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Extracellular matrix (ECM) proteins, collectively known as the matrisome, include collagens, glycoproteins, and proteoglycans. Alterations in the matrisome have been implicated in the neurodegenerative pathologies including Parkinson’s disease (PD). In this work, we utilized our previously published PD and control proteomics data from human prefrontal cortex and focused our analysis on the matrisome. Among matrisome proteins, we observed a significant enrichment in the expression of type I collagen in PD vs. control samples. We then performed histological analysis on the same samples used for proteomics study, and examined collagen expression using picrosirius red staining. Interestingly, we observed similar trends in collagen abundance in PD vs. control as in our matrisome analysis; thus, this and other histological analyses will be useful as a complementary technique in the future to study the matrisome in PD with a larger cohort, and it may aid in choosing regions of interest for proteomic analysis. Additionally, collagen hydroxyprolination was less variable in PD compared to controls. Glycoproteomic changes in matrisome molecules were also observed in PD relative to aged individuals, especially related to type VI collagen and versican. We further examined the list of differentially expressed matrisome molecules using network topology-based analysis and found that angiogenesis indicated by alterations in decorin and several members of the collagen family was affected in PD. These findings collectively identified matrisome changes associated with PD; further studies with a larger cohort are required to validate the current results.

INTRODUCTION: Approximately 20–25% of human breast tumors are found within an adipose, rather than fibrous, stroma. Adipose stroma is associated with an increased risk of lymph node metastasis, but the causal association between adipose stroma and metastatic progression in human breast cancer remains unclear. METHODS: We used micropatterned type I collagen gels to engineer ~3-mm-long microscale human breast tumors within a stroma that contains adipocytes and adipose-derived stem cells (ASCs) (collectively, "adipose cells"). Invasion and escape of human breast cancer cells into an empty 120-μm-diameter lymphatic-like cavity was used to model interstitial invasion and vascular escape in the presence of adipose cell-derived factors for up to 16 days. RESULTS: We found that adipose cells hasten invasion and escape by 1–2 days and 2–3 days, respectively. These effects were mediated by soluble factors secreted by the adipose cells, and these factors acted directly on tumor cells. Surprisingly, tumor invasion and escape were more strongly induced by ASCs than by adipocytes. CONCLUSIONS: This work reveals that both adipocytes and ASCs accelerate the interstitial invasion and escape of human breast cancer cells, and sheds light on the link between adipose stroma and lymphatic metastasis in human breast cancer. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00697-6.

Visceral obesity is associated with insulin resistance and higher risk of type 2 diabetes and metabolic diseases. A limited ability of adipose tissues to remodel through the recruitment and differentiation of adipose stem cells (ASCs) is associated with adipose tissue inflammation and fibrosis and the metabolic syndrome. We show that the lower adipogenesis of omental (Om) compared with abdominal subcutaneous (Abdsc) ASCs was associated with greater secretion of TGFβ ligands that acted in an autocrine/paracrine loop to activate SMAD2 and suppress adipogenesis. Inhibition of TGFβ signaling rescued Om ASC differentiation. In Abdsc ASCs, low concentrations of dexamethasone suppressed TGFβ signaling and enhanced adipogenesis, at least in part by increasing TGFBR3 protein that can sequester TGFβ ligands. Om ASCs were resistant to these dexamethasone effects; recombinant TGFBR3 increased their differentiation. Pericellular fibrosis, a hallmark of dysfunctional adipose tissue, was greater in Om and correlated with higher level of tissue TGFβ signaling activity and lower ASC differentiation. We conclude that glucocorticoids restrain cell-autonomous TGFβ signaling in ASCs to facilitate adipogenesis and healthy remodeling in Abdsc and these processes are impaired in Om. Therapies directed at overcoming glucocorticoid resistance in visceral adipose tissue may improve remodeling and help prevent metabolic complications of visceral obesity.

