Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene.

Liver directed gene transfer with adenoviral vectors is being considered for the treatment of several metabolic diseases, including familial hypercholesterolaemia (FH). Gene replacement therapy of human low density lipoprotein (LDL) receptor gene into the murine model of FH transiently corrected the dyslipidaemia; however, humoral and cellular immune responses to LDL receptor developed--possibly contributing to the associated hepatitis and extinguishing of transgene expression. We evaluated an alternative strategy of ectopic expression in the liver of the very low density lipoprotein (VLDL) receptor, which is homologous to the LDL receptor but has a different pattern of expression. Infusion of recombinant adenoviruses containing the VLDL receptor gene corrected the dsylipidaemia in the FH mouse and circumvented immune responses to the transgene leading to a more prolonged metabolic correction.

Recombinant adeno-associated virus for muscle directed gene therapy.

Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli beta-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

Analysis of temperature-sensitive functions of Fos: lack of a correlation between transformation and TRE-dependent trans-activation.

We have identified and characterized a mutant v-Fos protein (DN16G) that is temperature sensitive for transformation. This protein contains an asparagine to glycine substitution at position 156 in the basic region encompassing the DNA contact site. This point mutation also strongly decreases trans-activation in a transient expression assay, using the collagenase 12-O-tetradecanoyl phorbol 13-acetate (TPA)-responsive element (TRE) as the target element. However, the apparent correlation between trans-activation and transformation does not hold in view of the observation that under certain temperature conditions (DN16G at 39.5 degrees C and E300 at 37 degrees C) both proteins showed similarly poor transactivation properties, but dramatically differed in their transforming potential. These findings clearly suggest that the activation of transcription via TREs as analysed in this study is not a crucial mechanism in Fos-induced transformation.

fosB is a transforming gene encoding a transcriptional activator.

The fosB gene encodes a nuclear protein that shows a high degree of homology with c-Fos in several of the known functionally crucial domains, e.g., the leucine zipper and the DNA-binding site, but shows considerable divergence in other regions. Here, we report that FosB, when placed under the control of a constitutive promoter, exhibits clear transforming properties in focus assays using mouse NIH3T3 or rat 208F fibroblasts. The transforming potential of FosB is considerably stronger than that of a corresponding c-fos construct and resembles that of viral fos genes. Using chimeric fos/fosB constructs we show that the C-terminal half of FosB is responsible for these stronger transforming properties, apparently by giving rise to significantly higher levels of protein as compared with the corresponding c-fos sequence. Surprisingly, substitution of the N-terminus of Fos with that of FosB decreases its transforming potential. These differences in the transforming potential are not related to DNA or protein expression, but rather seem to reflect differences in the molecular function(s) encoded in the N-terminal halves of Fos and FosB protein. Both, fosB- and v-fos transformed cells show increased expression of a number of endogenous genes, including c-jun, transin, alpha 1(III) collagen and tissue plasminogen activator. Transactivation by FosB and v-fos of the c-jun and alpha 1(III) collagen gene promoters and of a 3 x TRE-tk chimeric promoter could be shown in transient CAT assays. v-Fos, but not FosB-transformed cells, also show elevated levels of urokinase and plasminogen activator inhibitor mRNAs, pointing to potential differences in the gene regulatory properties of the two Fos family members.

Deregulation of genes encoding microfilament-associated proteins during Fos-induced morphological transformation.

The mechanism of Fos-induced transformation is still poorly understood. In the present study, we have asked whether genes whose products play a role in determining cell morphology might become deregulated in the course of Fos-induced transformation. A clear up-regulation in Fos-transformed rat fibroblasts was seen with ezrin, as well as tropomyosin (TM) -3 and -5B, while TM-1 was down-regulated. Significantly, the same genes were deregulated in a very similar, but hormone-inducible way in cells expressing a Fos-estrogen receptor fusion protein. In agreement with these results, Fos-expressing cells showed decreased levels of two TM isoforms of 36 and 38 kDa, and showed an impaired TM network. The significance of these observations is strengthened by the fact that the deregulation of TM expression has been shown to contribute to morphological transformation in other experimental systems. Deregulation of the TM and ezrin genes preceeds the induction of morphological transformation suggesting that this deregulation is not merely a consequence of transformation. On the other hand, deregulation follows the induction of direct Fos target genes. We therefore propose that a cascade of regulatory events is triggered by Fos oncoproteins which eventually leads to the deregulation of genes encoding cytoskeleton-associated proteins.

