Inhibition of E-selectin gene expression by transforming growth factor beta in endothelial cells involves coactivator integration of Smad and nuclear factor kappaB-mediated signals.

Transforming growth factor (TGF)-beta(1) is a pleiotropic cytokine/growth factor that is thought to play a critical role in the modulation of inflammatory events. We demonstrate that exogenous TGF-beta(1) can inhibit the expression of the proinflammatory adhesion molecule, E-selectin, in vascular endothelium exposed to inflammatory stimuli both in vitro and in vivo. This inhibitory effect occurs at the level of transcription of the E-selectin gene and is dependent on the action of Smad proteins, a class of intracellular signaling proteins involved in mediating the cellular effects of TGF-beta(1). Furthermore, we demonstrate that these Smad-mediated effects in endothelial cells result from a novel competitive interaction between Smad proteins activated by TGF-beta(1) and nuclear factor kappaB (NFkappaB) proteins activated by inflammatory stimuli (such as cytokines or bacterial lipopolysaccharide) that is mediated by the transcriptional coactivator cyclic AMP response element-binding protein (CREB)-binding protein (CBP). Augmentation of the limited amount of CBP present in endothelial cells (via overexpression) or selective disruption of Smad-CBP interactions (via a dominant negative strategy) effectively antagonizes the ability of TGF-beta(1) to block proinflammatory E-selectin expression. These data thus demonstrate a novel mechanism of interaction between TGF-beta(1)-regulated Smad proteins and NFkappaB proteins regulated by inflammatory stimuli in vascular endothelial cells. This type of signaling mechanism may play an important role in the immunomodulatory actions of this cytokine/growth factor in the cardiovascular system.

The RNA processing enzyme RNase MRP is identical to the Th RNP and related to RNase P.

Sera from patients with autoimmune diseases often contain antibodies that bind ribonucleoproteins (RNPs). Sera from 30 such patients were found to immunoprecipitate ribonuclease P (RNase P), an RNP enzyme required to process the 5' termini of transfer RNA transcripts in nuclei and mitochondria of eukaryotic cells. All 30 sera also immunoprecipitated the nucleolar Th RNP, indicating that the two RNPs are structurally related. Nucleotide sequence analysis of the Th RNP revealed it was identical to the RNA component of the mitochondrial RNA processing enzyme known as RNase MRP. Antibodies that immunoprecipitated the Th RNP selectively depleted murine and human cell extracts of RNase MRP activity, indicating that the Th and RNase MRP RNPs are identical. Since RNase P and RNase MRP are not associated with each other during biochemical purification, we suggest that these two RNA processing enzymes share a common autoantigenic polypeptide.

Expression of the bumetanide-sensitive Na-K-Cl cotransporter BSC2 is differentially regulated by fluid mechanical and inflammatory cytokine stimuli in vascular endothelium.

In vascular endothelium, the electroneutral Na-K-Cl cotransport system is thought to function in the maintenance of a selective permeability barrier in certain vascular beds (e.g., brain), as well as in the preservation of endothelial homeostasis in the face of fluctuating osmotic conditions that may accompany certain pathophysiological conditions (e.g., diabetes mellitus). Here we demonstrate that the gene encoding the bumetanide-sensitive cotransporter BSC2, one of the two major isoforms of Na-K-Cl cotransporters present in mammalian cells, can be differentially regulated by inflammatory cytokines and fluid mechanical forces in cultured endothelium. Interleukin-1beta and tumor necrosis factor-alpha significantly upregulate expression of BSC2 mRNA and protein in human umbilical vein endothelial cells, a response that is inhibited by pretreatment with interferon-gamma. Steady laminar fluid shear stress, at a physiologic magnitude (10 dyn/cm2), is also able to induce and maintain elevated expression of BSC2 in cultured human umbilical vein endothelial cells, while a comparable time-averaged magnitude of turbulent fluid shear stress is not. In vivo, BSC2 mRNA is upregulated after intraperitoneal administration of bacterial endotoxin (LPS) in murine lung and kidney, but not in cardiac tissue. These results provide the first experimental evidence that the BSC2 gene can be selectively regulated by different inflammatory cytokine and fluid mechanical stimuli in endothelium, and support a role for BSC2 in vascular homeostasis and inflammation.