Adipose tissue fibrosis is regulated by the chronic and progressive metabolic imbalance caused by differences in caloric intake and energy expenditure. By exploring the cellular heterogeneity within fibrotic adipose tissue, we demonstrate that early adipocyte progenitor cells expressing both platelet derived growth factor receptor (PDGFR) α and β are the major contributors to extracellular matrix deposition. We show that the fibrotic program is promoted by senescent macrophages. These macrophages are highly enriched in the fibrotic stroma and exhibit a distinct expression profile . Furthermore, we demonstrate that these cells display a blunted phagocytotic capacity and acquire a senescence associated secretory phenotype. Finally, we determined that senescent macrophage-derived osteopontin in the fibrotic environment promoted progenitor cell proliferation, fibrotic gene expression, and inhibited adipogenesis. Our work reveals that obesity promotes macrophage senescence and provides a conceptual framework for the discovery of rational therapeutic targets for metabolic and inflammatory disease associated with obesity.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system., Graphical Abstract, Highlights • SARS-CoV-2 infection in induced lung cells is characterized by phosphoproteomics • Analysis of response reveals host cell signaling and protein expression profile • Comparison to studies in undifferentiated cell lines shows unique pathology in iAT2s • Systems-level predictions find druggable pathways that can impede viral life cycle, Hekman et al. describe how a layer of primary stem cells (iAT2s) recapitulating lung biology responds to infection with SARS-CoV-2. They compare their work to previous studies with immortalized cell lines. Their data predict what effect the virus has on a lung cell and which drugs may slow infection.

Background Pulmonary atelectasis is frequent in clinical settings. Yet there is limited mechanistic understanding and substantial clinical and biologic controversy on its consequences. The authors hypothesize that atelectasis produces local transcriptomic changes related to immunity and alveolar–capillary barrier function conducive to lung injury and further exacerbated by systemic inflammation. Methods Female sheep underwent unilateral lung atelectasis using a left bronchial blocker and thoracotomy while the right lung was ventilated, with (n = 6) or without (n = 6) systemic lipopolysaccharide infusion. Computed tomography guided samples were harvested for NextGen RNA sequencing from atelectatic and aerated lung regions. The Wald test was used to detect differential gene expression as an absolute fold change greater than 1.5 and adjusted P value (Benjamini–Hochberg) less than 0.05. Functional analysis was performed by gene set enrichment analysis. Results Lipopolysaccharide-unexposed atelectatic versus aerated regions presented 2,363 differentially expressed genes. Lipopolysaccharide exposure induced 3,767 differentially expressed genes in atelectatic lungs but only 1,197 genes in aerated lungs relative to the corresponding lipopolysaccharide-unexposed tissues. Gene set enrichment for immune response in atelectasis versus aerated tissues yielded negative normalized enrichment scores without lipopolysaccharide (less than –1.23, adjusted P value less than 0.05) but positive scores with lipopolysaccharide (greater than 1.33, adjusted P value less than 0.05). Leukocyte-related processes (e.g., leukocyte migration, activation, and mediated immunity) were enhanced in lipopolysaccharide-exposed atelectasis partly through interferon-stimulated genes. Furthermore, atelectasis was associated with negatively enriched gene sets involving alveolar–capillary barrier function irrespective of lipopolysaccharide (normalized enrichment scores less than –1.35, adjusted P value less than 0.05). Yes-associated protein signaling was dysregulated with lower nuclear distribution in atelectatic versus aerated lung (lipopolysaccharide-unexposed: 10.0 ± 4.2 versus 13.4 ± 4.2 arbitrary units, lipopolysaccharide-exposed: 8.1 ± 2.0 versus 11.3 ± 2.4 arbitrary units, effect of lung aeration, P = 0.003). Conclusions Atelectasis dysregulates the local pulmonary transcriptome with negatively enriched immune response and alveolar–capillary barrier function. Systemic lipopolysaccharide converts the transcriptomic immune response into positive enrichment but does not affect local barrier function transcriptomics. Interferon-stimulated genes and Yes-associated protein might be novel candidate targets for atelectasis-associated injury. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Abdominal aortic aneurysm (AAA) and intracranial aneurysm (IA) are serious arterial diseases in the aorta and brain, respectively. AAA and IA are associated with old age in males and females, respectively, and if rupture occurs, they carry high morbidity and mortality. Aneurysmal subarachnoid hemorrhage (SAH) due to IA rupture has a high rate of complication and fatality. Despite these severe clinical outcomes, preventing or treating these devastating diseases remains an unmet medical need. Inflammation and oxidative stress are shared pathologies of these vascular diseases. Therefore, therapeutic strategies have focused on reducing inflammation and reactive oxygen species levels. Interestingly, in response to cellular stress, the inducible heme oxygenase-1 (HO-1) is highly upregulated and protects against tissue injury. HO-1 degrades the prooxidant heme and generates molecules with antioxidative and anti-inflammatory properties, resulting in decreased oxidative stress and inflammation. Therefore, increasing HO-1 activity is an attractive option for therapy. Several HO-1 inducers have been identified and tested in animal models for preventing or alleviating AAA, IA, and SAH. However, clinical trials have shown conflicting results. Further research and the development of highly selective HO-1 regulators may be needed to prevent the initiation and progression of AAA, IA, or SAH.