Proto-oncogenic properties of the DP family of proteins.

The cellular transcription factor DRTF1/E2F is implicated in the control of cellular proliferation due to its interaction with key regulators of cell cycle progression, such as the retinoblastoma tumour suppressor gene product, cyclins and cyclin-dependent kinases. DRTF1/E2F is a heterodimeric DNA binding activity which arises when a member of two distinct families of proteins, DP and E2F, interact as DP/E2F heterodimers, for example, DP-1 and E2F-1. In DRTF1/E2F the activity of DP-1 is under cell cycle control, possibly by phosphorylation, and in many types of cells it is a frequent, if not general DNA binding component of DRTF1/E2F. The expression of other DP proteins, such as DP-2, is tissue-restricted. Here, we show that DP-1 and DP-2 are integrated with another growth regulating pathway which involves signal transduction emanating from activated Ras protein. Thus, activated Ha-ras can co-operate with DP-1 or DP-2 in the transformation of rat embryo fibroblasts, establishing for the first time that DP proteins are endowed with proto-oncogenic activity. Moreover, an analysis of a dominant-negative and mutant DP-1 proteins suggests that the primary target through which DP-1 mediates its oncogenic activity is unlikely to be due to the regulation of E2F site-transcription, suggesting an E2F-independent effector function for DP-1. These results therefore establish DP genes as proto-oncogenes and thus argue that deregulating the normal control of DP protein activity will be important in promoting aberrant cellular proliferation.

Functional domains in cyclin D1: pRb-kinase activity is not essential for transformation.

Although cyclin D1 plays a major role during cell cycle progression and is involved in human tumourigenesis, its domain structure is still poorly understood. In the present study, we have generated a series of cyclin D1 N- and C-terminal deletion constructs. These mutants were used to define the domains required for transformation of rat embryonal fibroblasts (REF) in cooperation with activated Ha-ras and and to establish correlations with defined biochemical properties of cyclin D1. Protein binding and REF assays showed that the region of the cyclin box required for the interaction with CDK4 as well as C-terminal sequences determining protein stability were crucial for transformation. Surprisingly, however, the N-terminal deletion of 20 amino acids which impaired pRb kinase activity did not affect the transforming ability of cyclin D1. Likewise, no effect on transformation was observed with mutants defective in p21CIP interaction. These observations argue against a crucial role of pRb inactivation or p21CIP squelching in cyclin D1-mediated transformation.

Cyclophosphamide diminishes inflammation and prolongs transgene expression following delivery of adenoviral vectors to mouse liver and lung.

Immune responses to adenovirus-mediated gene transfer contribute to the problems of transient recombinant gene expression, inflammation, and difficulties with vector readministration. Activation of CD4+ T cells is required for full realization of effector function of both CD8+ T cells (i.e., cytotoxic T cells) and B cells (i.e., neutralizing antibody). We evaluate in this study the effectiveness of a short course of high-dose cyclophosphamide to block immune responses in mice administered vector into lung and liver of C57BL/6 mice. Administration of cyclophosphamide with vector directed to liver blocked activation and mobilization of both CD4+ and CD8+ T cells. As a result, transgene expression was prolonged, inflammation was reduced, and, at the higher doses of cyclophosphamide, formation of neutralizing antibody was prevented and the vector was successfully readministered. Similar studies in the lung demonstrated an effective blockade of T and B cell responses. In contrast to the liver, where it was easier to stabilize transgene expression than to prevent neutralizing antibody, cyclophosphamide prevented the formation of neutralizing antibodies at all doses in the lung, whereas stabilization of transgene expression was only achieved at the highest dose. These experiments begin to define the parameters by which cyclophosphamide could be used as an adjunct in gene therapy.

Combination therapy with radiation or cisplatin enhances the potency of Ad5/35 chimeric oncolytic adenovirus in a preclinical model of head and neck cancer.

