Reciprocal regulation of RhoA and RhoC characterizes the EMT and identifies RhoC as a prognostic marker of colon carcinoma.

Understanding how RhoC expression and activation are regulated is essential for deciphering its contribution to tumorigenesis. Here, we report that RhoC expression and activation are induced by the epithelial to mesenchymal transition (EMT) of colon carcinoma. Using LIM 1863 colon cancer cells, RhoC protein expression and subsequent activation were detected coincident with the loss of E-cadherin and acquisition of mesenchymal characteristics. Several Ets-1 binding sites were identified in the RhoC promoter, and evidence was obtained using chromatin immunoprecipitation that Ets-1 can regulate RhoC expression during the EMT. Interestingly, a marked decrease in RhoA activation associated with the EMT was observed that corresponds to the increase in RhoC expression. Use of shRNA established that RhoA inhibits and RhoC promotes post-EMT cell migration, demonstrating functional significance for their coordinate regulation. To assess the importance of RhoC expression in colon cancer, immunohistochemistry was performed on 566 colorectal tumors with known clinical outcome. The level of RhoC ranged from no expression to high expression, and statistical analysis revealed that elevated RhoC expression correlates with poor outcome as well as aberrant expression and localization of E-cadherin. These data provide one mechanism for how RhoC expression is regulated in colon carcinoma and substantiate its utility as a prognostic marker.

Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor alpha.

OBJECTIVES: To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis. METHODS: Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)alpha. RESULTS: Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNF alpha. CONCLUSIONS: Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNF alpha. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.

Transcriptional regulation of vascular development.

Vascular development is a highly organized sequence of events that requires the correct spatial and temporal expression of specific sets of genes leading to the development of a primary vascular network. The first step in this process is the differentiation of pluripotent stem cells into endothelial cells. This is followed by endothelial proliferation, migration, and eventual formation of endothelial tubes. Maturation of these primitive tubes into fully developed blood vessels requires the recruitment of surrounding pericytes and their differentiation into vascular smooth muscle cells. Many of the events that occur during vasculogenesis are recapitulated during angiogenesis. Transcription factors have been shown to serve as master switches for regulating a number of developmental processes. Using a candidate gene approach, the genomic regulatory regions required to direct vascular-specific gene expression of several receptor tyrosine kinases that are critical for vasculogenesis have been characterized and some of the transcription factors that are involved in the regulation of these genes have recently been identified. Many of these factors are also involved in the regulation of hematopoiesis and may have overlapping functions in determining hematopoietic and endothelial differentiation. Targeted disruption of other transcription factors that were not previously thought to be involved in vascular development have also been recently shown to play a role in blood vessel development. The purpose of this review is to provide an update on the progress that has been made in our understanding of the transcriptional regulation of vascular development over the past few years.

Elf-1 is a transcriptional regulator of the Tie2 gene during vascular development.

Vascular development requires the tightly coordinated expression of several growth factors and their receptors. Among these are the Tie1 and Tie2 receptors, which are almost exclusively endothelial cell-specific. The critical transcriptional regulators of vascular-specific gene expression remain largely unknown. The Ets factors are a family of evolutionarily conserved transcription factors that regulate genes involved in cellular growth and differentiation. We have recently shown that the Ets factor NERF is a strong transactivator of the Tie1 and Tie2 genes. To extend these studies, we have begun to identify the Ets factors that are expressed in developing blood vessels of the chicken chorioallantoic membrane (CAM), a highly vascular embryonic network. RNA was extracted from microdissected CAM blood vessels, and reverse transcriptase-polymerase chain reaction was performed using oligonucleotides encoding conserved amino acids within the Ets domain. One of the polymerase chain reaction fragments was subcloned and identified as the chicken homologue of the Ets factor ELF-1, cELF-1. ELF-1 is most closely related to the Ets factor NERF. In situ hybridization and immunohistochemistry demonstrate that cELF-1 is enriched in developing chicken blood vessels. cELF-1 is also a strong transactivator of the Tie1 and Tie2 genes and can bind to conserved Ets sites within the promoters of these genes. A complex of similar size forms when gel shifts are performed with cellular extracts derived from the CAM blood vessels, which is recognized by an antibody against cELF-1. In summary, ELF-1 belongs to a subset of Ets factors that regulate vascular-specific gene expression during blood vessel development.

