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The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo fibroblasts bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal growth, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo fibroblasts, including 143 known genes. Of the 143 genes, program-assisted searching revealed 66 informative genes linked to Egr1. All 66 genes could be placed on a single regulatory network consisting of three branch points of known Egr1 target genes: TGFβ1, IL6, and IGFI. Moreover, 19 additional genes that are known targets of p53 were identified, indicating that p53 is a fourth branch point. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation confirmed that p53 is a direct target of Egr1. Because deficient p53 expression causes tumors in mice, we tested the role of Egr1 in a two-step skin carcinogenesis study (144 mice) that revealed a uniformly accelerated development of skin tumors in Egr1-null mice (P < 0.005). These studies reveal a new role for Egr1 as an in vivo tumor suppressor.

Supplementary Materials and Table 1 from Early Growth Response 1 Acts as a Tumor Suppressor In vivo and In vitro via Regulation of p53

DNA microarrays of promoter sequences have been developed in order to identify the profile of genes bound and activated by DNA regulatory proteins such as the transcription factors c-Jun and ATF2 as well as DNA-modifying methylases. The arrays contain 3083 unique human promoter sequences from +500 to -1000 nts from the transcription start site. Cisplatin-induced DNA damage rapidly leads to specific activation of the Jun kinase pathway leading to increased phosphorylation of c-Jun and ATF2-DNA complexes at hundreds of sites within 3 hours. Using three statistical criteria, approximately 269 most commonly phosphorylated c-Jun/ATF2-DNA complexes were identified and representative cases were verified by qPCR measurement of ChIP-captured DNA. Expression was correlated at the mRNA and protein levels. The largest functional cohort was 24 genes of known DNA repair function, most of which exhibited increased protein expression indicated coordinate gene regulation. In addition, cell lines of prostate cancer exhibit stable methylation or copy number changes that reflect the alterations of the corresponding primary tumors. 504 (18.5%) promoters showed differential hybridization between immortalized control prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, eight had previously been observed in prostate cancer, and 13 were previously determined methylation targets in other cancers. The vast majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes to study the role of DNA methylation in prostate tumors. Earlier studies using prototype promoter arrays examine approximately 7% of the proximal regulatory sequences while the current gene regulatory events surveyed here occur on a large scale and may rapidly effect the coordinated expression of a large number of genes.

Recent studies are reviewed indicating that the transcription factor early growth response-1 (Egr1) is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin. The downstream pathways of these factors display multiple nodes of interaction with each other, suggesting the existence of a functional network of suppressor factors that serve to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr1 is oncogenic in prostate cancer. In the majority of these cancers, PTEN or p53 is inactive. It is suggested that these defects in the suppressor network allow for the unopposed induction of TGFbeta1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, and explain the role of Egr1 in prostate cancer. Egr1 is a novel and logical target for intervention by gene therapy methods, and targeting methods are discussed.

DNA methylation and copy number in the genomes of three immortalized prostate epithelial and five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, and PC3M-LN4) were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme HpaII, followed by linker ligation, polymerase chain reaction (PCR) amplification, labeling, and hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5%) showed differential hybridization between immortalized prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, and TSPY) previously observed in prostate cancer and 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, and WIT-1). The majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, and GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

The Early Growth Response protein (Egr-1) is a C(2)H(2)-zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation-reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H(2)O(2) stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H(2)O(2) stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.

PTEN is a frequently mutated tumor suppressor in malignancies. Interestingly, some malignancies exhibit undetectable PTEN protein without mutations or loss of PTEN mRNA. The cause(s) for this reduction in PTEN is unknown. Cancer cells frequently exhibit loss of cadherin, beta-catenin, alpha-catenin and/or vinculin, key elements of adherens junctions. Here we show that F9 vinculin-null (vin(-/-)) cells lack PTEN protein despite normal PTEN mRNA levels. Their PTEN protein expression was restored by transfection with vinculin or by inhibition of PTEN degradation. F9 vin(-/-) cells express PTEN protein upon transfection with a vinculin fragment (amino acids 243-1066) that is capable of interacting with alpha-catenin but unable to target into focal adhesions. On the other hand, disruption of adherens junctions with an E-cadherin blocking antibody reduced PTEN protein to undetectable levels in wild-type F9 cells. PTEN protein levels were restored in F9 vin(-/-) cells upon transfection with an E-cadherin-alpha-catenin fusion protein, which targets into adherens junctions and interacts with beta-catenin in F9 vin(-/-) cells. beta-Catenin is known to interact with MAGI-2. MAGI-2 interaction with PTEN in the cell membrane is known to prevent PTEN protein degradation. Thus, MAGI-2 overexpression in F9 vin(-/-) cells restored PTEN protein levels. Moreover, expression of vinculin mutants that reinstated the disrupted interactions of beta-catenin with MAGI-2 in F9 vin(-/-) cells also restored PTEN protein levels. These studies indicate that PTEN protein levels are dependent on the maintenance of beta-catenin-MAGI-2 interaction, in which vinculin plays a critical role.

p300 and CBP are large (300 kDa) nuclear scaffold proteins that act as co-activators of transcription in most cell types and are over-expressed in prostate cancer. Recently, the Egr1 transcription factor was shown to up- or down-regulate p300 and CBP transcription based on the nature of its post-translational modification. Notably, interactions of the three proteins provide fine tuning for Egr1-induced growth or cell death responses. © 2005 Wiley-Liss, Inc.

Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin−/−) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal–regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin–FAK interactions. A vinculin fragment (amino acids 811–1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin−/− cells. Both vinY822F and vin−/− cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin−/− and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction.

The tumor suppressor PTEN is altered in many cancers, including breast cancer, but only a handful of factors are known to control its expression. PTEN plays a vital role in cell survival and proliferation by regulating Akt phosphorylation, a key component of the phosphatidylinositol 3 kinase (PI3K) pathway. Here we show that insulin-like growth factor-II (IGF-II), which signals through PI3K, regulates PTEN expression in the mammary gland. IGF-II injection into mouse mammary gland significantly increased PTEN expression. Transgenic IGF-II expression also increased mammary PTEN protein, leading to reductions in Akt phosphorylation, epithelial proliferation, and mammary morphogenesis. IGF-II induced PTEN promoter activity and protein levels and this involved the immediate early gene egr-1. Thus, we have identified a novel negative feedback loop within the PI3K pathway where IGF-II induces PTEN expression to modulate its physiologic effects.

