Cross-talk between steroids and NF-kappa B: what language?
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Animals↗
Biomedical subjects
P Herrlich
Publications and source records attributed to P Herrlich.
Synthesis and biological evaluation of aeroplysinin analogues: a new class of receptor tyrosine kinase inhibitors.
Receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR), are critically involved in the transduction of mitogenic signals across the plasma membrane and therefore in the regulation of cell growth and proliferation. Enhanced RTK activity is associated with proliferative diseases such as cancer, psoriasis and atherosclerosis, while decreased function may be associated for instance with diabetes. EGFR and PDGFR are selectively inhibited by analogues of the marine natural product aeroplysinin. The synthetic inhibitors display IC50 values in the low micromolar range and in contrast to the natural product show pronounced inhibitory activity in cultured cells in vivo. The mechanism of inhibition is likely based on a covalent modification of the target enzymes by reaction of epoxy ketone 8 with various nucleophiles.
Acetonitriles↗
The CD44 protein family.
The designation CD44 describes a group of type I transmembrane proteins which share N-terminal and C-terminal sequences. These molecules differ in the central extracellular domain by the use of sequences encoded by ten variant exons which may be completely absent or included in various combinations and by cell type specific addition of glycosaminoglycan and carbohydrate moieties. Expression of variant proteins is observed in normal tissues such as on keratinocytes, dendritic cells and activated lymphocytes in the adult organism and on morphogenetically active epithelium such as the apical ectodermal ridge (AER) in the embryo. Certain CD44 proteins expressed on the AER can act as low affinity fibroblast growth factor receptors and are vital for epithelial-mesenchymal cell communication. CD44 variant proteins have also been implicated in tumour growth and metastasis and we speculate that CD44 mediated growth factor presentation may also be decisive in metastasis formation. Molecular strategies designed to block growth factor presentation by CD44 may aid in the therapy of metastatic cancer.
Adult↗
Activation-dependent modulation of hyaluronate-receptor expression and of hyaluronate-avidity by human monocytes.
During inflammation, activated monocytes (Mo) migrate into tissues where they interact with extracellular matrix components such as hyaluronate (HA), produced in high amounts at inflammatory sites. We determined whether Mo that had invaded sites of cutaneous inflammation bind HA and express the putative HA receptors CD44 isoforms, ICAM-1, or receptor for hyaluronate-mediated motility (RHAMM). In cutaneous inflammation, activated infiltrating Mo displayed high HA avidity and expressed epitopes encoded by CD44s, CD44 variant exons v3, v4, v5, v6, v7, and v9, and ICAM-1, but not RHAMM. We further investigated how activation affects the avidity of Mo for HA and which receptors were responsible for such binding. Mo freshly purified from human peripheral blood bound little HA and expressed CD44s but no epitopes encoded by CD44v exons, ICAM-1, or RHAMM. During short-term tissue culture, Mo upregulated their HA avidity and expression of ICAM-1, CD44s, and epitopes encoded by CD44v, all of which were further augmented by IFN-gamma or lipopolysaccharide, whereas RHAMM was not detectable. Thus in vitro activated Mo resembled Mo that had migrated to inflammatory sites in vivo. Lipolysaccharide or IFN-gamma-induced HA binding was inhibited by more than 90% with monoclonal antibodies directed against N-terminal HA binding domains of CD44s, but not by monoclonal antibodies against CD44v epitopes or ICAM-1. In conclusion, we show that upon in vitro or in vivo activation, Mo enhance their capacity to bind HA. This is critically dependent upon the expression ofCD44s epitopes. Regulated CD44-HA interactions may be important for the ability of Mo to migrate into and within sites of inflammation and for Mo effector functions.
Humans↗
Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein.
The extracellular matrix component hyaluronan is believed to play important roles in various processes of organogenesis, cell migration and cancer. Recognition of and binding to hyaluronan is mediated by cell surface receptors. Three of them, CD44, ICAM-1 and RHAMM (receptor for hyaluronic acid mediated motility), have been identified. A cDNA clone designated RHAMM turned out to possess transforming capacity. Based on this published sequence, we isolated the complete cDNA of the murine gene. The cDNA comprises an open reading frame of 2.3 kb and encodes a 95 kDa protein. The protein carries a hyaluronan binding motif which binds to hyaluronan in vitro but not to heparin or chondroitin sulphate. It is ubiquitously expressed in normal cells and in all tumour cell lines irrespective of their metastatic properties. One tumour cell line, the metastatic Lewis lung carcinoma, expresses a larger 105 kDa variant form of the protein due to a genomic rearrangement. Antibodies raised against the 95 kDa protein were used for subcellular localization studies. The hyaluronan binding protein is not detectable at the cell surface but is rather localized exclusively intracellularly. Clearly, the sequence we have identified encodes a protein with properties substantially different to the RHAMM protein. We tentatively name the protein intracellular hyaluronic acid binding protein, IHABP.
