Absence of heme oxygenase-1 expression in the lung parenchyma exacerbates endotoxin-induced acute lung injury and decreases surfactant protein-B levels.

Acute respiratory distress syndrome (ARDS) is a devastating disease process characterized by severe acute lung injury, inflammatory cell recruitment to the lung, upregulation of pro-inflammatory cytokines and increased oxidative stress. Epithelial cell injury, diffuse alveolar damage and surfactant dysfunction ensue leading to refractory hypoxemic respiratory failure. There are no specific effective therapies for ARDS and novel therapeutic approaches are desperately needed. In this study we assessed the role of the cytoprotective and anti-inflammatory enzyme heme oxygenase (HO)-1 in a model of nebulized endotoxin-induced acute lung injury. HO-1 null (HO-1(-/-)) mice exhibited severe physiologic lung dysfunction following lipopolysaccharide (LPS) nebulization, but had similar inflammatory responses as wild-type (WT) mice. However, a dramatic reduction in surfactant protein-B (SP-B) expression was observed in the lungs of LPS-treated HO-1(-/-) mice compared with similarly treated WT mice. Using reciprocal bone marrow transplantation (BMT) to generate HO-1-chimeric mice, we found that absence of HO-1 in the lung parenchyma, not in bone marrow-derived inflammatory cells, was responsible for enhanced SP-B downregulation and severe physiologic lung dysfunction. These findings have implications for our understanding of the pathophysiology of ARDS and may guide future therapeutic strategies.

Pre-emptive gene therapy using recombinant adeno-associated virus delivery of extracellular superoxide dismutase protects heart against ischemic reperfusion injury, improves ventricular function and prolongs survival.

In high-risk patients, the ideal cardiovascular gene therapy requires a strategy that provides long-term protection of myocardium against episodes of ischemic/reperfusion injury. We report the development of an efficient, long-lasting pre-emptive gene therapy strategy in a rat model of ischemic-reperfusion (I/R) injury of heart. At 6 weeks prior to myocardial injury, the human extracellular superoxide dismutase (Ec-SOD) gene was delivered by direct intramyocardial injections, using a recombinant adeno-associated virus vector. Significant myocardial protection was documented by the decrease in infarct size at 24 h post I/R, improved left ventricular function at 7 weeks postinjury, and enhanced long-term survival in the SOD treated group. This concept of preinjury delivery and 'pre-emptive' gene therapy via the expression of a secreted protein that renders paracrine therapeutic action can be an effective strategy for organ protection against future injury.

Role of macrophage-expressed adipocyte fatty acid binding protein in the development of accelerated atherosclerosis in hypercholesterolemic mice.

Atherosclerosis is an inflammatory disease process associated with elevated levels of plasma cholesterol, especially low-density lipoproteins. The latter become trapped within the arterial wall and are oxidized and taken up by macrophages to form foam cells. This process is an initiating event for atherosclerosis. Fatty acid binding proteins (FABP) are involved in fatty acid metabolism and cellular lipid transport, and adipocyte FABP (aP2) is also expressed in macrophages. We recently generated mice lacking both apolipoprotein (Apo)E and aP2 (ApoE-/-aP2-/-) and found that these mice, compared with ApoE-/- mice, developed markedly smaller atherosclerotic lesions that contained fewer macrophages. Here we investigated the mechanism(s) responsible for this prevention of atherosclerotic lesion formation. Bone marrow transplantations were performed in ApoE-/- mice, receiving cells from either ApoE-/- or ApoE-/-aP2-/- mice. The lack of aP2 in donor marrow cells led to the development of smaller (5.5-fold) atherosclerotic lesions in the recipient mice. No differences were found in plasma cholesterol, glucose, or insulin levels between recipients of bone marrow cells from ApoE-/- or ApoE-/-aP2-/- mice. However, the expression of chemoattractant and inflammatory cytokines was decreased in macrophages from ApoE-/-aP2-/- mice compared with ApoE-/- mice, which may contribute to the decrease in atherosclerotic lesion formation. Taken together, we demonstrate the importance of macrophage aP2 in the development of atherosclerotic lesions.

Upstream stimulatory factors regulate aortic preferentially expressed gene-1 expression in vascular smooth muscle cells.