Egr1 is a multifunctional transcription factor regulating a remarkable spectrum of cellular responses from survival to apoptosis, growth to growth arrest, differentiation to transformation, senescence as well as memory and learning effects. In prostate cancer, Egr1 levels are constitutively high and closely linked to cancer development and progression. This zinc-finger protein is a short-lived, immediate early growth response gene known to be induced by a large number of extracellular stimuli such as irradiation (all wavelengths tested), hypoxia, hyperoxia, chemotherapy agents, and more. Therefore the target genes that Egr1 regulates in prostate cancer cells play an important role in generating many of the cellular responses that characterize these cells. After Egr1 binds to its binding sites on gene promoters, specificity of response is determined by whether Egr1 transcriptionally up- or downregulates the target genes. Expression microarray analyses combined with binding data promise new ways to identify stage specific cancer markers, to aid in patient risk assessment and in therapeutic choices.

In the majority of aggressive tumorigenic prostate cancer cells, the transcription factor Egr1 is overexpressed. We provide new insights of Egr1 involvement in proliferation and survival of TRAMP C2 prostate cancer cells by the identification of several new target genes controlling growth, cell cycle progression, and apoptosis such as cyclin D2, P19ink4d, and Fas. Egr1 regulation of these genes, identified by Affymetrix microarray, was confirmed by real-time PCR, immunoblot, and chromatin immunoprecipitation assays. Furthermore we also showed that Egr1 is responsible for cyclin D2 overexpression in tumorigenic DU145 human prostate cells. The regulation of these genes by Egr1 was demonstrated using Egr1 antisense oligonucleotides that further implicated Egr1 in resistance to apoptotic signals. One mechanism was illustrated by the ability of Egr1 to inhibit CD95 (Fas/Apo) expression, leading to insensitivity to FasL. The results provide a mechanistic basis for the oncogenic role of Egr1 in TRAMP C2 prostate cancer cells.

Egr1, an immediate early transcription factor, responds to diverse stimuli and affects gene transcription to accomplish its biological effects. One important effect of Egr1 expression is to decrease the growth and tumorigenic potential of several tumor cell types. To identify important Egr1 target genes, we have adapted a methodology involving formaldehyde-induced protein-DNA cross-linking, chromatin immunoprecipitation, and multiplex PCR. Using this approach, we report the cloning of a new Egr1 target gene that is able to account, at least in part, for the growth inhibitory activity of Egr1. We have named this new protein TOE1 fortarget of E gr1.

Cripto, a growth factor with an EGF-like domain, and the first member of the EGF-CFC family of genes to be sequenced and characterized, contributes to deregulated growth of cancer cells. A role for Cripto in tumor development has been described in the human and the mouse. Members of the EGF-CFC family are found only in vertebrates: CFC proteins in zebrafish, Xenopus, chick, mouse and human have been characterized and indicate some common general functions in development. Cripto expression was first found in human and mouse embryonal carcinoma cells and male teratocarcinomas, and was demonstrated to be over-expressed in breast, cervical, ovarian, gastric, lung, colon, and pancreatic carcinomas in contrast to normal tissues where Cripto expression was invariably low or absent. Cripto may play a role in mammary tumorigenesis, since in vitro, Cripto induces mammary cell proliferation, reduces apoptosis, increases cell migration, and inhibits milk protein expression. This prediction is strengthened by observations of Cripto expression in 80% of human and mouse mammary tumors. At least three important roles for Cripto in development have created considerable interest, and each activity may be distinct in its mechanism of receptor signaling. One role is in the patterning of the anterior-posterior axis of the early embryo, a second is a crucial role in the development of the heart, and a third is in potentiating branching morphogenesis and modulating differentiation in the developing mammary gland. Whether these properties are functions of different forms of Cripto, different Cripto receptors or the distinct domains within this 15-38 kDa glycoprotein are examined here, but much remains to be revealed about this evolutionarily conserved gene product. Since all Cripto receptors have not yet been determined with certainty, future possible uses as therapeutic targets remain to be developed. Cripto is released or shed from expressing cells and may serve as an accessible marker gene in the early to mid-progressive stages of breast and other cancers. Meanwhile some speculations on possible receptor complexes for Cripto signaling in mammary cells are offered here as a spur to further discoveries.

The PTEN tumour suppressor and pro-apoptotic gene is frequently mutated in human cancers. We show that PTEN transcription is upregulated by Egr-1 after irradiation in wild-type, but not egr-1-/-, mice in vivo. We found that Egr-1 specifically binds to the PTEN 5' untranslated region, which contains a functional GCGGCGGCG Egr-1-binding site. Inducing Egr-1 by exposing cells to ultraviolet light upregulates expression of PTEN messenger RNA and protein, and leads to apoptosis. egr-1-/- cells, which cannot upregulate PTEN expression after irradiation, are resistant to ultraviolet-light-induced apoptosis. Therefore, Egr-1 can directly regulate PTEN, triggering the initial step in this apoptotic pathway. Loss of Egr-1 expression, which often occurs in human cancers, could deregulate the PTEN gene and contribute to the radiation resistance of some cancer cells.