Ad5/35 chimeric oncolytic viruses (OVs) have earlier been shown to increase the level of vector transduction, intratumoral virus spread and survival in a number of xenograft models when compared with Ad5-based viruses. Because radiation and chemotherapy are the current standards of care for most cancer indications, Ad5/35 OVs have been tested here in combination with either radiation or chemotherapy in a head and neck cancer (HNC) xenograft model to determine whether such combination therapies enhance the potency of the virus, thereby leading to a greater therapeutic effect. In mice treated with either Ad5 OV (OV-5) or Ad5/35 chimeric OV (OV-5T35H), there was a delay in tumor progression compared with animals treated with phosphate-buffered saline (PBS) alone. When Ad5/35 chimeric OV and radiation were used in combination, there was a further delay in tumor progression, which resulted in a significant increase in the mean survival time of tumor-bearing mice compared with Ad5/35 or Ad5 OV monotherapy or to the combination of Ad5 OV with radiation, which was significantly less potent (P<0.0001) compared with the Ad5/35 OV plus radiation combination. Similarly, a combination of Ad5/35 chimeric OV with cisplatin significantly delayed tumor progression compared with Ad5/35 OV or Ad5 OV virus alone or with the combination of Ad5 virus with cisplatin (P<0.01). In summary, the combination of the potent Ad5/35 chimeric OV with either radiation or chemotherapy leads to significantly increased survival of mice bearing highly aggressive tumors, and may therefore offer an effective treatment strategy for patients with difficult to treat HNC.

Comparison of the E3 and L3 regions for arming oncolytic adenoviruses to achieve a high level of tumor-specific transgene expression.

Arming oncolytic adenoviral vectors with anticancer transgenes that can be expressed in a tumor-selective manner may enable the engineering of vectors with increased potency, while retaining their safety profile. Armed oncolytic adenoviral vectors were constructed in which transgene expression has been linked via modified splice acceptor sequences that did not necessitate the deletion of any part of the adenoviral genome. Several oncolytic adenoviral vectors were compared in which the transgene was inserted in place of either the E3 or the L3 region. While all vectors had similar viral growth and cytotoxicity characteristics, the highest level of transgene expression was observed from a vector in which the transgene had been inserted downstream of the L3 23K protease gene, the Ad-23K-GM vector. Notably, no transgene expression occurred with this vector in the absence of DNA replication either in vitro or in vivo. In contrast, viruses in which the transgene was inserted into E3 locations exhibited a low level of transgene expression even in the absence of DNA replication. In summary, by utilizing the L3 region for arming oncolytic viruses, higher levels of tumor-specific transgene expression can be obtained without the need to delete any parts of the viral genome.

Immunity to adenovirus and adeno-associated viral vectors: implications for gene therapy.

Viral vectors have provided effective methods for in vivo gene delivery for therapeutic purposes. The ability of viruses to infect a wide variety of cell types in vivo has been exploited for several applications, such as liver, lung, muscle, brain, eye and many others. Immune responses directed towards the viral capsids and the transgene products have severely affected the ability of these vectors to induce long-term gene expression. This paper reviews the influence of viral vectors on antigen-presenting cells (APC), which are central to the induction of innate as well as adaptive immune responses. In this respect, we have focused on adenovirus and adeno-associated viruses because of the polar responses these vector systems induce in vivo. While adenovirus vector can induce significant inflammatory responses, adeno-associated viral vectors are characterized by their inability to consistantly induce immune responses to the transgene product. Understanding the mechanism of infection, transduction and activation of APC by viral vectors will provide strategies to develop safe vectors and prevent immune responses in gene therapies.

Cytotoxic T-lymphocyte target proteins and their major histocompatibility complex class I restriction in response to adenovirus vectors delivered to mouse liver.