ESE-1 is a novel transcriptional mediator of inflammation that interacts with NF-kappa B to regulate the inducible nitric-oxide synthase gene.

Inflammation is a hallmark of several vascular diseases. The nuclear factor kappaB (NF-kappaB) transcription factors are dimeric proteins involved in the activation of a large number of genes in response to inflammatory stimuli. We report the involvement of a novel member of the ETS transcription factor, ESE-1, in mediating vascular inflammation. ESE-1 is induced in response to inflammatory cytokines and lipopolysaccharide in vascular smooth muscle cells, endothelial cells, and cells of the monocyte-macrophage lineage. This induction occurs within hours of stimulation and is mediated by NF-kappaB transactivation of the ESE-1 promoter. We have identified the inducible form of nitric-oxide synthase (NOS2) as a putative target for ESE-1. ESE-1 can bind to the p50 subunit of NF-kappaB, and cotransfection of ESE-1 with the p50 and p65 subunits of NF-kappaB synergistically enhances transactivation of the NOS2 promoter by ESE-1. An ESE-1-binding site within the NOS2 promoter has been identified, the site-directed mutagenesis of which completely abolishes the ability of ESE-1 to transactivate the NOS2 promoter. Finally, in a mouse model of endotoxemia, associated with acute vascular inflammation, ESE-1 is strongly expressed in vascular endothelium and smooth muscle cells. In summary, ESE-1 represents a novel mediator of vascular inflammation.

Tel-2 is a novel transcriptional repressor related to the Ets factor Tel/ETV-6.

We report here the isolation of Tel-2, a novel member of the Ets transcription factor family, with high homology to Tel/ETV-6. Tel-2 is the second mammalian member of the Tel Ets family subclass whose prototype Tel is involved in various chromosomal translocations in human cancers. Six differentially expressed alternative splice products of Tel-2 were characterized encoding different Tel-2 isoforms which either contain or lack the amino-terminal Pointed domain and also vary at the carboxyl terminus. In contrast to Tel, which is highly expressed in several different cell types and tissues, Tel-2 is only weakly expressed in a variety of tissues and cell types, including placenta, prostate, spleen, liver, and lung. Tel-2 binds to functionally relevant Ets-binding sites of several genes and only the Tel-2 isoform containing the Pointed domain and the DNA-binding domain acts as a strong repressor of transcription. The retinoic acid receptor alpha and bone morphogenetic protein-6B (BMP-6) genes are specifically repressed by Tel-2 indicating a function for Tel-2 as an inhibitor of differentiation. Due to the important involvement of Tel in human cancer and the location of Tel-2 within the MHC cluster region, Tel-2 might be involved in chromosomal translocations in human cancer as well.

PDEF, a novel prostate epithelium-specific ets transcription factor, interacts with the androgen receptor and activates prostate-specific antigen gene expression.

Prostate cancer, the most frequent solid cancer in older men, is a leading cause of cancer deaths. Although proliferation and differentiation of normal prostate epithelia and the initial growth of prostate cancer cells are androgen-dependent, prostate cancers ultimately become androgen-independent and refractory to hormone therapy. The prostate-specific antigen (PSA) gene has been widely used as a diagnostic indicator for androgen-dependent and -independent prostate cancer. Androgen-induced and prostate epithelium-specific PSA expression is regulated by a proximal promoter and an upstream enhancer via several androgen receptor binding sites. However, little progress has been made in identifying androgen-independent regulatory elements involved in PSA gene regulation. We report the isolation of a novel, prostate epithelium-specific Ets transcription factor, PDEF (prostate-derived Ets factor), that among the Ets family uniquely prefers binding to a GGAT rather than a GGAA core. PDEF acts as an androgen-independent transcriptional activator of the PSA promoter. PDEF also directly interacts with the DNA binding domain of androgen receptor and enhances androgen-mediated activation of the PSA promoter. Our results, as well as the critical roles of other Ets factors in cellular differentiation and tumorigenesis, strongly suggest that PDEF is an important regulator of prostate gland and/or prostate cancer development.