3T3 Cells↗
How tumor cells make use of CD44.
A variant of CD44 containing exon v3 sequences is expressed in the apical ectodermal ridge of the limb bud during embryogenesis. This variant is modified by heparan sulfate moieties and acts as low affinity receptor for FGFs. These FGFs are presented by CD44 to mesenchymal cells which induces their proliferation and limb outgrowth. We suggest that a similar growthfactor presentation mechanism accounts for the function of CD44 variants on metastasizing tumor cells.
Animals↗
CD44 variant isoforms are essential for the function of epidermal Langerhans cells and dendritic cells.
Upon antigen encounter epidermal Langerhans cells (LC) and dendritic cells (DC) emigrate from peripheral organs and invade lymph nodes through the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Part of this process is mimicked by metastasizing tumor cells. Since splice variants of CD44 promote metastasis to lymph nodes we explored the expression of CD44 proteins on migrating LC and DC. We show that following antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6 and v9. Antibodies against CD44 epitopes arrest LC in the epidermis, prevent the binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.
Cell Movement↗
Variant exons v6 and v7 together expand the repertoire of glycosaminoglycans bound by CD44.
Isoforms of the glycoprotein CD44 are cell surface receptors for the glycosaminoglycan hyaluronate. They have been implicated in many biological processes, but their function in these is poorly understood and cannot be explained solely by hyaluronate binding. In the present work we examine the ligand binding properties of alternatively spliced CD44 variant isoforms which are functionally involved in the immune system, embryonic development, and tumor behavior. We show that these isoforms bind directly to the purified glycosaminoglycans chondroitin sulfate, heparin, and heparin sulfate, in addition to being able to bind to hyaluronate. Binding to this extended repertoire of glycosaminoglycans by CD44 depends on the inclusion of peptide sequences encoded by the alternatively spliced exons v6 and v7, and occurs both when the CD44 is solubilized from the plasma membrane and when it is expressed on intact cells. A single point mutation in the most N-terminal hyaluronate binding motif of CD44 ablates both hyaluronate and chondroitin sulfate binding, suggesting that glycosaminoglycans are bound through a common motif, and that only one of the hyaluronate binding motifs is responsible for the majority of glycosaminoglycan binding by CD44 on the cell surface. Taken together, these observations indicate that alternative splicing regulates the ligand binding specificity of CD44 and suggest that structural changes in the CD44 protein have a profound effect on the range of ligands to which this molecule can bind with potentially wide-ranging functional consequences.
Alternative Splicing↗
Interdomain binding mediates tumor growth suppression by the NF2 gene product.
The neurofibromatosis 2 (NF2) tumor suppressor gene encodes an intracellular membrane-associated protein, called merlin (or schwannomin), that belongs to the band 4.1 family of cytoskeleton-associated proteins. Inactivating NF2 mutations occur in several sporadic tumor types and have been linked to the NF2 disease, whose hallmark is the development of bilateral Schwann cell tumors (schwannomas) of the eighth cranial nerve. Two major alternatively spliced NF2 variants are expressed in normal tissues: 'NF2-17' lacking exon 16 and 'NF2-16' that contains exon 16 and encodes a merlin protein truncated at the C-terminus. We report that overexpression of NF2-17 in rat schwannoma cells inhibits their growth in vitro and in vivo, while NF2-16 fails to influence schwannoma growth. Tumor growth inhibition by merlin depends on an interdomain association occurring either in cis or in trans between the N- and C-termini. This association does not occur in the truncated NF2-16 protein nor in a mutant NF2-17 protein lacking C-terminal sequences. These data indicate that merlin has a unique mechanism of tumor suppression, inhibiting cell proliferation via self-association.
Animals↗
CD44 expression is aberrant in benign Schwann cell tumors possessing mutations in the neurofibromatosis type 2, but not type 1, gene.
Atypical expression of CD44 splice variants has been implicated in the progression of numerous tumors. This abnormal CD44 expression is presumed to result from gene alterations that cause tumorigenic transformation. Two tumor types that have been linked to specific gene alterations are schwannomas, which have mutations in the neurofibromatosis (NF) type 2 (NF2) gene, and neurofibromas, which characteristically possess NF type 1 (NF1) gene mutations. We examined CD44 expression in normal sciatic nerves, in schwannomas with confirmed NF2 mutations, and in neurofibromas and malignant peripheral nerve sheath tumor tissue and cell lines from NF1 patients. Compared to normal nerves, schwannomas express higher total levels of CD44 and additional splice variants, whereas CD44 expression in neurofibromas is unaltered. Malignant peripheral nerve sheath tumor tissue and cell lines express the CD44v6 epitope, which is not expressed by normal Schwann cells or by other Schwann cell tumors. These data indicate that altered CD44 expression correlates strictly with mutations in the NF2 but not NF1 gene and suggest that CD44v6 might be a marker for the malignant transformation of Schwann cells.