Identification of transcriptional regulatory elements in human mitochondrial DNA by linker substitution analysis.

Human mitochondrial DNA contains two major promoters, one for transcription of each strand of the helix. Previous mapping and mutagenesis data have localized these regulatory elements and have suggested regions important to their function. In order to define, at high resolution, the sequences critical for accurate and efficient transcriptional initiation, a linker substitution analysis of the entire promoter region was performed. Each promoter was shown to consist of approximately 50 base pairs comprising two functionally distinct elements. These and previous data strongly support a mode of transcription initiation requiring minimal sequences surrounding the initiation sites that are likely interactive with core polymerase and upstream regulatory domains capable of binding a transcription factor that modulates the efficiency of transcription initiation. Furthermore, in at least one case, this upstream regulatory domain is capable of operating bidirectionally.

TGF-beta in the cardiovascular system: molecular mechanisms of a context-specific growth factor.

Transforming growth factor beta-1 is the prototypical member of a class of growth factors whose actions have been strongly implicated in a number of pathophysiologic processes including chronic vascular diseases such as atherosclerosis and hypertension. One of the hall-marks of this class of growth factors is the diverse nature of their actions; a characteristic that is thought to arise from the fact that the effects of these factors are very dependent upon the particular cellular context in which they operate. There has been substantial progress in understanding the molecular signaling mechanisms utilized by these factors. These findings are beginning to provide a mechanistic framework with which to understand the complex and pleiotropic actions of these factors on cells and tissues of the cardiovascular system.

Endothelial dysfunction, hemodynamic forces, and atherogenesis.

Phenotypic modulation of endothelium to a dysfunctional state contributes to the pathogenesis of cardiovascular diseases such as atherosclerosis. The localization of atherosclerotic lesions to arterial geometries associated with disturbed flow patterns suggests an important role for local hemodynamic forces in atherogenesis. There is increasing evidence that the vascular endothelium, which is directly exposed to various fluid mechanical forces generated by pulsatile blood flow, can discriminate among these stimuli and transduce them into genetic regulatory events. At the level of individual genes, this regulation is accomplished via the binding of certain transcription factors, such as NF kappa B and Egr-1, to shear-stress response elements (SSREs) that are present in the promoters of biomechanically inducible genes. At the level of multiple genes, distinct patterns of up- and downregulation appear to be elicited by exposure to steady laminar shear stresses versus comparable levels of non-laminar (e.g., turbulent) shear stresses or cytokine stimulation (e.g., IL-1 beta). Certain genes upregulated by steady laminar shear stress stimulation (such as eNOS, COX-2, and Mn-SOD) support vasoprotective (i.e., anti-inflammatory, anti-thrombotic, anti-oxidant) functions in the endothelium. We hypothesize that the selective and sustained expression of these and related "atheroprotective genes" in the endothelial lining of lesion-protected areas represents a mechanism whereby hemodynamic forces can influence lesion formation and progression.

Blood flow and vascular gene expression: fluid shear stress as a modulator of endothelial phenotype.

Vascular endothelium, the cellular monolayer lining the entire cardiovascular system, is exposed to a variety of biochemical and biomechanical stimuli. Fluid shear stresses generated by blood flow in the vasculature can profoundly influence the phenotype of the endothelium by regulating the activity of certain flow-sensitive proteins (for example, enzymes), as well as by modulating gene expression. The finding that specific fluid mechanical forces can alter endothelial structure and function has provided a framework for a mechanistic understanding of flow-dependent processes, ranging from vascular remodeling in response to hemodynamic changes, to the initiation and localization of chronic vascular diseases such as atherosclerosis.

Characterization of human MRP/Th RNA and its nuclear gene: full length MRP/Th RNA is an active endoribonuclease when assembled as an RNP.