ESE-3, a novel member of an epithelium-specific ets transcription factor subfamily, demonstrates different target gene specificity from ESE-1.

Most cancers originate as a result of aberrant gene expression in mainly glandular epithelial tissues leading to defects in epithelial cell differentiation. The latter is governed by distinct sets of transcriptional regulators. Here we report the characterization of epithelium-specific Ets factor, family member 3 (ESE-3), a novel member of the ESE subfamily of Ets transcription factors. ESE-3 shows highest homology to two other epithelium restricted Ets factors, ESE-1 and ESE-2. ESE-3, like ESE-1 and ESE-2, is exclusively expressed in a subset of epithelial cells with highest expression in glandular epithelium such as prostate, pancreas, salivary gland, and trachea. A potential role in branching morphogenesis is suggested, since ESE-3 transactivates the c-MET promoter via three high affinity binding sites. Additionally, ESE-3 binding to DNA sequences in the promoters of several glandular epithelium-specific genes suggests a role for ESE-3 in later stages of glandular epithelium differentiation. Although ESE-3 and ESE-1 bind with similar affinity to various Ets binding sites, ESE-3 and ESE-1 differ significantly in their ability to transactivate the promoters containing these sites. Our results support the notion that ESE-1, ESE-2, and ESE-3 represent a unique epithelium-specific subfamily of Ets factors that have critical but distinct functions in epithelial cell differentiation and proliferation.

Characterization of ESE-2, a novel ESE-1-related Ets transcription factor that is restricted to glandular epithelium and differentiated keratinocytes.

Epithelial cell differentiation is tightly controlled by distinct sets of transcription factors that regulate the expression of stage-specific genes. We recently isolated the first epithelium-specific Ets transcription factor (ESE-1). Here we describe the characterization of ESE-2, a second epithelium-restricted ESE-1-related Ets factor. Like ESE-1, ESE-2 is induced during keratinocyte differentiation. However, whereas ESE-1 is expressed in the majority of epithelial cell types, ESE-2 expression is restricted to differentiated keratinocytes and glandular epithelium such as salivary gland, prostate, mammary gland, and kidney. In contrast to ESE-1, full-length ESE-2 binds poorly to DNA due to the presence of a negative regulatory domain at the amino terminus. Furthermore, although ESE-1 and the amino-terminally deleted ESE-2 bind with similar affinity to the canonical E74 Ets site, ESE-2 and ESE-1 differ strikingly in their relative affinity toward binding sites in the c-MET and PSMA promoters. Similarly, ESE-1 and ESE-2 drastically differ in their ability to transactivate epithelium-specific promoters. Thus, ESE-2, but not ESE-1, transactivates the parotid gland-specific PSP promoter and the prostate-specific PSA promoter. In contrast, ESE-1 transactivates the keratinocyte-specific SPRR2A promoter Ets site and the prostate-specific PSMA promoter significantly better than ESE-2. Our results demonstrate the existence of a unique class of related epithelium-specific Ets factors with distinct functions in epithelial cell gene regulation.

Role of the Ets transcription factors in the regulation of the vascular-specific Tie2 gene.