EGR-1, a transcription factor with important functions in the regulation of growth and differentiation, is highly expressed in brain. Previous studies have shown that EGR-1 suppresses the transformed phenotype. However, the expression and role of EGR-1 in human glioblastoma cells are not yet determined. In this study, we found that the basal expression of the EGR-1 protein is undetectable, but is inducible in four human glioblastoma cell lines. To determine EGR-1 functions, we re-expressed EGR-1 in human glioblastoma U251 cells and found that the secretion of transforming growth factor-beta1 (TGF-beta1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin (FN) was greatly enhanced. Addition of anti-TGF-beta antibodies completely inhibited the secretion of PAI-1, but had little effect on secretion of FN, indicating that PAI-1 is under the control of EGR-1-induced TGF-beta1. An examination of the promoter of the FN gene revealed two EGR-1-binding sites between positions -75 and -52 and positions -4 and +14 that specifically bound EGR-1 in gel mobility shift experiments. Utilizing wild-type and mutant FN promoter/luciferase reporter genes, we demonstrated that EGR-1 positively regulated the activity of the FN gene. In addition, cell adhesion and migration were greatly increased in the EGR-1-expressing cells, and adhesion was reversed by addition of RGD-containing peptides. These results suggest that EGR-1 may regulate cell interaction with the extracellular matrix by coordinated induction of TGF-beta1, FN, and PAI-1 in human glioblastoma cells.

A methodology is described that allows the in vivo trapping of transcription factors to their target regulatory elements in multiple genes simultaneously. Cross-linking using formaldehyde is the first of several steps to isolate, purify, clone and characterize multiple gene promoter DNA fragments. The example that we use indicates that the TGFβ1 gene is a direct target induced by Egr-1 in HT1080 cells that express constitutive Egr-1, thus explaining the growth retardation that follows Egr-1 expression. The genes identified using this procedure reflect the specific activities of Egr-1 at that moment in the cell and provide a method for confirmation of genes that are the direct targets of Egr-1 action.

Mounting evidence supports the role of truncated vinculin in the intracellular actin-based motility of Shigella flexneri. Vinculin's role was recently questioned by Goldberg [1997: Cell Motil Cytoskeleton 37:44–53] who observed Shigella motility in mouse embryonal carcinoma 5.51 cells, a genetically modified cell line that reputedly lacked vinculin. That challenge implicitly relied on the assumption that 5.51 cells had no detectable vinculin polypeptide and lacked full-length vinculin mRNA. Despite the appearance of being an unambiguous test of vinculin's role in Shigella motility, 5.51 cells were shown to contain adequate amounts of truncated vinculin (as well as the corresponding mRNA transcript) to support bacterial locomotion. We also examined Shigella locomotion in γ229 cells, a related embryonal carcinoma cell line containing approximately one-half the vinculin content found in 5.51 cells. We observed that there was a commensurate twofold decrease in the Shigella motility rate, as compared to 5.51 cells; this finding raises the possibility that vinculin can become a rate-limiting factor under some circumstances. Immunofluorescence microscopy using vin 11-5 monoclonal antibody directed against the vinculin head domain showed intense staining of Shigella rocket tails in both γ229 and 5.51 cells. Our findings clearly demonstrate that motility in 5.51 cells cannot be regarded as a valid criterion for evaluating the role of truncated vinculin in Shigella motility. Cell Motil. Cytoskeleton 45:272–278, 2000 © 2000 Wiley-Liss, Inc.

Amphiregulin (Ar) and Cripto (Cr) are autocrine growth factors for mammary cells and both have been observed to exhibit high expression in human mammary tumors, in contrast with adjacent tissues. To investigate whether Ar and Cr play roles in the progression of mammary cell proliferation to unregulated growth and tumor formation, the levels of expression were examined in transgenic mice (TGM) that over-express several different oncogenes: MMTV-Polyoma virus middle T antigen (MMTV-PyMT), MMTV-c-ErbB2 (c-neu, HER2) and MT-hTGF alpha. These transgenic mice all produce mammary tumors but with different rates of progression. The levels of Ar were induced up to 10-fold in association with hyperplasia in 2 of the TGM. Cr overexpression was consistently observed in hyperplastic mammary glands in all the animal models, decreasing in overt tumors in 2 of the TGM models. In MMTV-PyMT mammary glands, the levels of Cr expression rose 7- to 10-fold in hyperplastic tissue and 25-fold the levels in tumors compared to age-matched transgene negative mice. Ar and especially Cr thus should have potential value as markers of preneoplastic change in mammary tissue.

Re-expression of EGR-1 in fibrosarcoma HT1080 suppresses transformation including tumorigenicity (Huang, R.-P., Liu, C., Fan, Y., Mercola, D., and Adamson, E. (1995) Cancer Res. 55, 5054-5062) owing in part to up-regulation of the transforming growth factor (TGF)-beta1 promoter by EGR-1 which suppresses growth by an autocrine mechanism (Liu, C., Adamson, E., and Mercola, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11831-11836). Here we show that enhanced cell attachment contributes to the suppression via increased secretion of fibronectin (FN) and also of plasminogen activator inhibitor-1 (PAI-1). The secretion of FN and PAI-1 is strongly correlated with EGR-1 expression (RPEARSON = 0.971 and 0. 985, respectively). Addition of authentic TGF-beta1 to parental cells greatly stimulated secretion of PAI-1 but not FN, whereas addition of TGF-beta antibody or lipofection with specific antisense TGF-beta1 oligonucleotides to EGR-1-regulated cells completely inhibits the secretion of PAI-1 but not FN. However, in gel mobility shift assays pure EGR-1 or nuclear extracts of EGR-1-regulated cells specifically bind to two GC-rich elements of the human FN promoter at positions -75/-52 and -4/+18, indicating that the increased secretion of FN is likely due to direct up-regulation by EGR-1. Moreover, adhesion was greatly enhanced in EGR-1-regulated cells and was reversed by treatment with Arg-Gly-Asp (RGD) or PAI-1 antibody indicating that the secreted proteins are functional. We conclude that EGR-1 regulates the coordinated expression of gene products important for cell attachment ("oikis" factor) and normal growth control.

Previously, we showed that the transcription factor Egr-1 suppressed the proliferation of v-sis transformed NIH3T3 cells and also a number of human tumor cells. Here, we investigate the possible mechanisms responsible for this function. We show that transfected Egr-1 in human fibrosarcoma cells HT1080 leads to down-regulation of Bcl-2. Transient CAT transfection assays reveal that expression of Egr-1 suppresses Bcl-2 promoter activity in a dose-dependent manner. Furthermore, overexpression of Bcl-2 in Egr-1-expressing HT1080 cells enhanced cell proliferation in monolayer culture and increased anchorage-independent growth. Our results suggest that suppression of tumor cell proliferation by Egr-1 may be at least partially mediated through the down-regulation of Bcl-2.