Vertebrate cells contain a site-specific endoribonuclease (RNase MRP) that cleaves mitochondrial RNA transcribed from the origin of leading-strand mitochondrial DNA replication. This report presents the characterization of the human enzyme and its essential RNA component. Human RNase MRP is a ribonucleoprotein with a nucleus-encoded RNA of 265 nucleotides. As expected, the single-copy RNA coding region is homologous (84%) to the corresponding mouse gene; surprisingly, at least 700 nucleotides of the immediate 5'-flanking region are conserved. The 265-nucleotide MRP RNA and an MRP RNA cleavage product representing the 3'-terminal 108 nucleotides exist in nuclear and mitochondrial RNA isolates; the larger MRP RNA is present in greatest abundance in the nucleus. The putative processing site within the 265-nucleotide MRP RNA is offset from that of mouse MRP RNA, but in each case cleavage is precise and occurs at the sequence ANCCCGC. Oligonucleotide-mediated inhibition experiments reveal that both the 5' and 3' portions of the MRP RNA are involved in cleavage by RNase MRP; this implies that full length MRP RNA complexed with proteins is an active species in vertebrate cells.

Secondary structure of the RNA component of a nuclear/mitochondrial ribonucleoprotein.

RNase mitochondrial RNA processing (MRP) is a site-specific endoribonuclease located in both the nucleus and mitochondria of vertebrate cells. The enzyme is a ribonucleoprotein whose RNA component has been shown to be encoded by a nuclear gene. Because RNase MRP is particular in its substrate requirement, RNA-RNA interaction has been proposed as important for the cleavage reaction. A secondary structure of this RNA from mouse cells has been derived by chemical modification of in vivo MRP RNA in ribonucleoprotein form, as isolated free RNA, and as RNA synthesized in vitro. Full-length MRP RNA appears to adopt a conformation containing a significant number of single-stranded residues and may form a pseudoknot. The data are consistent with both the RNA within the ribonucleoprotein and the free RNA possessing comparable secondary structures and suggest a possible site of interaction between enzyme and substrate. The human MRP RNA can be folded into a conformation very similar to that predicted for the mouse MRP RNA. A more limited analysis of human MRP RNA is consistent with the structure proposed for the mouse species.

Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements.

Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.

Early atherosclerotic lesions develop in a topographical pattern that strongly suggests involvement of hemodynamic forces in their pathogenesis. We hypothesized that certain endothelial genes, which exhibit differential responsiveness to distinct fluid mechanical stimuli, may participate in the atherogenic process by modulating, on a local level within the arterial wall, the effects of systemic risk factors. A differential display strategy using cultured human endothelial cells has identified two genes, manganese superoxide dismutase and cyclooxygenase-2, that exhibit selective and sustained up-regulation by steady laminar shear stress (LSS). Turbulent shear stress, a nonlaminar fluid mechanical stimulus, does not induce these genes. The endothelial form of nitric oxide synthase also demonstrates a similar LSS-selective pattern of induction. Thus, three genes with potential atheroprotective (antioxidant, antithrombotic, and antiadhesive) activities manifest a differential response to distinct fluid mechanical stimuli, providing a possible mechanistic link between endothelial gene expression and early events in atherogenesis. The activities of these and other LSS-responsive genes may have important implications for the pathogenesis and prevention of atherosclerosis.

MEKK-1, a component of the stress (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, can selectively activate Smad2-mediated transcriptional activation in endothelial cells.

Smad proteins are essential components of the intracellular signaling pathways utilized by members of the transforming growth factor-beta (TGF-beta) superfamily of growth factors. Certain Smad proteins (e.g. Smad1, -2, and -3) can act as regulated transcriptional activators, a process that involves phosphorylation of these proteins by activated TGF-beta superfamily receptors. We demonstrate that the intracellular kinase mitogen-activated protein kinase kinase kinase-1 (MEKK-1), an upstream activator of the stress-activated protein kinase/c-Jun N-terminal kinase pathway, can participate in Smad2-dependent transcriptional events in cultured endothelial cells. A constitutively active form of MEKK-1 but not mitogen-activated protein kinase kinase-1 (MEK-1) or TGF-beta-activated kinase-1, two distinct intracellular kinases, can specifically activate a Gal4-Smad2 fusion protein, and this effect correlates with an increase in the phosphorylation state of the Smad2 protein. These effects do not require the presence of the C-terminal SSXS motif of Smad2 that is the site of TGF-beta type 1 receptor-mediated phosphorylation. Activation of Smad2 by active MEKK-1 results in enhanced Smad2-Smad4 interactions, nuclear localization of Smad2 and Smad4, and the stimulation of Smad protein-transcriptional coactivator interactions in endothelial cells. Overexpression of Smad7 can inhibit the MEKK-1-mediated stimulation of Smad2 transcriptional activity. A physiological level of fluid shear stress, a known activator of endogenous MEKK-1 activity in endothelial cells, can stimulate Smad2-mediated transcriptional activity. These data demonstrate a novel mechanism for activation of Smad protein-mediated signaling in endothelial cells and suggest that Smad2 may act as an integrator of diverse stimuli in these cells.