The phenotypic modulation of vascular smooth muscle cells (VSMC) plays a central role in the pathogenesis of arteriosclerosis. Aortic preferentially expressed gene-1 (APEG-1), a VSMC-specific gene, is expressed highly in differentiated but not in dedifferentiated VSMC. Previously, we identified an E-box element in the mouse APEG-1 proximal promoter, which is essential for VSMC reporter activity. In this study, we investigated the role of upstream stimulatory factors (USF) in the regulation of APEG-1 transcription via this E-box element. By electrophoretic mobility shift assays, recombinant USF1 and USF2 homo- and heterodimers bound specifically to the APEG-1 E-box. Nuclear extracts prepared from primary cultures of rat aortic smooth muscle cells exhibited specific USF1 and USF2 binding to the APEG-1 E-box. To investigate the binding properties of USF during VSMC differentiation, nuclear extracts were prepared from the neural crest cell line, MONC-1, which differentiates into VSMC in culture. Maximal USF1 and USF2 protein levels and binding to the APEG-1 E-box occurred 3 h after the differentiation of MONC-1 cells was initiated. Co-transfection experiments demonstrated that dominant negative USF repressed APEG-1 promoter activity, and USF1, but not USF2, transactivated the APEG-1 promoter. Our studies demonstrate that USF factors contribute to the regulation of APEG-1 expression and may influence the differentiation of VSMC.

Impaired abdominal wall development and deficient wound healing in mice lacking aortic carboxypeptidase-like protein.

Aortic carboxypeptidase-like protein (ACLP) is a member of a diverse group of proteins that contain a domain with similarity to that of the Dictyostelium discoideum protein discoidin I. The discoidin domain has been identified in mammalian milk fat globule membrane proteins, blood coagulation factors, and receptor tyrosine kinases, where it may facilitate cell aggregation, adhesion, or cell-cell recognition. Here we show that ACLP is a secreted protein that associates with the extracellular matrix (ECM). During mouse embryogenesis, ACLP is abundantly expressed in the ECM of collagen-rich tissues, including the vasculature, dermis, and the developing skeleton. We deleted the ACLP gene in mice by homologous recombination. The majority of ACLP(-/-) mice die perinatally due to gastroschisis, a severe disruption of the anterior abdominal wall and herniation of the abdominal organs. ACLP(-/-) mice that survived to adulthood developed nonhealing skin wounds. Following injury by a dermal punch biopsy, ACLP(-/-) mice exhibited deficient wound healing compared with controls. In addition, dermal fibroblasts isolated from ACLP(-/-) 18.5-day-postconception embryos exhibited a reduced proliferative capacity compared with wild-type cells. These results indicate that ACLP is an ECM protein that is essential for embryonic development and dermal wound healing processes.

Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia.

Chronic hypoxia causes pulmonary hypertension with smooth muscle cell proliferation and matrix deposition in the wall of the pulmonary arterioles. We demonstrate here that hypoxia also induces a pronounced inflammation in the lung before the structural changes of the vessel wall. The proinflammatory action of hypoxia is mediated by the induction of distinct cytokines and chemokines and is independent of tumor necrosis factor-alpha signaling. We have previously proposed a crucial role for heme oxygenase-1 (HO-1) in protecting cardiomyocytes from hypoxic stress, and potent anti-inflammatory properties of HO-1 have been reported in models of tissue injury. We thus established transgenic mice that constitutively express HO-1 in the lung and exposed them to chronic hypoxia. HO-1 transgenic mice were protected from the development of both pulmonary inflammation as well as hypertension and vessel wall hypertrophy induced by hypoxia. Significantly, the hypoxic induction of proinflammatory cytokines and chemokines was suppressed in HO-1 transgenic mice. Our findings suggest an important protective function of enzymatic products of HO-1 activity as inhibitors of hypoxia-induced vasoconstrictive and proinflammatory pathways.

Cardiac-specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice.

Heme oxygenase (HO)-1 degrades the pro-oxidant heme and generates carbon monoxide and antioxidant bilirubin. We have previously shown that in response to hypoxia, HO-1-null mice develop infarcts in the right ventricle of their hearts and that their cardiomyocytes are damaged by oxidative stress. To test whether HO-1 protects against oxidative injury in the heart, we generated cardiac-specific transgenic mice overexpressing different levels of HO-1. By use of a Langendorff preparation, hearts from transgenic mice showed improved recovery of contractile performance during reperfusion after ischemia in an HO-1 dose-dependent manner. In vivo, myocardial ischemia and reperfusion experiments showed that infarct size was only 14.7% of the area at risk in transgenic mice compared with 56.5% in wild-type mice. Hearts from these transgenic animals had reduced inflammatory cell infiltration and oxidative damage. Our data demonstrate that overexpression of HO-1 in the cardiomyocyte protects against ischemia and reperfusion injury, thus improving the recovery of cardiac function.

Exacerbation of chronic renovascular hypertension and acute renal failure in heme oxygenase-1-deficient mice.