Aortic carboxypeptidase-like protein (ACLP) is a collagen-binding extracellular matrix protein that has important roles in wound healing and fibrosis. ACLP contains thrombospondin repeats, a collagen-binding discoidin domain, and a catalytically inactive metallocarboxypeptidase domain. Recently, mutations in the ACLP-encoding gene, AE-binding protein 1 (AEBP1), have been discovered, leading to the identification of a new variant of Ehlers–Danlos syndrome causing connective tissue disruptions in multiple organs. Currently, little is known about the mechanisms of ACLP secretion or the role of post-translational modifications in these processes. We show here that the secreted form of ACLP contains N-linked glycosylation and that inhibition of glycosylation results in its intracellular retention. Using site-directed mutagenesis, we determined that glycosylation of Asn-471 and Asn-1030 is necessary for ACLP secretion and identified a specific N-terminal proteolytic ACLP fragment. To determine the contribution of secreted ACLP to extracellular matrix mechanical properties, we generated and mechanically tested wet-spun collagen ACLP composite fibers, finding that ACLP enhances the modulus (or stiffness), toughness, and tensile strength of the fibers. Some AEBP1 mutations were null alleles, whereas others resulted in expressed proteins. We tested the hypothesis that a recently discovered 40-amino acid mutation and insertion in the ACLP discoidin domain regulates collagen binding and assembly. Interestingly, we found that this protein variant is retained intracellularly and induces endoplasmic reticulum stress identified with an XBP1-based endoplasmic reticulum stress reporter. Our findings highlight the importance of N-linked glycosylation of ACLP for its secretion and contribute to our understanding of ACLP-dependent disease pathologies.

The vascular adventitia contains numerous cell types including fibroblasts, adipocytes, inflammatory cells, and progenitors embedded within a complex extracellular matrix (ECM) network. In response to vascular injury, adventitial progenitors and fibroblasts become activated and exhibit increased proliferative capacity and differentiate into contractile cells that remodel the ECM. These processes can lead to vascular fibrosis and disease progression. Our previous work established that the ECM protein aortic carboxypeptidase-like protein (ACLP) promotes fibrotic remodeling in the lung and is activated by vascular injury. It is currently unknown what controls vascular adventitial cell differentiation and if ACLP has a role in this process. Using purified mouse aortic adventitia Sca1+ progenitors, ACLP repressed stem cell markers (CD34, KLF4) and upregulated smooth muscle actin (SMA) and collagen I expression. ACLP enhanced myocardin-related transcription factor A (MRTFA) activity in adventitial cells by promoting MRTFA nuclear translocation. Sca1 cells from MRTFA-null mice exhibited reduced SMA and collagen expression induced by ACLP, indicating Sca1 cell differentiation is regulated in part by the ACLP-MRTFA axis. We determined that ACLP induced vessel contraction and increased adventitial collagen in an explant model. Collectively these studies identified ACLP as a mediator of adventitial cellular differentiation, which may result in pathological vessel remodeling.