The Tie2 gene encodes a vascular endothelium-specific receptor tyrosine kinase that is required for normal vascular development and is also upregulated during angiogenesis. The regulatory regions of the Tie2 gene that are required for endothelium-specific gene expression in vivo have been identified. However, the transcription factors required for Tie2 gene expression remain largely unknown. We have identified highly conserved binding sites for Ets transcription factors in the Tie2 promoter. Mutations in 2 particular binding sites lead to a 50% reduction in the endothelium-specific activity of the promoter. We have compared the ability of several members of the Ets family to transactivate the Tie2 promoter. Our results demonstrate that 1 of 3 distinct isoforms of the novel Ets transcription factor NERF, NERF2, is expressed in endothelial cells and can strongly transactivate the regulatory regions of the Tie2 gene in comparison to other Ets factors, which have little or no effect. NERF2 can bind to the Tie2 promoter Ets sites in electrophoretic mobility shift assays. These studies support a role for Ets factors in the regulation of vascular-specific gene expression and suggest that the novel Ets factor NERF2 may be a critical transcription factor in specifying the expression of the Tie2 gene in vascular endothelial cells.

Genomic organization of the human ELF3 (ESE-1/ESX) gene, a member of the Ets transcription factor family, and identification of a functional promoter.

We recently isolated a novel member of the Ets transcription factor/oncogene family, ESE-1/ESX/ELF3, with features distinct from any other Ets-related factor. ELF3 is the prototype of a new subclass of Ets factors, contains two DNA-binding domains, and, in contrast to any known Ets factor, is expressed exclusively in epithelial cells. ELF3 expression is induced during differentiation of the epidermis, indicating a role in the regulation of terminal differentiation genes in the epidermis. Due to the important role that other Ets factors play in cellular differentiation, ELF3 is expected to be a critical regulator of epithelial gene expression. We report here the cloning and the structural organization of the human ELF3 gene. The human ELF3 gene contains nine exons, which span approximately 5.8 kb of genomic DNA. Intron/exon borders and number of exons are almost identical to those in the mouse ELF3 gene. Comparison of the immediate promoter regions of the human and mouse ELF3 genes demonstrates the presence of TATA and CCAAT boxes as well as potential binding sites for Ets factors and NF-kappaB. Transfection experiments demonstrate that a 1.5-kb fragment of the 5' upstream region acts as a strong promoter in two epithelial cell lines.

Role of ets factors in the activity and endothelial cell specificity of the mouse Tie gene promoter.

The Tie gene encodes an endothelial cell receptor tyrosine kinase necessary for normal vascular development. The Tie gene promoter targets expression of heterologous genes specifically to endothelial cells in transgenic mice. Here we have characterized the promoter sequences critical for endothelial cell-specific activity in cultured cells and transgenic mice. Progressive deletions and site-directed mutations of the promoter showed that the critical endothelial cell-specific elements are an octamer transcription factor binding site and several Ets binding sites located in two clusters within 300 bp upstream of the major transcription initiation site. Among members of the Ets transcription factor family tested, NERF-2 (a novel transcription factor related to the ets factor ELF-1), which is expressed in endothelial cells, and ETS2 showed the strongest transactivation of the Tie promoter; ETS1 gave lower levels of stimulation and the other Ets factors gave little or no transactivation. Furthermore, the Tie promoter directed the production of high amounts of human growth hormone into the circulation of transgenic mice. The secreted amounts correlated with transgene copy number, being relatively insensitive to the effects of the transgene integration site. These properties suggest that Tie promoter activity is controlled by endothelial cell Ets factors and that it has potential for use in vectors for endothelial cell-specific gene expression.

Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response.

Proliferation of vascular smooth muscle cells (VSMCs) in response to injury plays a key role in the pathogenesis of vascular disorders. Fas ligand (FasL) induces apoptosis in Fas-bearing cells, and its expression on activated T cells contributes to the regulation of the immune response and physiological cell turnover. Here, we show that a replication-defective adenovirus encoding FasL (Ad-FasL) induced apoptosis in Fas-bearing VSMCs. When introduced locally to balloon-injured rat carotid arteries, a well characterized model of a VSMC-derived lesion, Ad-FasL functioned as a potent inhibitor of neointima formation. In rats immunized with an empty adenoviral vector, robust T cell infiltration of the vessel wall was detected after local delivery of a beta-galactosidase-expressing virus (Ad-betagal), whereas T cell infiltrates were not detected after local delivery of Ad-FasL. Prior immunization prevented beta-galactosidase expression from Ad-betagal, whereas the expression of the FasL transgene was unaffected. When Ad-betagal and Ad-FasL were delivered together to preimmunized animals, T cell infiltration was reduced and beta-galactosidase expression was restored. These data demonstrate that Fas ligand gene transfer can effectively inhibit injury-induced vessel lesion formation and can allow adenovirus-harboring cells to evade immune destruction.