Heme oxygenase (HO) is a cytoprotective enzyme that degrades heme (a potent oxidant) to generate carbon monoxide (a vasodilatory gas that has anti-inflammatory properties), bilirubin (an antioxidant derived from biliverdin), and iron (sequestered by ferritin). Because of properties of HO and its products, we hypothesized that HO would be important for the regulation of blood pressure and ischemic injury. We studied chronic renovascular hypertension in mice deficient in the inducible isoform of HO (HO-1) using a one kidney-one clip (1K1C) model of disease. Systolic blood pressure was not different between wild-type (HO-1(+/+)), heterozygous (HO-1(+/-)), and homozygous null (HO-1(-/-)) mice at baseline. After 1K1C surgery, HO-1(+/+) mice developed hypertension (140+/-2 mm Hg) and cardiac hypertrophy (cardiac weight index of 5.0+/-0.2 mg/g) compared with sham-operated HO-1(+/+) mice (108+/-5 mm Hg and 4.1+/-0.1 mg/g, respectively). However, 1K1C produced more severe hypertension (164+/-2 mm Hg) and cardiac hypertrophy (6.9+/-0.6 mg/g) in HO-1(-/-) mice. HO-1(-/-) mice also experienced a high rate of death (56%) within 72 hours after 1K1C surgery compared with HO-1(+/+) (25%) and HO-1(+/-) (28%) mice. Assessment of renal function showed a significantly higher plasma creatinine in HO-1(-/-) mice compared with HO-1(+/-) mice. Histological analysis of kidneys from 1K1C HO-1(-/-) mice revealed extensive ischemic injury at the corticomedullary junction, whereas kidneys from sham HO-1(-/-) and 1K1C HO-1(+/-) mice appeared normal. Taken together, these data suggest that chronic deficiency of HO-1 does not alter basal blood pressure; however, in the 1K1C model an absence of HO-1 leads to more severe renovascular hypertension and cardiac hypertrophy. Moreover, renal artery clipping leads to an acute increase in ischemic damage and death in the absence of HO-1.

Akt participation in the Wnt signaling pathway through Dishevelled.

Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.

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.

Down-regulation of high mobility group-I(Y) protein contributes to the inhibition of nitric-oxide synthase 2 by transforming growth factor-beta1.

The inducible isoform of nitric-oxide synthase (NOS2) catalyzes the production of nitric oxide (NO), which participates in the pathophysiology of systemic inflammatory diseases such as sepsis. NOS2 is transcriptionally up-regulated by endotoxin and inflammatory cytokines, and down-regulated by transforming growth factor (TGF)-beta1. Recently we have shown that high mobility group (HMG)-I(Y) protein, an architectural transcription factor, contributes to NOS2 gene transactivation by inflammatory mediators. The aim of the present study was to determine whether regulation of HMG-I(Y) by TGF-beta1 contributes to the TGF-beta1-mediated suppression of NOS2. By Northern blot analysis, we show that TGF-beta1 decreased cytokine-induced HMG-I(Y) mRNA levels in vascular smooth muscle cells and macrophages in vitro and in vivo. Western analysis confirmed the down-regulation of HMG-I(Y) protein by TGF-beta1. To determine whether the down-regulation of HMG-I(Y) contributed to a decrease in NOS2 gene transactivation by TGF-beta1, we performed cotransfection experiments. Overexpression of HMG-I(Y) was able to restore cytokine inducibility of the NOS2 promoter that was suppressed by TGF-beta1. The effect of TGF-beta1 on NOS2 gene transactivation was not related to a decrease in binding of HMG-I(Y) to the promoter of the NOS2 gene, but due to a decrease in endogenous HMG-I(Y) protein. These data provide the first evidence that cytokine-induced HMG-I(Y) can be down-regulated by TGF-beta1. This down-regulation of HMG-I(Y) contributes to the TGF-beta1-mediated decrease in NOS2 gene transactivation by proinflammatory stimuli.

CLIF, a novel cycle-like factor, regulates the circadian oscillation of plasminogen activator inhibitor-1 gene expression.