AEBP1 encodes the aortic carboxypeptidase-like protein (ACLP) that associates with collagens in the extracellular matrix (ECM) and has several roles in development, tissue repair, and fibrosis. ACLP is expressed in bone, the vasculature, and dermal tissues and is involved in fibroblast proliferation and mesenchymal stem cell differentiation into collagen-producing cells. Aebp1−/− mice have abnormal, delayed wound repair correlating with defects in fibroblast proliferation. In this study, we describe four individuals from three unrelated families that presented with a unique constellation of clinical findings including joint laxity, redundant and hyperextensible skin, poor wound healing with abnormal scarring, osteoporosis, and other features reminiscent of Ehlers-Danlos syndrome (EDS). Analysis of skin biopsies revealed decreased dermal collagen with abnormal collagen fibrils that were ragged in appearance. Exome sequencing revealed compound heterozygous variants in AEBP1 (c.1470delC [p.Asn490_Met495delins(40)] and c.1743C>A [p.Cys581∗]) in the first individual, a homozygous variant (c.1320_1326del [p.Arg440Serfs∗3]) in the second individual, and a homozygous splice site variant (c.1630+1G>A) in two siblings from the third family. We show that ACLP enhances collagen polymerization and binds to several fibrillar collagens via its discoidin domain. These studies support the conclusion that bi-allelic pathogenic variants in AEBP1 are the cause of this autosomal-recessive EDS subtype.

White adipose tissue expands through both adipocyte hypertrophy and hyperplasia and it is hypothesized that fibrosis or excess accumulation of extracellular matrix within adipose tissue may limit tissue expansion contributing to metabolic dysfunction. The pathways that control adipose tissue remodeling are only partially understood, however it is likely that adipose tissue stromal and perivascular progenitors participate in fibrotic remodeling and also serve as adipocyte progenitors. The goal of this study was to investigate the role of the secreted extracellular matrix protein aortic carboxypeptidase-like protein (ACLP) on adipose progenitor differentiation in the context of adipose tissue fibrosis. Treatment of 10T1/2 mouse cells with recombinant ACLP suppressed adipogenesis and enhanced myofibroblast differentiation, which was dependent on transforming growth factor-β receptor kinase activity. Mice fed a chronic high fat diet exhibited white adipose tissue fibrosis with elevated ACLP expression and cellular fractionation of these depots revealed that ACLP was co-expressed with collagens primarily in the inflammatory cell depleted stromal-vascular fraction (SVF). SVF cells isolated from mice fed a high fat diet secreted increased amounts of ACLP compared to low fat diet control SVF. These cells also exhibited reduced adipogenic differentiation capacity in vitro. Importantly, differentiation studies in primary human adipose stromal cells revealed that mature adipocytes do not express ACLP and exogenous ACLP administration blunted their differentiation potential while upregulating myofibroblastic markers. Collectively, these studies identify ACLP as a stromal derived mediator of adipose progenitor differentiation that may limit adipocyte expansion during white adipose tissue fibrosis.

Perlecan is a major heparan sulfate (HS) proteoglycan in the arterial wall. Previous studies have linked it to atherosclerosis. Perlecan contains a core protein and three HS side chains. Its core protein has five domains (DI-DV) with disparate structures and DII is highly homologous to the ligand-binding portion of LDL receptor (LDLR). The functional significance of this domain has been unknown. Here, we show that perlecan DII interacts with LDL. Importantly, the interaction largely relies on O-linked glycans that are only present in the secreted DII. Among the five repeat units of DII, most of the glycosylation sites are from the second unit, which is highly divergent and rich in serine and threonine, but has no cysteine residues. Interestingly, most of the glycans are capped by the negatively charged sialic acids, which are critical for LDL binding. We further demonstrate an additive effect of HS and DII on LDL binding. Unlike LDLR, which directs LDL uptake through endocytosis, this study uncovers a novel feature of the perlecan LDLR-like DII in receptor-mediated lipoprotein retention, which depends on its glycosylation. Thus, perlecan glycosylation may play a role in the early LDL retention during the development of atherosclerosis.