Isolation and characterization of a novel epithelium-specific transcription factor, ESE-1, a member of the ets family.

We report here the isolation of a novel, highly tissue-restricted member of the ets transcription factor/oncogene family, ESE-1 (for epithelium-specific Ets), which has features distinct from those of any other ets-related factor. ESE-1 contains two putative DNA binding domains: an ETS domain, which is unique in that the 5' half shows relatively weak homology to known ets factors, and an A/T hook domain, found in HMG proteins and various other nuclear factors. In contrast to any known ets factors, ESE-1 is expressed exclusively in epithelial cells. ESE-1 expression is induced during terminal differentiation of the epidermis and in a primary human keratinocyte differentiation system. The keratinocyte terminal differentiation marker gene, SPRR2A, is a putative target for ESE-1, since SPRR2A expression during keratinocyte differentiation correlates with induction of ESE-1 expression, and ESE-1 binds with high affinity to and transactivates the ets binding site in the SPRR2A promoter. ESE-1 also binds to and transactivates the enhancer of the Endo A gene, a potential target for ESE-1 in simple epithelia. Due to the important role that other ets factors play in cellular differentiation, ESE-1 is expected to be a critical regulator of epithelial cell differentiation.

ELF-1 interacts with and transactivates the IgH enhancer pi site.

We previously identified a B-cell-specific regulatory element in the immunoglobulin heavy chain (IgH) enhancer, pi, with striking similarity to binding sites for ets-related transcription factors. Whereas the ability of ets-related factors to bind to and transactivate the pi site has been substantiated, the identification of the particular member of the ets family responsible for B-cell-specific regulation of the pi site has remained controversial. We have used antibodies specific for individual members of the ets family to evaluate which ets-related factor in B-cell nuclear extracts interacts with the IgH pi site. We present strong evidence that ELF-1 is highly expressed in B-cells and is one of two major factors specifically interacting with the murine IgH enhancer pi site in B-cell nuclear extracts. Binding of ELF-1 correlates with activity of the pi site, since mutations abolishing function of pi also inhibit binding of ELF-1. Furthermore, we demonstrate that ELF-1 can transactivate the IgH enhancer in HeLa cells, suggesting a role for ELF-1 in B-cell-specific IgH gene expression.

Characterization of NERF, a novel transcription factor related to the Ets factor ELF-1.

We have cloned the gene for a novel Ets-related transcription factor, new Ets-related factor (NERF), from human spleen, fetal liver, and brain. Comparison of the deduced amino acid sequence of NERF with those of other members of the Ets family reveals that the level of homology to ELF-1, which is involved in the regulation of several T- and B-cell-specific genes, is highest. Homologies are clustered in the putative DNA binding domain in the middle of the protein, a basic domain just upstream of this domain, and several shorter stretches of homology towards the amino terminus. The presence of two predominant NERF transcripts in various fetal and adult human tissues is due to at least three alternative splice products, NERF-1a, NERF-1b, and NERF-2, which differ in their amino termini and their expression in different tissues. Only NERF-2 and ELF-1, and not NERF-1a and NERF-1b, function as transcriptional activators of the lyn and blk gene promoters, although all isoforms of NERF bind with affinities similar to those of ELF-1 to a variety of Ets binding sites in, among others, the blk, lck, lyn, mb-1, and immunoglobulin H genes and are expressed at similar levels. Since NERF and ELF-1 are coexpressed in B and T cells, both might be involved in the regulation of the same genes.