The activation of cytotoxic T lymphocytes (CTLs) to cells infected with adenovirus vectors contributes to problems of inflammation and transient gene expression that attend their use in gene therapy. The goal of this study was to identify in a murine model of liver gene therapy the proteins that provide targets to CTLs and to characterize the major histocompatibility complex (MHC) class I restricting elements. Mice of different MHC haplotypes were infected with an E1-deleted adenovirus expressing human alkaline phosphatase (ALP) or beta-galactosidase as a reporter protein, and splenocytes were harvested for in vitro CTL assays to aid in the characterization of CTL epitopes. A library of vaccinia viruses was created to express individual viral open reading frames, as well as the ALP and lacZ transgenes. The MHC haplotype had a dramatic impact on the distribution of CTL targets: in C57BL/6 mice, the hexon protein presented by both H-2Kb and H2Db was dominant, and in C3H mice, H-2Dk-restricted presentation of ALP was dominant. Adoptive transfer of CTLs specific for various adenovirus proteins or transgene products into either Rag-I or C3H-scid mice infected previously with an E1-deleted adenovirus verified the in vivo relevance of the adenovirus-specific CTL targets identified in vitro. The results of these experiments illustrate the impact of lr gene control on the response to gene therapy with adenovirus vectors and suggest that the efficacy of therapy with adenovirus vectors may exhibit considerable heterogeneity when applied in human populations.

Blunting of immune responses to adenoviral vectors in mouse liver and lung with CTLA4Ig.

Adenoviral vectors deleted of E1 are attractive vehicles for in vivo gene therapy because efficient gene transfer can be achieved. Immune responses to the vector and vector-transduced cells lead to destruction of target cells, inflammation and difficulties with vector readministration. Immune effectors have been identified as CD8+ cytotoxic T lymphocytes, which destroy vector-transduced cells, as well as B cells which secrete neutralizing antibodies and block repeated gene transfer. The central role of CD4+ T cells in the activation of both of these effector functions has focused immunosuppressive strategies towards blockade of costimulatory molecules. We describe in this study a strategy which aims to inhibit CD4+ T cell activation by transiently administering an inhibitor of the CD28/B7 pathway, ie CTLA4Lg, at the time an E1-deleted adenovirus is administered to liver or lung. In lung, CTLA4Ig treatment significantly blocked the formation of neutralizing antibodies, allowing efficient readministration of virus, whereas transgene expression was only moderately prolonged. In contrast, CTLA4Ig did not suppress neutralizing antibody formation in the context of liver gene therapy, but resulted in more stable gene expression. In vitro assays revealed suppression of T cell activation in either organ. This observation suggests that transient inhibition of the CD28/B7 pathway at the time of virus instillation can partially interfere with both arms of the immune response to adenovirus-mediated gene transfer circumventing the need for chronic immune suppression.

An autonomous N-terminal transactivation domain in Fos protein plays a crucial role in transformation.

To date, three functional domains have been defined in c-Fos and v-Fos proteins and have been shown to play a role in transactivation: the leucine zipper mediating hetero-dimerization, the basic DNA contact site, and a C-terminally located transactivation domain (C-TA) harbouring the HOB1 and HOB2 motifs. While the bZip region, consisting of the leucine zipper and the DNA contact site, is indispensable for transformation, the C-TA domain is not required and is actually altered by internal deletions in the FBR-MuSV. We now show that the N-terminal regions of c-Fos and v-Fos contain a second transactivation domain (N-TA). A functionally crucial motif within the N-TA domain, termed NTM, was pinpointed to a approximately 25 amino acid stretch around positions 60-84 which is highly conserved in FosB. Analysis of LexA fusion proteins showed that the N-TA domains of both c-Fos and FosB function in an autonomous fashion in both fibroblasts and yeast. Most importantly, deletion of the NTM motif impairs the transforming properties of v-Fos. Apart from the bZip region, the N-TA domain is the only functional domain required for transformation by v-Fos, at least when its expression is driven by the strong FBR-MuSV-LTR promoter.

Factors influencing cross-presentation of non-self antigens expressed from recombinant adeno-associated virus vectors.