The onset of myocardial infarction occurs frequently in the early morning, and it may partly result from circadian variation of fibrinolytic activity. Plasminogen activator inhibitor-1 activity shows a circadian oscillation and may account for the morning onset of myocardial infarction. However, the molecular mechanisms regulating this circadian oscillation remain unknown. Recent evidence indicates that basic helix-loop-helix (bHLH)/PAS domain transcription factors play a crucial role in controlling the biological clock that controls circadian rhythm. We isolated a novel bHLH/PAS protein, cycle-like factor (CLIF) from human umbilical vein endothelial cells. CLIF shares high homology with Drosophila CYCLE, one of the essential transcriptional regulators of circadian rhythm. CLIF is expressed in endothelial cells and neurons in the brain, including the suprachiasmatic nucleus, the center of the circadian clock. In endothelial cells, CLIF forms a heterodimer with CLOCK and up-regulates the PAI-1 gene through E-box sites. Furthermore, Period2 and Cryptochrome1, whose expression show a circadian oscillation in peripheral tissues, inhibit the PAI-1 promoter activation by the CLOCK:CLIF heterodimer. These results suggest that CLIF regulates the circadian oscillation of PAI-1 gene expression in endothelial cells. In addition, the results potentially provide a molecular basis for the morning onset of myocardial infarction.

Transforming growth factor-beta 1 inhibition of macrophage activation is mediated via Smad3.

Activated macrophages are critical cellular participants in inflammatory disease states. Transforming growth factor (TGF)-beta1 is a growth factor with pleiotropic effects including inhibition of immune cell activation. Although the pathway of gene activation by TGF-beta1 via Smad proteins has recently been elucidated, suppression of gene expression by TGF-beta1 remains poorly understood. We found that of Smad1-Smad7, Smad3 alone was able to inhibit expression of markers of macrophage activation (inducible nitric-oxide synthase and matrix metalloproteinase-12) following lipopolysaccharide treatment in gene reporter assays. Transient and constitutive overexpression of a dominant negative Smad3 opposed the inhibitory effect of TGF-beta1. Domain swapping experiments suggest that both the Smad MH-1 and MH-2 domains are required for inhibition. Mutation of a critical amino acid residue required for DNA binding in the MH-1 of Smad3 (R74A) resulted in the loss of inhibition. Transient overexpression of p300, an interactor of the Smad MH-2 domain, partially alleviated the inhibition by TGF-beta1/Smad3, suggesting that inhibition of gene expression may be due to increased competition for limiting amounts of this coactivator. Our results have implications for the understanding of gene suppression by TGF-beta1 and for the regulation of activated macrophages by TGF-beta1.

Striated muscle preferentially expressed genes alpha and beta are two serine/threonine protein kinases derived from the same gene as the aortic preferentially expressed gene-1.

Aortic preferentially expressed gene (APEG)-1 is a 1.4-kilobase pair (kb) mRNA expressed in vascular smooth muscle cells and is down-regulated by vascular injury. An APEG-1 5'-end cDNA probe identified three additional isoforms. The 9-kb striated preferentially expressed gene (SPEG)alpha and the 11-kb SPEGbeta were found in skeletal muscle and heart. The 4-kb brain preferentially expressed gene was detected in the brain and aorta. We report here cloning of the 11-kb SPEGbeta cDNA. SPEGbeta encodes a 355-kDa protein that contains two serine/threonine kinase domains and is homologous to proteins of the myosin light chain kinase family. At least one kinase domain is active and capable of autophosphorylation. In the genome, all four isoforms share the middle three of the five exons of APEG-1, and they differ from each other by using different 5'- and 3'-ends and alternative splicing. We show that the expression of SPEGalpha and SPEGbeta is developmentally regulated in the striated muscle during C2C12 myoblast to myotube differentiation in vitro and cardiomyocyte maturation in vivo. This developmental regulation suggests that both SPEGalpha and SPEGbeta can serve as sensitive markers for striated muscle differentiation and that they may be important for adult striated muscle function.

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.

Prevention of hypoxia-induced pulmonary hypertension by enhancement of endogenous heme oxygenase-1 in the rat.

We investigated the role of heme oxygenase (HO)-1 in the development of hypoxia-induced pulmonary hypertension. HO catalyzes the breakdown of heme to the antioxidant bilirubin and the vasodilator carbon monoxide. Hypoxia is a potent but transient inducer of HO-1 in vascular smooth muscle cells in vitro and in the lung in vivo. By using agonists of HO-1, we sustained a high expression of HO-1 in the lungs of rats for 1 week. We report that this in vivo enhancement of HO-1 in the lung prevented the development of hypoxic pulmonary hypertension and inhibited the structural remodeling of the pulmonary vessels. The mechanism(s) underlying this effect may involve a direct vasodilating and antiproliferative action of endogenous carbon monoxide, as well as an indirect effect of carbon monoxide on the production of vasoconstrictors. These results provide evidence that enhancement of endogenous adaptive responses may be used to prevent hypoxia-induced pulmonary hypertension.

Transforming growth factor-beta 1 inhibits cytokine-mediated induction of human metalloelastase in macrophages.