The molecular events of cardiac lineage specification and differentiation are largely unknown. Here we describe the involvement of a growth factor with an EGF-like domain, Cripto-1 (Cr-1), in cardiac differentiation. During embryonic development, Cr-1 is expressed in the mouse blastocyst, primitive streak, and later is restricted to the developing heart. To investigate the role of Cr-1, we have generated Cr-1-negative embryonic stem (ES) cell lines by homologous recombination. The resulting double "knockout" ES cells have selectively lost the ability to form beating cardiac myocytes, a process that can be rescued by reintroducing Cr-1 gene back into the Cr(-/-) cells. Furthermore, the lack of functional Cr-1 is correlated with absence of expression of cardiac-specific myosin light and heavy chain genes during differentiation. Differentiation into other cell types including skeletal muscle is not disrupted. These results suggest that Cr-1 is essential for contractile cardiomyocyte formation in Vitro. (C) 1998 Academic Press.

Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, gamma 227 and gamma 229, and a reconstituted gamma 229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins alpha- and beta-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines. BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculin-negative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted gamma 229 and gamma 227 cells that were formed in suspension during a two-hour static incubation at 37 degrees C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.

Many ubiquitously expressed genes, including oncogenes, lack a proximal TATA or CAAT box but have a region of G + C-rich sequences that appears to replace the usual promoter initiation site. The zinc-finger protein Sp1 is one of the prevalent activators of these genes. The Egr-1 zinc-finger protein has a similar binding site and if the two sites occur in the same region, a variety of activation or inhibitory responses may be obtained. We show that competition between the two factors for overlapping sites on growth-promoting genes could explain why the overexpression of Egr-1 suppresses transformed growth in a number of cell types [Huang et al. (1995): Cancer Res 55:5054–5062; Huang et al. (1997): Int J Cancer]. We demonstrate here that Egr-1 and Sp1 can bind to the same G + C-rich sites and that Egr-1 can displace Sp1 and hence inhibit its activity. We measured the responses of synthetic consensus binding sites and natural promoter sequences linked to a reporter gene and showed that Egr-1 inhibited the activation of transcription by Sp1 on overlapping Sp1/Egr-1 sites. In contrast, Sp1 activity could be augmented by Egr-1 at nonoverlapping sites in the Egr-1 gene promoter, in transient reporter gene studies in Drosophila SL2 cells. In addition, over-expression of exogenous Sp1 in mammalian cells, also leads to increased Egr-1 protein expression, which further inhibits Sp1 transactivation of numerous genes. Therefore, we can account for some of the complex responses of G + C-rich enhancer/promoters by a form of “facilitated inhibition” of Sp1 by Egr-1 at overlapping sites. J. Cell. Biochem. 66:489–499, 1997. © 1997 Wiley-Liss, Inc.

We have examined several types of tumor cell lines and shown that they invariably expressed little or no Egr-1, in contrast to their normal counterparts. We have previously shown that the expression of exogenous Egr-1 in human breast and other tumor cells markedly reduces transformed growth and tumorigenicity. We therefore hypothesized that the loss of Egr-1 expression plays a role in transformation. All human and mouse breast cancer cell lines and tumors examined had reduced Egr-1 expression compared with their normal counterparts. Reduced Egr-1 expression was also observed in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, and this level increased to normal levels in tumors that regressed after tamoxifen treatment. We concluded, therefore, that loss of Egr-1 expression may play a role in the deregulation of normal growth in the tumorigenic process and that Egr-1 acts as a tumor suppressor gene.

Amphiregulin (Ar) is an EGF receptor ligand that functions to modulate the growth of both normal and malignant epithelial cells. We asked whether mouse preimplantation embryos express Ar, and if so, what the function of Ar is during preimplantation development. We used RT-PCR to show expression of Ar mRNA in mouse blastocysts, and using a polyclonal anti-Ar antibody and indirect immunofluorescence, we detected the presence of Ar protein in morula- and blastocyst-stage embryos. Ar protein was present in both the cytoplasm and nucleus in both morulae- and blastocyst-stage embryos, which is similar to Ar distribution in other cell types. Embryos cultured in Ar developed into blastocysts more quickly and also exhibited increased cell numbers compared to control embryos. In addition, 4-cell stage embryos cultured in an antisense Ar phosphorothioate-modified oligodeoxynucleotide (S-oligo) for 48 hr exhibited slower rates of blastocyst formation and reduced embryo cell numbers compared to embryos exposed to a random control S-oligo. TGF-α significantly improved blastocyst formation, but not cell numbers, for embryos cultured in the antisense Ar S-oligo. From these observations, we propose that Ar may function as an autocrine growth factor for mouse preimplantation embryos by promoting blastocyst formation and embryo cell number. We also propose that blastocyst formation is stimulated by Ar and TGF-α, while Ar appears to exert a greater stimulatory effect on cell proliferation than does TGF-α in these embryos. Mol. Reprod. Dev. 47:271–283, 1997. © 1997 Wiley-Liss, Inc.

Retinoids cause differentiation in embryonal carcinoma (EC) cells, thus mimicking events in mammalian development. Here, we show that retinoids also cause apoptosis in P19 EC cells. Characteristic DNA fragmentation was observed within 36 h after addition of retinoic acid (RA). Synthetic retinoids that are selective for RA receptors (RAR) were also effective in inducing apoptosis, whereas RXR selective ligands were without effect. The combination of RAR and RXR ligands resulted in a synergistic increase in apoptotic cell death. As with apoptosis, neuronal differentiation of P19 cells was synergistically induced by the combination of RAR and RXR ligands. Data obtained with an RAR antagonist and with P19 cells carrying a dominant negative RXR indicate that the two processes are receptor mediated. Together, our results indicate that retinoid-induced apoptosis and neuronal differentiation are closely coupled, and that both RAR and RXR play a role in these processes as active receptors for their respective ligands.