Alternative Splicing↗
I kappaB alpha-independent downregulation of NF-kappaB activity by glucocorticoid receptor.
I kappaB alpha is an inhibitor protein that prevents nuclear transport-and activation of the transcription factor NF-kappaB. In acute inflammation, NF-kappaB is activated and increases the expression of several pro-inflammatory cytokine and chemokine genes. Glucocorticoids counteract this process. It has been proposed that the glucocorticoid-dependent inhibition of NF-kappaB activity is mediated by increased synthesis of I kappaB alpha which should then sequester NF-kappaB in an inactive cytoplasmic form. Here, we show by the use of a mutant glucocorticoid receptor and steroidal ligands that hormone-induced I kappaB alpha synthesis and inhibition of NF-kappaB activity are separable biochemical processes. A dimerization-defective glucocorticoid receptor mutant that does not enhance the I kappaB alpha level is still able to repress NF-kappaB activity. Conversely, glucocorticoid analogues competent in enhancing I kappaB alpha synthesis do not repress NF-kappaB activity. These results demonstrate that increased synthesis of I kappaB alpha is neither required nor sufficient for the hormone-mediated downmodulation of NF-kappaB activity.
Animals↗
An essential role for CD44 variant isoforms in epidermal Langerhans cell and blood dendritic cell function.
Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.
Animals↗
CREB is activated by UVC through a p38/HOG-1-dependent protein kinase.
Changes in environmental conditions such as the addition of growth factors or irradiation of cells in culture first affect immediate response genes. We have shown previously that short wavelength UV irradiation (UVC) elicits massive activation of several growth factor receptor-dependent pathways. At the level of the immediate response gene c-fos, these pathways activate the transcription factor complex serum response factor (SRF)-p62TCF which mediates part of the UV-induced transcriptional response. These studies have, however, suggested that more that one pathway is required for full UV responsiveness of c-fos. Using appropriate promoter mutations and dominant-negative cAMP response element (CRE)-binding protein (CREB), we now find that UVC-induced transcriptional activation depends also on the CRE at position -60 of the c-fos promoter and on the functionality of a CREB. Upon UV irradiation, CREB and ATF-1 are phosphorylated at serines 133 and 63, respectively, preceded by and dependent on activation of p38/RK/HOG-1 and of a p38/RK/HOG-1-dependent p108 CREB kinase. Although p90RSK1 and MAPKAP kinase 2 are also activated by UV, p90RSK1 does not, at least not decisively, participate in this signalling pathway to CREB and ATF-1 as it is not p38/RK/HOG-1 dependent, and CREB is a poor substrate for MAPKAP kinase 2 in vitro. On the basis of resistance to the growth factor receptor inhibitor suramin and of several types of cross-refractoriness experiments, the UVC-induced CREB/ATF-1 phosphorylation represents an as yet unrecognized route of UVC-induced signal transduction, independent of suramin-inhibitable growth factor receptors and different from the Erk 1,2-p62TCF pathway.
Activating Transcription Factor 1↗
Efficient lysis of CD44v7/8-presenting target cells by genetically engineered cytotoxic T-lymphocytes--a model for immunogene therapy of cervical cancer.
Variant proteins of the CD44 surface glycoprotein family are expressed on many different human tumors and their lymph node metastases. An epitope encoded by sequences of variant exons CD44v7 and v8 and recognized by the monoclonal antibody VFF17 is frequently detected in cervical cancer, whereas the normal cervical epithelium lacks expression of this epitope. We have developed an immunotherapeutic approach for cervical cancer based on the expression of this CD44v7/8 epitope. The single chain antigen-binding fragment of VFF17 was fused to a signal transducing protein (zeta-chain) of the T-cell receptor complex (TCR) and was introduced into a retroviral gene transfer vector. Gene transfer was applied to the murine cytotoxic T-cell line cl96. All recombinant clones expressed the fusion protein on their cell surface. Functionality of the recombinant fusion protein was tested by subjection of several recombinant clones to in vitro cytotoxicity assays. CD44v7/8-expressing target cells were killed efficiently by reprogrammed cl96 in an MHC-independent fashion, whereas CD44v7/ 8-negative cells were not affected. These transfected T cell lines will now be tested in vivo using immune-deficient mice bearing CD44v7/8-expressing tumors.
Amino Acid Sequence↗
UV-induced signal transduction.