Matrix metalloproteinases (MMP) have been identified in vulnerable areas of atherosclerotic plaques and may contribute to plaque instability through extracellular matrix degradation. Human metalloelastase (MMP-12) is a macrophage-specific MMP with broad substrate specificity and is capable of degrading proteins found in the extracellular matrix of atheromas. Despite its potential importance, little is known about the regulation of MMP-12 expression in the context of atherosclerosis. In this study, we report that in human peripheral blood-derived macrophages, MMP-12 mRNA was markedly up-regulated by several pro-atherosclerotic cytokines and growth factors including interleukin-1beta, tumor necrosis factor-alpha, macrophage colony-stimulating factor, vascular endothelial growth factor, and platelet-derived growth factor-BB. In contrast, the pleiotropic anti-inflammatory growth factor transforming growth factor-beta1 (TGF-beta1) inhibited cytokine-mediated induction of MMP-12 mRNA, protein, and enzymatic activity. Analyses of MMP-12 promoter through transient transfections and electrophoretic mobility shift assays indicated that both its induction by cytokines and its inhibition by TGF-beta1 depended on signaling through an AP-1 site at -81 base pairs. Moreover, the inhibitory effect of TGF-beta1 on MMP-12 was dependent on Smad3. Taken together, MMP-12 is induced by several factors implicated in atherosclerosis. The inhibition of MMP-12 expression by TGF-beta1 suggests that TGF-beta1, acting via Smad3, may promote plaque stability.

Thioredoxin facilitates the induction of heme oxygenase-1 in response to inflammatory mediators.

Heme oxygenase (HO)-1 is a stress response protein that is regulated by oxidative stress. HO-1 catalyzes the generation of biliverdin, carbon monoxide, and iron from heme. Lipopolysaccharide (LPS) and interleukin (IL)-1beta induce HO-1 through the binding of nuclear proteins to AP-1 motifs in enhancer regions upstream from the transcription start site. The DNA binding activity of AP-1 proteins depends on the reduction of cysteines in their DNA-binding domains. We found that agents that disrupt free sulfhydryl groups abolish AP-1 binding activity in nuclear proteins obtained from rat aortic smooth muscle cells and macrophages stimulated with IL-1beta or LPS. Thioredoxin (TRX) may regulate the redox status of nuclear transcription factors in response to oxidative stimuli, thus we determined the role of TRX in the physiologic regulation of HO-1. TRX underwent nuclear translocation in cells stimulated with IL-1beta and LPS. We transfected macrophages with a heterologous promoter construct containing two AP-1 sites from an upstream enhancer region in the HO-1 promoter. Recombinant TRX induced promoter activity to a level analogous to that induced by LPS, and this TRX response was abolished by mutation of the AP-1 sites. An inhibitor of TRX reductase, used to prevent TRX translocation in the reduced state, decreased HO-1 induction by IL-1beta and LPS. These data provide the first evidence that TRX contributes to the induction of HO-1 by inflammatory mediators.

Role of activating protein-1 and high mobility group-I(Y) protein in the induction of CD44 gene expression by interleukin-1beta in vascular smooth muscle cells.

CD44 is a multifunctional cell adhesion molecule that participates in pathological states such as inflammation and tumorigenesis. CD44 is induced on vascular smooth muscle cells after arterial wall injury and may mediate their proliferation and migration into the neointima during arteriosclerosis. We have demonstrated elsewhere that the proinflammatory cytokine interleukin (IL)-1beta up-regulates CD44 mRNA and protein expression in cultured rat aortic smooth muscle cells (RASMC) by increasing gene transcription. By transient transfection of 5'-deletion constructs into RASMC, we show in the present study that a conserved AP-1 site 110 base pairs from the transcription start site of the mouse CD44 promoter is important for basal activity. Mutation of the AP-1 site significantly reduced induction of promoter activity by IL-1beta, and electrophoretic mobility shift assays demonstrated that Fos and c-Jun were present in the CD44 AP-1 binding complex after IL-1beta stimulation. In addition, cotransfection of the architectural transcription factor high mobility group (HMG)-I(Y) protein with c-Fos and c-Jun markedly increased trans-activation of the CD44 promoter. Taken together, our studies demonstrate that AP-1 proteins are a central regulatory component used by IL-1beta to modulate expression of CD44 during an inflammatory response in vascular smooth muscle cells and that transcription of CD44 by AP-1 proteins is enhanced by HMG-I(Y). -Foster, L. C., Wiesel, P., Huggins, G. S, Pañares, R., Chin, M. T., Pellacani, A., Perrella, M. A. Role of activating protein-1 and high mobility group-I(Y) protein in the induction of CD44 gene expression by interleukin-1beta in vascular smooth muscle cells.