated in a second wave starting at 4 to 5 h after irradiation, reaching a maximum at 8 h, and persisting for several more hours. It was accompanied by an increase in Fos protein synthesis. The second peak of c-fos RNA was caused by an UV dose-dependent increase in mRNA half-life from about 10 to 60 min. With similar kinetics, the mRNAs of other UV target genes (i.e., the Kin17 gene, c-jun, IkappaB, and c-myc) were stabilized (e.g., Kin17 RNA from 80 min to more than 8 h). The delayed response was not due to autocrine cytokine secretion with subsequent autostimulation of the secreting cells or to UV-induced growth factor receptor activation. Cells unable to repair UVC-induced DNA damage responded to lower doses of UVC with an even greater accumulation of c-fos and Kin17 mRNAs than repair-proficient wild-type cells, suggesting that a process in which a repair protein is involved regulates mRNA stability. Although resembling the induction of p53, a DNA damage-dependent increase in p53 was not a necessary intermediate in the stabilization reaction, since cells derived from p53 knockout mice showed the same pattern of c-fos and Kin17 mRNA accumulation as wild-type cells. The data indicate that the signal flow induced by UV radiation addresses not only protein stability (p53) and transcription but also RNA stability, a hitherto-unrecognized level of UV-induced regulation.

The CD44 receptor of lymphoma cells: structure-function relationships and mechanism of activation.

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.

Redox regulation of signal transduction in mammalian cells.

This mini-review addresses the mechanism of ultraviolet-light-induced activation of receptor tyrosine kinases. The experimental approach into this mechanism revealed the existence of redox regulation of signal transduction in mammalian cells. It is postulated that, in addition to responsiveness to oxidative attacks from outside, redox regulation of specific redox-sensitive proteins likely represents an important physiological mechanism.

Overexpression of activated neu/erbB2 initiates immortalization and malignant transformation of immature Schwann cells in vitro.

The neu/erbB2 protooncogene is overexpressed in numerous human cancers and is mutationally activated in N-ethyl-N-nitrosourea (ENU)-induced rodent tumors of the Schwann cell lineage. We investigated whether expression of activated neu in Schwann cells is sufficient to initiate their immortalization and transformation. Clones of embryonic dorsal root ganglia cells infected with a retrovirus bearing activated neu (NID cells) were selected based on their expression of Schwann cell-specific markers. Compared to embryonic Schwann cells infected with a virus encoding empty vector, we found that NID cells have altered shapes and disorganized cytoskeletons, grow in the absence of growth factors required for normal Schwann cell survival and proliferation, and can be repeatedly passaged. Furthermore, NID cells are invasive in an in vitro matrix invasion assay and form metastatic tumors when injected into syngeneic animals. The neu-induced growth and invasive phenotypes could be reversed by drugs that inhibit Ras and Src activity. Interestingly, later stage Schwann cells infected with activated neu failed to become immortalized. These findings indicate that constitutive activation of erbB2 is sufficient to initiate the immortalization and transformation of immature Schwann cells, and support the notion that Schwann cells have particular developmental stages during which they are susceptible to immortalizing and transforming events.

Inactivation of protein-tyrosine phosphatases as mechanism of UV-induced signal transduction.

UV irradiation of cells causes ligand-independent activation of receptor tyrosine kinases. On the basis of dephosphorylation kinetics, UV-induced inactivation of receptor-directed tyrosine phosphatases (PTP) has been proposed as the mechanism of receptor activation (Knebel, A., Rahmsdorf, H. J., Ullrich, A., and Herrlich, P. (1996) EMBO J. 15, 5314-5325). Here we show that four defined protein-tyrosine phosphatases (PTPs), SHP-1, RPTPalpha, RPTPsigma, and DEP-1, are partially inactivated upon UV irradiation of PTP-overexpressing cells. The dephosphorylation of coexpressed platelet-derived growth factor beta (PDGFbeta) receptor by RPTPalpha is inhibited upon UV irradiation. UV converts RPTPalpha into a substrate-trapping enzyme which can coprecipitate PDGFbeta receptor, similarly to the PTP mutant at the active-center cysteine: C433S. In agreement with the proposed mechanism that inactivation of PTPs accounts for receptor tyrosine kinase activation, no evidence for a UV-induced receptor cross-linking could be obtained in PDGFbeta receptor-enriched membrane micelle preparations and in PDGFbeta receptor overexpressing 293 cells. The intrinsic activity of PDGFbeta receptor kinase was required for the UV-induced enhancement of receptor phosphorylation, but was not changed upon UV irradiation. The data support a mechanism of UV-induced signal transduction involving inactivation of PTPs through an unknown reactive intermediate that oxidizes the conserved cysteine in the active sites of PTPs.

Inhibition of MT-450 rat mammary tumour growth by antibodies recognising subtypes of blood group antigen B.

Using subtractive immunization to identify cell surface epitopes expressed in a metastasis-specific fashion on cells of the rat MT-W9 mammary carcinoma model, we generated a monoclonal antibody called M-N#1. This antibody binds specifically to metastasizing cells of the MT-W9 series and also to certain other metastasizing rat mammary carcinoma cell lines. We demonstrate that the M-N#1 antibody recognizes a fucosylated N-glycosyl sugar modification, and furthermore show that the epitope specificity of the M-N#1 antibody is for blood group antigen B subtypes 2, 3 and 4 with slight cross-reactivity with blood group antigen A subtype 2. The expression of these carbohydrate epitopes on MT-450 cells is functionally important, because the M-N#1 antibody efficiently inhibits MT-450 tumour growth in spontaneous metastasis assays. These results suggest that expression of the subtypes of blood group antigen B recognized by the M-N#1 antibody does not directly participate in the metastatic cascade but rather confers a growth or survival advantage on the tumour cells.