Exposure of mammalian cells to UV irradiation induces rapid and transient expression of early growth response-1 gene (Egr-1) encoding a transcription factor that plays a role in cell survival. These signals from the irradiated cell surface are likely to involve more than one pathway, and we show here that an essential pathway involves activation of several growth factor receptors by reactive oxygen intermediates (ROI). UVC irradiation causes the tyrosine phosphorylation of EGF receptor (EGFR) in mouse NIH 3T3 fibroblasts and HC11 mouse mammary cells. EGFR activation by irradiation of cells is abrogated by suramin, by antioxidants, and by the presence of a dominant negative EGFR. UV induces the formation of complexes between activated EGFR and SOS, Grb2, PLC gamma, and SHC that can be precipitated with antibodies to EGFR. The activation of EGFR by UV is mimicked by H2O2, suggesting that ROI may function upstream of EGFR activation. Our observations support the hypothesis that ROI and growth factor receptors operate in the early steps of the UV signal that lead to the enhanced expression and activity of Egr-1.

Tissue factor (TF) gene expression is rapidly induced in epithelial cells by phorbol 12-myristate 13-acetate and serum. We have shown that this induction is mediated by a novel serum response region (SRR) (-111 to +14 bp) within the human TF promoter. In this study, we characterized cis-acting genetic elements within the SRR that regulated basal and inducible expression of the TF gene in HeLa cells. Gel mobility shift assays using oligonucleotides spanning the entire SRR identified three 12-base pair (bp) motifs within subregions 1, 2, and 3 that bound constitutively expressed Sp1 and inducibly expressed EGR-1. Analysis of protein binding to these 12-bp motifs by competition with Sp1 and EGR-1 sites, mutation, and antibody supershift experiments indicated that they each contained distinct EGR-1 and Sp1 sites that overlapped by 6 bp. Functional studies using HeLa cells transfected with plasmids containing the wild-type TF promoter (-111 to +14 bp) or derivatives containing mutations in the three Sp1 and/or EGR-1 sites examined basal and inducible expression. The Sp1 sites mediated basal promoter activity, and both Sp1 and EGR-1 sites were required for maximal induction of the TF promoter by phorbol 12-myristate 13-acetate or serum. These data indicated that TF gene expression in HeLa cells was regulated by both Sp1 and EGR-1.

The transcription factor EGR-1 is a potential regulator of over 30 genes and plays a role in growth, development, and differentiation and, in addition, has significant transformation suppression activity. The regulatory properties are reviewed and a hypothesis for the transformation suppression activity is proposed. EGR-1 contains three "zinc-finger" motifs in the C-terminal portion of the molecule that constitute the DNA-binding domain and interact with the promoters by virtue of two classes of GC-rich elements: single GC-elements (GCEs) with the consensus 5'-T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T-3' and overlapping sites consisting of an Sp-1 binding site and the GCE consensus or close homolog of these sequences. The Wilm's tumor suppressor gene product WT1 interacts with the same GCE and, owing in part to four alternate splice products, interacts with a broader range of GC-rich elements with the consensus 5'-GNGNGGGNG-3' and 5'-TCCTCCTCCTCCTC-3'. WT1 commonly but not invariably acts as repressor of transcription, whereas EGR-1, in the absence of overlapping Sp-1 binding sequences, is often an activator. The well-known rapid response of the EGR-1 gene following mitogenic stimulation together with the occurrence of GCEs in the promoters of many growth factors and protooncogenes suggests a role of EGR-1 in growth. Moreover, EGR-1 is constitutively expressed in several viral-transformed systems. On the other hand, studies of model and human tumor lines reveal that EGR-1 has significant growth and transformation suppression roles. Recent studies show that this effect can be accounted for by the ability of EGR-1 to induce the expression and secretion of TGF-beta1, a potent growth suppressor of many cell types, by binding to a single GCE of the TGF-beta1 promoter. Although the effects of EGR- at overlapping Sp1/EGR-1 DNA binding sites are not predictable, known cases fall into two loose groups. Sp1 is usually activating and increasing concentrations of EGR-1 lead to displacement that results in either inhibition of transactivation or EGR-1-dependent transactivation. Moreover, recent studies suggest that displaced Sp1 binds to and activates the endogenous Egr-1 gene, thereby leading to "facilitated inhibition" of Sp1 function by the resulting increased EGR-1. This effect may augment the growth suppressive function of EGR-1 based on induction of TGF-beta1.

The degree of phosphorylation of c-Jun, Jun-B, Jun-D and Egr-1 transcription factors was examined during normal growth and during a prolonged period of defined transformation of NIH-3T3 cells which conditionally express v-sis [Mercola, D. et al. (1992) Oncogene, 7, 1793-1803]. During the asynchronous growth of normal cells phosphorylation of all factors was low and constant at all stages of growth from low density (c. 25 x 10(3) cells/cm2) through log-phase of growth to saturation density (c. 100 x 10(3) cells/cm2). Upon induction of v-sis, a marked and coordinate increase in phosphorylation occurred for c-Jun, Jun-B and Egr-1 to approximately 320%, 230% and 420% respectively above basal levels which was stable for the 2.5 day transformation period. The phosphorylation of Jun-D increased to over 600% and, after about 20 h, steadily declined to near basal levels at 54 h post-induction. Moreover, at any time phosphorylation and v-sis expression were fully reversible upon removal of the inducer. It appears that increased phosphorylation of the Jun family members and Egr-1 is not necessary for normal growth of NIH-3T3 but is dependent upon the expression of v-sis. Thus, normal and transformed cells may be distinguished. For c-Jun, the v-sis enhanced phosphorylation occurs at serines 63 and 73 and is required for transformation by several oncogenes [Smeal, T. et al. (1992) Mol. Cell. Biol., 12, 3507-3513]. The results described here show that the phosphorylation of additional factors is a stable and specific correlate of transformation which have have regulatory significance during transformation.