Prevalence of residual left atrial thrombi among patients with acute thromboembolism and newly recognized atrial fibrillation.

BACKGROUND: Thromboembolism related to atrial fibrillation (AF) is a major cause of morbidity and mortality. Patients with acute thromboembolism and AF are at high risk for early recurrent events. OBJECTIVE: To determine the prevalence of left atrial thrombi in patients who had acute thromboembolism and newly diagnosed AF. PATIENTS AND METHODS: Adult inpatients with AF were screened to identify those with acute (< 36 hours) systemic thromboembolism and newly recognized AF. Of 41 qualifying patients, 31 (76%) agreed to undergo transesophageal echocardiographic study, including 24 with acute neurologic events and seven with peripheral thromboembolism. A control population consisted of 88 adults with newly recognized AF without clinical thromboembolism. RESULTS: Transesophageal echocardiography identified left atrial thrombi in 13 (43%) of the 30 study patients who underwent transesophageal echocardiography compared with nine (10%) of 87 controls (P < .001). Spontaneous echo contrast was identified in 27 (87%) of the study population vs 42 (48%) of controls (P < .001). The prevalence of this marker of blood stasis did not differ between patients with left atrial thrombi without thromboembolism (P = .69). Duration of AF, prevalence of abnormal left ventricular function, left atrial size, and mitral regurgitation were similar in both groups. CONCLUSIONS: Left atrial thrombi were identified in more than 40% of patients with acute thromboembolism and newly recognized AF. These data suggest that a major source of recurrent thromboembolism in this group may be residual thrombus migration. Among patients with AF and atrial thrombi, clinical thromboembolism seems to occur randomly, or is related to an unidentified process.

Transesophageal echocardiographically facilitated early cardioversion from atrial fibrillation using short-term anticoagulation: final results of a prospective 4.5-year study.

OBJECTIVES: We sought to validate the safety of transesophageal echocardiographically guided early cardioversion in conjunction with short-term anticoagulation as a strategy for guiding early cardioversion in hospitalized patients with atrial fibrillation. BACKGROUND: Because atrial thrombi are poorly seen by conventional imaging techniques, several weeks of prophylactic anticoagulation is routinely prescribed before cardioversion. Transesophageal echocardiography is a superior test for identifying atrial thrombi; preliminary feasibility studies have supported its use to guide early cardioversion for patients in whom no thrombus is observed, but safety has not been validated in any large series. METHODS: All patients admitted to hospital with atrial fibrillation during a 4.5-year period were screened. The inclusion criterion was a clinical duration of atrial fibrillation > 2 days or of unknown duration. Patients received anticoagulation with heparin/warfarin and underwent conventional transthoracic echocardiography followed by transesophageal study. Patients in whom transesophageal echocardiography revealed no atrial thrombus underwent pharmacologic or electrical cardioversion followed by warfarin therapy for 1 month. Cardioversion was deferred in patients with evidence of atrial thrombi, and they received prolonged warfarin treatment. RESULTS: Two hundred thirty-three patients (86% of those eligible) agreed to participate, and 230 underwent transesophageal echocardiography. Transesophageal echocardiography identified 40 atrial thrombi (left atrium 34, right atrium 6) in 34 patients (15%). One hundred eighty-six (95%) of 196 patients without thrombi had successful cardioversion to sinus rhythm, all without prolonged anticoagulation, and none (0%, 95% confidence interval 0% to 1.6%) experienced a clinical thromboembolic event. Eighteen patients with atrial thrombi underwent uneventful cardioversion after prolonged anticoagulation. CONCLUSIONS: Compared with smaller series that have shown only feasibility, this large prospective and consecutive study of patients undergoing transesophageal echocardiographically facilitated early cardioversion in conjunction with short-term anticoagulation validates the safety of this strategy. This treatment algorithm has a safety profile similar to conventional therapy and minimizes both the period of anticoagulation and the overall duration of atrial fibrillation.