Epidermal growth factor induces Egr-1 promoter activity in hepatocytes in vitro and in vivo.

Early growth response-1 (Egr-1) is a transcription factor that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene has been shown to respond to many extracellular signals. In most cases, these findings have not been extended to the in vivo setting. The goal of the present study was to explore the role of epidermal growth factor (EGF) in mediating Egr-1 expression in hepatocytes under both in vitro and in vivo conditions. In HepG2 cells, Egr-1 protein and mRNA were upregulated in the presence of EGF. In stable transfections of HepG2 cells, a 1,200-bp Egr-1 promoter contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent signaling pathway. A promoter region containing the two most proximal serum response elements was sufficient to transduce the EGF signal. In transgenic mice that carry the Egr-1 promoter coupled to the LacZ reporter gene, systemic delivery of EGF by intraperitoneal injection resulted in an induction of the endogenous Egr-1 gene and the Egr-1-lacZ transgene in hepatocytes. Together, these results suggest that the 1,200-bp promoter contains information for EGF response in hepatocytes both in vitro and in intact animals.

The Egr-1 promoter contains information for constitutive and inducible expression in transgenic mice.

Egr-1 is an immediate early gene that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene is expressed in many cell types and is induced by a wide variety of extracellular signals. The mechanisms by which the Egr-1 gene is regulated in vivo remain poorly understood. In this study, we have generated transgenic mice with a construct containing 1200 bp of the mouse Egr-1 promoter coupled to nuclear localized LacZ. In multiple independent lines of mice, reporter gene expression was detected in subsets of endothelial cells, vascular smooth-muscle cells, cardiomyocytes, neurons, and hepatocytes. This pattern closely resembled that of the endogenous gene. After partial hepatectomy, reporter gene activity was upregulated between two- and fivefold in regenerating livers. Taken together, these findings suggest that the Egr-1 promoter contains information for appropriate spatial and temporal expression in vivo.

Human prostaglandin transporter gene (hPGT) is regulated by fluid mechanical stimuli in cultured endothelial cells and expressed in vascular endothelium in vivo.

BACKGROUND: biomechanical forces generated by blood flow within the cardiovascular system have been proposed as important modulators of regional endothelial phenotype and function. This process is thought to involve the regulation of vascular gene expression by physiological fluid mechanical stimuli such as fluid shear stresses. METHODS AND RESULTS: We demonstrate sustained upregulation of a recently identified gene encoding a human prostaglandin transporter (hPGT) in cultured human vascular endothelium exposed to a physiological fluid mechanical stimulus in vitro. This biomechanical induction is selective in that steady laminar shear stress is sufficient to upregulate the hPGT gene at the level of transcriptional activation, whereas a comparable level of turbulent shear stress (a nonphysiological stimulus) is not. Various biochemical stimuli, such as bacterial endotoxin and the inflammatory cytokines recombinant human interleukin 1beta cytokines (rhIL-1beta) and tumor necrosis factor-alpha (TNF-alpha), did not significantly induce hPGT. Using a specific antiserum to hPGT, we demonstrate endothelial expression within the arterial vasculature and the microcirculation of highly vascularized tissues such as the heart. CONCLUSIONS: Our results identify hPGT as an inducible gene in vascular endothelium and suggest that biomechanical stimuli generated by blood flow in vivo may be important determinants of hPGT expression. Furthermore, this demonstration of regulated endothelial expression of hPGT implicates this molecule in the regional metabolism of prostanoids within the cardiovascular system.