The development of the mammalian preimplantation embryo in vitro occurs more slowly and less successfully compared to development in the uterus. The fact that it can occur at all in a defined protein-free medium suggests that the process is autonomous. Accumulated evidence indicates that a number of peptide growth factors contribute in an autocrine fashion to preimplantation development. Other growth factors are maternally derived and act in a paracrine manner on the embryo. Some of these factors such as insulin-related factors stimulate growth preferentially, but others such as epidermal growth factor (EGF) play more important roles in differentiation. Several cytokines appear to be implicated in peri-implantation events and in maternal-fetal interactions. At this stage, the data are mostly descriptive. Are all these different growth factors and receptors necessary for early development? Some implications of apparent redundancy of gene expression are discussed and future studies are predicted.

Murine embryonal carcinoma cells do not express detectable cell-surface epidermal growth factor receptors (EGF-R) but after 2 days of differentiation induced by retinoic acid (RA) increasingly express mRNA and protein encoded by the EGF receptor gene (Joh et al., Cell Growth and Differentiation 3, 315, 1992). The effect on morphology, growth, and differentiation of the introduction of expression vectors that produce either a truncated, kinase-negative mouse EGF receptor or an antisense mRNA was studied in P19 embryonal carcinoma (EC) cells before and after differentiation. The presence of either construct should lead to the reduction of EGF-R expression by either the dominant negative effects of a truncated protein or the inhibition of endogenous EGF-R mRNA production/translation by complementary RNA, respectively. Cells were cotransfected with the bacterial neomycin resistance gene and constitutively expressing clones were selected with G418. The cytomegalovirus LTR promoter/enhancer was found to be very inefficiently activated in Pl9 EC cells. After RA addition, changes in gene expression included induction of both the exogenous truncated constructs and endogenous EGF-R. Differentiation was gauged by the expression of tissue-type plasminogen activator and intermediate filament protein markers of neural tissues, as well as EGF-R. The expression of 120-kDa truncated EGF-Rs was high in four clones, but all 10 clones examined had diminished abilities to differentiate after RA induction compared to four control cell lines. Similarly, the majority of antisense transfected clones was unable to differentiate normally. The results indicate that the reduced expression of EGF-Rs in differentiating EC cells inhibits the rate, frequency, and extent of differentiation after RA induction. We conclude that the expression of EGF-Rs plays a role in the stimulation of differentiation and we speculate that the mechanism involves the tyrosine kinase activity of the receptor.

Two-cell mouse preimplantation embryos were cultured for 48 h in four different reagents to modulate epidermal growth factor (EGF) receptor function. These were rabbit polyclonal and mouse monoclonal antibodies to EGF receptor, EGF receptor antisense RNA, and EGF receptor antisense deoxyoligonucleotides. Embryos were scored for two endpoints: onset of cavitation as a measure of trophectoderm differentiation and mean embryo cell number as a measure of cell proliferation. The consistent observations were that cavitation was significantly accelerated by antibodies and delayed by antisense RNA and antisense deoxyoligonucleotides. None of these reagents exerted a significant effect on mean embryo cell number, with one exception the polyclonal antibody. Our interpretation of these observations is that the antibody binding facilitated cavitation by mimicking natural ligand-receptor binding and inducing the signal transduction cascade that is typical for the EGF receptor. In the case of antisense RNA or deoxyoligonucleotide, we propose that they delayed onset of cavitation by interfering with EGF receptor production. We hypothesize that during this period of development, EGF receptor is concerned predominantly with the regulation of differentiation more than with cell proliferation. © 1993Wiley-Liss, Inc.

Epidermal growth factor receptors (EGF-Rs) are expressed at increasing levels on mouse preimplantation embryos. Immunofluorescence assays were used to show that unfertilized eggs and 2-cell embryos have a very low level of reactivity to antimouse EGF-R antibodies, but by the 4-cell stage and later the reactivity increases. The synthesis of EGF-R protein was verified at the blastocyst stage by immunoprecipitation of a 170-kDa metabolically labeled protein. The EGF-R protein is expressed on cell plasma membrane surfaces, but after compaction at the 8-cell stage concentrates on the apical cell surfaces. We also find a low level of expression of EGF-R protein on inner cell mass cells; thus, all cell lineages express receptors from the beginning of gestation. The receptor protein synthesized by the 8-cell embryo and later is probably translated from embryonic transcription, since reverse transcription-polymerase chain reaction indicates increasing levels of mRNA starting after the 4-cell stage. However, we also detected maternal mRNA in zygotes and 2-cell embryos. The pervasive nature of EGF-R expression throughout development suggests important roles for these receptors which could include autocrine and paracrine stimulation.

The pluripotent murine embryonal carcinoma cell line, P19, differentiates along at least three main pathways under the inductive influence of retinoic acid (RA). The events most critical to the establishment of a particular differentiation pathway must occur early since P19 cells are committed to differentiation pathways after 30 min of exposure to RA (M. W. McBurney, personal communication and our unpublished results). We have, therefore, looked for genes that are induced (or repressed) within 30 min of RA addition and find that Egr-1 is one of these genes. Egr-1 is a transcription factor of the zinc-finger class and is known to transactivate genes after binding to specific oligonucleotide sequences. We describe here the extremely rapid and transient increase of Egr-1 transcript and protein levels in P19 cells after RA addition. Stable induction of Egr-1 transcripts occurred in the presence of protein synthesis inhibitors. Simultaneous addition of RA and cycloheximide did not result in an additive effect. The mechanism of induction with either drug appears to involve relief of a block to transcriptional elongation. The response was more rapid at high RA concentrations and this suggests that the Egr-1 transcription factor could play a role in initiation of differentiation pathways of P19 EC cells.