BACKGROUND: We have previously demonstrated that recombinant adeno-associated virus vectors expressing the influenza virus hemagglutinin (rAAV-HA) in skeletal muscle results in T-cell priming and muscle fiber destruction due to cross-presentation of HA by dendritic cells (DC). Based on controversial observations concerning the stability of non-self proteins expressed from rAAV vectors it is important to understand the factors influencing cross-presentation of transgene products following rAAV mediated gene transfer, in order to be able to use this vector safely in the clinic. METHODS: In order to understand the factors influencing in vivo cross-presentation of non-self proteins, we have retargeted the immunogenic lacZ protein in the context of rAAV from the cytoplasm to the cell surface and studied the activation of LacZ specific immune responses following intramuscular mediated gene transfer. In addition, using tools available for studying in vitro HA-specific T-cell activation, our aim was to identify the cell types involved in class I and class II restricted cross-presentation as well as the nature of the cross-presented material. RESULTS: By retargeting the lacZ protein in the context of rAAV to the cell membrane, we found that one of the factors influencing the efficiency of cross-presentation of non-self antigens is the localization of the transgene product within the target cells. Following rAAV-LacZ mediated gene transfer to the muscle we demonstrated that the membrane-bound form of LacZ resulted in target cell destruction, which is in stark contrast to the stability observed with rAAV-LacZ vectors expressing the cytoplasmic form of LacZ. Using an in vitro assay, we were able to show that dendritic cells (DC) in addition to B-cells cross-presented HA to class II restricted T-cells whereas only the former were able to activate class I restricted CD8+ T-cells. High-dose antigens were needed for efficient class I restricted T-cell priming, whereas class II restricted T-cells were activated by less antigen. CONCLUSION: The present results indicate that immune responses to non-self antigens expressed from rAAV vectors depend on the accessibility of such antigens to different local antigen-presenting cells.

Successful interference with cellular immune responses to immunogenic proteins encoded by recombinant viral vectors.

Vectors derived from the adeno-associated virus (AAV) have been successfully used for the long-term expression of therapeutic genes in animal models and patients. One of the major advantages of these vectors is the absence of deleterious immune responses following gene transfer. However, AAV vectors, when used in vaccination studies, can result in efficient humoral and cellular responses against the transgene product. It is therefore important to understand the factors which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies. We have compared T-cell activation against a strongly immunogenic protein, the influenza virus hemagglutinin (HA), which is synthesized in skeletal muscle following gene transfer with an adenovirus (Ad) or an AAV vector. In both cases, cellular immune responses resulted in the elimination of transduced muscle fibers within 4 weeks. However, the kinetics of CD4(+) T-cell activation were markedly delayed when AAV vectors were used. Upon recombinant Ad (rAd) gene transfer, T cells were activated both by direct transduction of dendritic cells and by cross-presentation of the transgene product, while upon rAAV gene transfer T cells were only activated by the latter mechanism. These results suggested that activation of the immune system by the transgene product following rAAV-mediated gene transfer might be easier to control than that following rAd-mediated gene transfer. Therefore, we tested protocols aimed at interfering with either antigen presentation by blocking the CD40/CD40L pathway or with the T-cell response by inducing transgene-specific tolerance. Long-term expression of the AAV-HA was achieved in both cases, whereas immune responses against Ad-HA could not be prevented. These data clearly underline the importance of understanding the mechanisms by which vector-encoded proteins are recognized by the immune system in order to specifically interfere with them and to achieve safe and stable gene transfer in clinical trials.

Asymmetrical recognition of the palindromic AP1 binding site (TRE) by Fos protein complexes.

Fos and Jun proteins form a tight complex which binds specifically to the AP1 recognition sequence, a palindromic DNA element also referred to as the TPA responsive element (TRE). To elucidate the mechanism of Fos-Jun interaction with the TRE we have performed UV cross-linking studies using oligonucleotides where thymines were replaced with bromouracil. Our results indicate that both Fos and Jun directly contact the TRE but that the interaction of Fos and Jun with thymines in structurally equivalent positions in the two half sites of the TRE is different. In addition, we have carried out a comprehensive mutagenesis study of the TRE by introducing all possible point mutations plus thymine----uracil substitutions into the palindromic TRE core sequences and the adjacent nucleotides on both sides. The results of this analysis clearly show that the palindromic TRE is asymmetrical with respect to binding of Fos-Jun. We also show that a Fos protein complex with a homodimeric DNA binding site binds considerably less efficiently to TRE mutants with a perfect dyad symmetry compared with the binding to the wild-type TRE. This demonstrates that the asymmetrical recognition of the TRE is not due to the heterodimeric nature of the Fos/Jun complex but directly related to an asymmetry in the TRE sequence. The methyl groups of all four thymine residues within the TRE seem to be functionally crucial since thymine----uracil substitutions strongly reduce or abolish binding to Fos/Jun. The relevance of structurally equivalent methyl groups in the TRE core sequence is different, lending further support to the conclusion that the TRE is asymmetrical.