Irradiation of cells with wavelength ultraviolet (UVA, B and C) induces the transcription of many genes. The program overlaps with that induced by oxidants and alkylating agents and has both protective and other functions. Genes transcribed in response to UV irradiation include genes encoding transcription factors, proteases and viral proteins. While the transcription factor encoding genes is initiated in minutes after UV irradiation (immediate response genes) and depends exclusively on performed proteins, the transcription of protease encoding occurs only many hours after UV irradiation. Transcription factors controlling the activity of immediate response genes are activated by protein kinases belonging to the group of proline directed protein kinases immediately after UV irradiation. Experimental evidence suggests that these kinases are activated in UV irradiated cells through pathways which are used by growth factors. In fact, the first cellular reaction detectable in UV irradiated cells is the phosphorylation of several growth factor receptors at tyrosine residues. This phosphorylation does not depend on UV induced DNA damage, but is due to an inhibition of the activity of tyrosine phosphatases. In contrast, for late cellular reactions to UV, an obligatory role of DNA damage in transcribed regions of the genome can be demonstrated. Thus, UV is absorbed by several target molecules relevant for cellular signaling, and it appears that numerous signal transduction pathways are stimulated. The combined action of these pathways establishes the genetic program that determines the fate of UV irradiated cells.
Animals↗
Nuclear and non-nuclear targets of genotoxic agents in the induction of gene expression. Shared principles in yeast, rodents, man and plants.
The interplay between environmental cues and the genetic response is decisive for the development, health and well-being of an organism. For some environmental factors a narrow margin separates beneficial and toxic impacts. With the increasing exposure to UV-B this dichotomy has reached public attention. This review will be concerned with the mechanisms that mediate a cellular genetic response to noxious agents. The toxic stimuli find access to the regulatory network inside cells by interacting at several points with cellular molecules - a process that converts the 'outside information' into 'cellular language'. As a consequence of such interactions, many adverse agents cause massive signal transduction and changes of gene expression. There is an interesting conservation of the mechanisms from yeast to man. An understanding of the genetic programs and of their phenotypic consequences is lagging behind.
Animals↗
Growth retardation of tumors by adoptive transfer of cytotoxic T lymphocytes reprogrammed by CD44v6-specific scFv:zeta-chimera.
Variants of the CD44 protein family containing sequences encoded by variant exon 6 (v6) are involved in the metastatic spread of rat and human tumors. The rat-specific antibody 1.1ASML, which recognizes a v6 epitope, interferes with metastatic dissemination of a rat pancreatic carcinoma. The single-chain antigen-binding fragment of this monoclonal antibody was fused to the zeta-chain of the T-cell receptor complex. The appropriate fusion gene was incorporated into a retroviral gene transfer vector. Murine cytotoxic T lymphocytes (CTLs) were infected, and cellular clones which express the single-chain zeta-chain fusion protein on their cell surface were selected. These CTLs are not MHC-restricted in their CD44v6 recognition and exhibit in vitro lytic activity toward cells expressing CD44 variants comprising exon v6. Tumor cell xenografts grown in athymic nude mice are suppressed in their growth upon infusion of the genetically manipulated CTLs. Our data indicate that the CD44v6 epitope is an effective target for immune tumor therapy and demonstrate the efficacy of genetically engineered CTLs in targeting tumors expressing such epitopes.
Adoptive Transfer↗
Dephosphorylation of receptor tyrosine kinases as target of regulation by radiation, oxidants or alkylating agents.
Several non-physiologic agents such as radiation, oxidants and alkylating agents induce ligand-independent activation of numerous receptor tyrosine kinases (RTKs) and of protein tyrosine kinases at the inner side of the plasma membrane (e.g. Dévary et al., 1992; Sachsenmaier et al., 1994; Schieven et al., 1994; Coffer et al., 1995). Here we show additional evidence for the activation of epidermal growth factor receptor (EGFR), and we show activation of v-ErbB, ErbB2 and platelet-derived growth factor receptor. As a common principle of action the inducing agents such as UVC, UVB, UVA, hydrogen peroxide and iodoacetamide inhibit receptor tyrosine dephosphorylation in a thiol-sensitive and, with the exception of the SH-alkylating agent, reversible manner. EGFR dephosphorylation can also be modulated by these non-physiologic agents in isolated plasma membranes in the presence of Triton X-100. Further, substrate (EGFR) and phosphatase have been separated: a membrane preparation of cells that have been treated with epidermal growth factor (EGF) and whose dephosphorylating enzymes have been permanently destroyed by iodoacetamide can be mixed with a membrane preparation from untreated cells which re-establishes EGFR dephosphorylation. This dephosphorylation can be modulated in vitro by UV and thiol agents. We conclude that RTKs exhibit significant spontaneous protein kinase activity; several adverse agents target (an) essential SH-group(s) carried by (a) membrane-bound protein tyrosine phosphatase(s).
Acetylcysteine↗