The mutant F9 cell line F9att-5.51 synthesizes reduced amounts of uvomorulin (UM) protein and we hypothesized earlier (Adamson, Baribault, and Kemler, Dev. Biol. (1990), 138, 338) that this may account for its inability to compact into tightly aggregated balls of cells. Subsequently, when 5.51 cells are treated with retinoic acid to stimulate their differentiation, they are unable to form embryoid bodies as do wild-type cells which form an outer epithelial layer of visceral endoderm cells. We have now examined the possibility that the UM protein made in the mutant line is defective, but find that it is normal in structure and stability. The gene coding for UM appears to be normal as does the mRNA which is synthesized at a normal rate but is severely reduced in steady-state measurements of mutant cells. A rescue experiment was performed by increasing levels of UM in mutant cells by means of transfection with a UM expression vector. The resulting cells expressed abundant UM mRNA and protein but were still unable to form compacted aggregates and did not differentiate into embryoid bodies. Interestingly, the stability of endogenous UM mRNA was improved in the presence of exogenous UM; therefore, a positive feedback mechanism contributes to low mRNA levels in mutant cells. The accumulated data suggest that UM in 5.51 cells is unable to mount a compaction activity because a distal connecting link in the multicomponent process initiated by UM is missing or or aberrant. The missing component is likely to connect UM to actin and the cytoskeleton of the cell.

The PTEN tumour suppressor gene is induced by the early growth response 1 (EGR1) transcription factor, which also transactivates p53, p73, and p300/CBP as well as other proapoptotic and anti-cancer genes. Here, we describe a novel Akt-EGR1-alternate reading frame (ARF)-PTEN axis, in which PTEN activation in vivo requires p14ARF-mediated sumoylation of EGR1. This modification is dependent on the phosphorylation of EGR1 at S350 and T309 by Akt, which promotes interaction of EGR1 with ARF at K272 in its repressor domain by the ARF/Ubc9/SUMO system. EGR1 sumoylation is decreased by ARF reduction, and no EGR1 sumoylation is detected in ARF(-/-) mice, which also exhibit reduced amounts of PTEN. Our model predicts that perturbation of any of the clinically important tumour suppressors, PTEN, EGR1, and ARF, will cause some degree of dysfunction of the others. These results also explain the known negative feedback regulation by PTEN on its own synthesis through PI3 kinase inhibition.

WOS: 000261273100011, PubMed ID: 19032775, Background: UV irradiation activates the EGF receptor, induces Egr1 expression and promotes apoptosis in a variety of cell types. We examined the hypothesis that Egr1 regulates genes that mediate this process by use of a chip-on-chip protocol in human tumorigenic prostate M12 cells. Results: UV irradiation led to significant binding of 288 gene promoters by Egr1. A major functional subgroup consisted of apoptosis related genes. The largest subgroup of 24 genes; belong to the EGFR-signal transduction pathway. Egr1 promoter binding had a significant impact on gene expression of target genes. Conventional ChIP and qRTPCR were used to validate promoter binding and expression changes. siRNA experiments were used to demonstrate the specific role of Egr1 in gene regulation. UV-stimulation promotes growth arrest and apoptosis of M12 cells and our data clearly show that downstream target of EGFR, namely Egr1, mediates this apoptotic response. Our study also identified numerous previously unknown targets of Egr1. These include FasL, MAX and RRAS2, which may play a role in the apoptotic response/growth arrest. Conclusion: Our results indicate that M12 cells undergo Egr1-dependent apoptotic response upon UV-stimulation and led to the identification of downstream targets of Egr1, which mediate EGFR function., NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [U01 CA114810, R01 CA68822]; Mary Kay Ash Foundation [DAMD17-03-1-0022]; US Department of DefenseUnited States Department of Defense; PCRP [W81XWH-04-1-0029], We are thankful to Nima Aghdam for providing technical support and Veronique Baron for comments on the manuscript. We also thank Xiao-Qin Xia for printing the promoter arrays and Stephen Plymate for the gift of M12 cells. This work was supported in part by grants from the NIH (SPECS program 1 U01 CA114810) and the Mary Kay Ash Foundation to DM. DOD PCRP DAMD17-03-1-0022, NIH grant R01 CA68822 and a prostate cancer foundation grant to MMCC. SA was partially supported by US Department of Defense, PCRP Grant #W81XWH-04-1-0029.

The receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGF alpha) is ubiquitous, implying quite general roles for EGF/TGF alpha in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5-day blastocyst (trophectoderm) surface and since TGF alpha-like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre- and peri-implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGF alpha are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed.

Uvomorulin (E-cadherin) is a cell adhesive molecule analogous to L-CAM in the chicken. Uvomorulin is important in the process of compaction in eight-cell mouse embryos and plays a role in F9 embryonal carcinoma cell interactions but it is not clear if it mediates aggregation or compaction, the closer interaction that also occurs in F9 cells cultured in suspension. This paper describes the finding of reduced levels of uvomorulin in a mutant cell line of F9 (5.51 att−) that is consistent with a role for uvomorulin in both aggregation and compaction. The mutant line expresses 40–50% of normal levels of uvomorulin as measured by surface radioiodination, immunoblotting, and biosynthetic labeling and immunoprecipitation with two different antisera. The mutant cell line expresses only abnormally unstable uvomorulin transcripts at very low levels. In addition, karyotypic analyses revealed an abnormal chromosome 8 on which the uvomorulin gene is located and therefore could account for aberrant uvomorulin expression. F9 5.51 att− cells aggregate loosely but do not compact (A. Grover, M. J. Rosenstraus, B. Sterman, M. E. Snook, and E. D. Anderson, 1987, Dev. Biol.119, 1–11) . The conclusion is that reduced levels of uvomorulin are sufficient for aggregation but insufficient for compaction.

p73, a transcription factor rarely mutated in cancer, regulates a subset of p53 target genes that cause cells to respond to genotoxic stress by growth arrest and apoptosis. p73 is produced in two main forms; only TAp73 reiterates the roles of p53, while DeltaNp73 can be oncogenic in character. We show that the TAp73 form produced by TP73 P1 promoter has five distinct Egr1-binding sites, each contributing to the transcriptional upregulation of TAp73 by Egr1 in several cell types. In contrast, TP73 P2 promoter transcribes DeltaNp73, is not induced by Egr1, but is induced by TAp73 and p53. Induction of TAp73 by genotoxic stress requires Egr1 in mouse in vivo. Newly discovered non-consensus p53-binding sites in p73, p53 and Egr1 promoters reveal inter-regulating networks and sustained expression by feedback loops in response to stress, resulting in prolonged expression of the p53 family of genes and efficient apoptosis.