Immune responses to viral antigens versus transgene product in the elimination of recombinant adenovirus-infected hepatocytes in vivo.

Human adenoviruses have been developed as an attractive vehicle for in vivo liver-directed gene therapy. Problems with the application of first generation recombinant adenoviruses to liver-directed gene therapy have been transient expression of the recombinant gene and development of hepatitis. Previous studies in mouse models of gene transfer to liver and lung suggested that MHC class I-restricted cytotoxic T lymphocytes (CTLs) to viral antigens may be effectors in the elimination of transgene expression. The goal of this study was to evaluate the importance of viral antigens versus transgene product in inducing CTL mediated hepatocyte destruction in vivo. Immunization of C57BL/6 mice with a lacZ-expressing adenovirus elicited CTL responses to both viral antigens and the transgene product, beta-galactosidase (beta-gal). Adoptive transfer experiments, as well as studies involving lacZ-transgenic mice (ROSA-26) revealed that CTLs to viral antigens are sufficient to destroy virus-infected hepatocytes, indicating that CTLs to beta-gal can not solely account for the observed hepatocyte destruction that has characterized the use of first generation viruses. In addition, we confirmed that B cell-mediated events do not participate in destruction of hepatocytes in vivo, despite the production of virus- and beta-gal-specific antibodies. These data confirm the hypothesis that viral gene expression elicits host responses that contribute to the problem of transgene instability. Recombinant adenoviruses must be redesigned to diminish viral gene expression if they are to be used in the treatment of chronic diseases.

Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers.

Immune responses to vector-corrected cells have limited the application of gene therapy for treatment of chronic disorders such as inherited deficiency states. We have found that recombinant adeno-associated virus (AAV) efficiently transduces muscle fibers in vivo without activation of cellular and humoral immunity to neoantigenic transgene products such as beta-galactosidase, which differs from the experience with recombinant adenovirus, where vibrant T-cell responses to the transgene product destroy the targeted muscle fibers. T cells activated following intramuscular administration of adenovirus expressing lacZ (AdlacZ) can destroy AAVlacZ-transduced muscle fibers, indicating a prior state of immunologic nonresponsiveness in the context of AAV gene therapy. Adoptive transfer of dendritic cells infected with AdlacZ leads to immune mediated elimination of AAVlacZ-transduced muscle fibers. AAVlacZ-transduced antigen-presenting cells fail to demonstrate beta-galactosidase activity and are unable to elicit transgene immunity in adoptive transfer experiments. These studies indicate that vector-mediated transduction of dendritic cells is necessary for cellular immune responses to muscle gene therapy, a step which AAV avoids, providing a useful biological niche for its use in gene therapy.

Mutual transrepression of Fos and the glucocorticoid receptor: involvement of a functional domain in Fos which is absent in FosB.

In this study, we show that Fos protein can repress transactivation by the glucocorticoid receptor (GR). In addition, we demonstrate that GR is capable of inhibiting, in a hormone-dependent fashion, Fos-mediated transactivation of AP-1 dependent transcription. Moreover, repression of the serum response element by Fos is abolished by the GR in the presence of hormone. Transrepression of glucocorticoid mediated induction involves a region of Fos, located between amino acids 40 and 111, to which no function has been previously assigned, and which is poorly conserved among Fos, FosB and Fra-1. In agreement with this finding, FosB is not capable of transrepressing GR activation of transcription, representing the first functional difference between Fos and FosB. We have mapped the domain of the GR which is required for repression of AP-1 dependent transcription, to the region of central DNA binding domain. Our results suggest that Fos and the GR may form transcriptionally inactive complexes and point to a regulatory interrelationship between different signal transduction pathways.