The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo fibroblasts bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal growth, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo fibroblasts, including 143 known genes. Of the 143 genes, program-assisted searching revealed 66 informative genes linked to Egr1. All 66 genes could be placed on a single regulatory network consisting of three branch points of known Egr1 target genes: TGFβ1, IL6, and IGFI. Moreover, 19 additional genes that are known targets of p53 were identified, indicating that p53 is a fourth branch point. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation confirmed that p53 is a direct target of Egr1. Because deficient p53 expression causes tumors in mice, we tested the role of Egr1 in a two-step skin carcinogenesis study (144 mice) that revealed a uniformly accelerated development of skin tumors in Egr1-null mice (P < 0.005). These studies reveal a new role for Egr1 as an in vivo tumor suppressor.

p300 and CBP are large (300 kDa) nuclear scaffold proteins that act as co-activators of transcription in most cell types and are over-expressed in prostate cancer. Recently, the Egr1 transcription factor was shown to up- or down-regulate p300 and CBP transcription based on the nature of its post-translational modification. Notably, interactions of the three proteins provide fine tuning for Egr1-induced growth or cell death responses.

Vinculin and its muscle splice variant metavinculin link focal adhesions and cell-to-cell contact sites to the actin cytoskeleton. We hypothesized that normal expression of vinculin isoforms would be essential for integrity of cardiomyocytes and preservation of normal cardiac function. We studied heterozygous vinculin knockout mice (Vin+/-) that develop and breed normally. The Vin+/- mice displayed: 1) a 58% reduction of vinculin and a 63% reduction of metavinculin protein levels versus wild-type littermates; 2) normal basal cardiac function and histology but abnormal electrocardiograms, intercalated disks, and ICD-related protein distribution; 3) increased mortality following acute hemodynamic stress imposed by transverse aortic constriction (TAC); 4) cardiac dysfunction by 6 weeks post-TAC; and 5) misalignment of alpha-actinin containing Z-lines and abnormal myocardial ultrastructure despite preserved cardiac function. Decreased expression of vinculin/metavinculin leads to abnormal myocyte structure without baseline physiological evidence of cardiac dysfunction. These structural changes predispose to stress-induced cardiomyopathy.

The lipid phosphatase, phosphatase and tensin homolog (PTEN), is a key element in controlling cell growth and survival and has a well established role as tumor suppressor protein in many neoplasia. Several data indicate that silencing of PTEN gene expression may be relevant in follicular thyroid cell transformation. Thus, in the present study regulation of PTEN gene expression in thyroid cells was investigated. Cotransfection experiments indicated that in normal FRTL-5 rat thyroid cells, PTEN promoter activity was increased by overexpression of the transcription factor early growth response protein-1 (Egr-1). Moreover, Western blot experiments indicated that when Egr-1 expression was up-regulated by treating FRTL-5 cells with H2O2, an increase in PTEN expression was also observed. TSH induced opposite modifications on PTEN and Egr-1 protein levels. Moreover, acute or chronic TSH stimulation determined distinct effects. In fact, acute TSH stimulation (30 and 60 min) induced a decrease in PTEN, but an increase in Egr-1 protein levels. These effects were cAMP dependent; in fact, they were mimicked by forskolin. A chronic TSH treatment (5 d) stimulated PTEN protein expression, whereas Egr-1 protein was down-regulated. In contrast to normal thyroid cells, when the thyroid tumor cell lines ARO and BCPAP were exposed to H2O2, neither Egr-1 nor PTEN protein levels were increased. Acute stimulation of ARO and BCPAP cells with forskolin increased Egr-1, but not PTEN, protein levels. Therefore, thyroid tumor cell lines show alteration of PTEN gene expression regulation. RT-PCR experiments performed on human thyroid tumors showed that the absence of Egr-1 mRNA is always paralleled by the absence of PTEN mRNA. Thus, modification of the Egr-1-dependent mechanisms may play a role in the silencing of PTEN gene expression occurring during thyroid cell transformation.

Related coactivators p300 and CBP affect the transcriptional activities of many transcription factors (TF), producing multiple downstream effects. Here we show that immediate early response TF, Egr1, acts upstream of p300/CBP to induce or to repress transcription, depending on the stimulus. Cells induced with serum to increase endogenous Egr1 increase the transcription of p300/CBP only when Egr1 binding sites in the promoter are not mutated, causing the expression of downstream targets of Egr1 which leads to survival and growth. Induction of p300/CBP by Egr1 results in acetylation and stabilization of Egr1 and transactivation of survival genes but repression of Egr1 and p300/CBP in negative feedback loops. In contrast, induction of Egr1 by UV-C irradiation leads to repression of p300/CBP transcription: Egr1 is preferentially phosphorylated, leading to regulation of target genes that cause cell death. This complex balance of opposing effects appears to finely modulate important cellular life and death responses.

In the majority of aggressive tumorigenic prostate cancer cells, the transcription factor Egr1 is overexpressed. We provide new insights of Egr1 involvement in proliferation and survival of TRAMP C2 prostate cancer cells by the identification of several new target genes controlling growth, cell cycle progression, and apoptosis such as cyclin D2, P19ink4d, and Fas. Egr1 regulation of these genes, identified by Affymetrix microarray, was confirmed by real-time PCR, immunoblot, and chromatin immunoprecipitation assays. Furthermore we also showed that Egr1 is responsible for cyclin D2 overexpression in tumorigenic DU145 human prostate cells. The regulation of these genes by Egr1 was demonstrated using Egr1 antisense oligonucleotides that further implicated Egr1 in resistance to apoptotic signals. One mechanism was illustrated by the ability of Egr1 to inhibit CD95 (Fas/Apo) expression, leading to insensitivity to FasL. The results provide a mechanistic basis for the oncogenic role of Egr1 in TRAMP C2 prostate cancer cells.