Summary Adipose tissue is an essential regulator of metabolic homeostasis. In contrast with white adipose tissue, which stores excess energy in the form of triglycerides, brown adipose tissue is thermogenic, dissipating energy as heat via the unique expression of the mitochondrial uncoupling protein UCP1. A subset of UCP1+ adipocytes develops within white adipose tissue in response to physiological stimuli; however, the developmental origin of these "brite" or "beige" adipocytes is unclear. Here, we report the identification of a BMP7-ROCK signaling axis regulating beige adipocyte formation via control of the G-actin-regulated transcriptional coactivator myocardin-related transcription factor A, MRTFA. White adipose tissue from MRTFA –/– mice contains more multilocular adipocytes and expresses enhanced levels of brown-selective proteins, including UCP1. MRTFA –/– mice also show improved metabolic profiles and protection from diet-induced obesity and insulin resistance. Our study hence unravels a central pathway driving the development of physiologically functional beige adipocytes.

High mobility group (HMG) proteins are a family of architectural transcription factors, with HMGA1 playing a role in the regulation of genes involved in promoting systemic inflammatory responses. We speculated that blocking HMGA1-mediated pathways might improve outcomes from sepsis. To investigate HMGA1 further, we developed genetically modified mice expressing a dominant negative (dn) form of HMGA1 targeted to the vasculature. In dnHMGA1 transgenic (Tg) mice, endogenous HMGA1 is present, but its function is decreased due to the mutant transgene. These mice allowed us to specifically study the importance of HMGA1 not only during a purely pro-inflammatory insult of endotoxemia, but also during microbial sepsis induced by implantation of a bacterial-laden fibrin clot into the peritoneum. We found that the dnHMGA1 transgene was only present in Tg and not wild-type (WT) littermate mice, and the mutant transgene was able to interact with transcription factors (such as NF-κB), but was not able to bind DNA. Tg mice exhibited a blunted hypotensive response to endotoxemia, and less mortality in microbial sepsis. Moreover, Tg mice had a reduced inflammatory response during sepsis, with decreased macrophage and neutrophil infiltration into tissues, which was associated with reduced expression of monocyte chemotactic protein-1 and macrophage inflammatory protein-2. Collectively, these data suggest that targeted expression of a dnHMGA1 transgene is able to improve outcomes in models of endotoxin exposure and microbial sepsis, in part by modulating the immune response and suggest a novel modifiable pathway to target therapeutics in sepsis. High mobility group A1 is a modifiable pathway for therapeutics in sepsis, using a dominant negative approach to regulate the immune response.

Vascular smooth muscle cells (VSMCs) of the arterial wall normally display a differentiated and contractile phenotype. In response to arterial injury, VSMCs switch to a synthetic phenotype, contributing to vascular remodeling. Cysteine-rich protein 2 (CRP2) is a cytoskeletal protein expressed in VSMCs and blunts VSMC migration in part by sequestering the scaffolding protein p130Cas at focal adhesions. CRP2 deficiency in mice increases neointima formation following arterial injury. The goal of this study was to use Csrp2 promoter-lacZ transgenic mice to analyze CRP2 expression during VSMC phenotypic modulation. In a neointima formation model after carotid artery cessation of blood flow, lacZ reporter activity and smooth muscle (SM) α-actin expression in the media were rapidly downregulated 4 days after carotid ligation. Fourteen days after ligation, there was a high level expression of both Csrp2 promoter activity and SM α-actin protein expression in neointimal cells. In atherosclerosis prone mice fed an atherogenic diet, Csrp2 promoter activity was detected within complex atherosclerotic lesions. Interestingly, Csrp2 promoter activity was also present in the fibrous caps of complicated atherosclerotic lesions, indicating that CRP2 might contribute to plaque stability. These findings support the concept that CRP2 contributes to the phenotypic modulation of VSMCs during vascular disease. Modulating transcription to increase CRP2 expression during vascular injury might attenuate vascular remodeling. In addition, increased CRP2 expression at the fibrous caps of advanced lesions might also serve to protect atherosclerotic plaques from rupture.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway.

Pulmonary artery hypertension (PAH) patients exhibit elevated levels of inflammatory cytokines and infiltration of inflammatory cells in the lung. Concurrently, mutations of bmpr2, the gene encoding the type II receptor of bone morphogenetic proteins (BMP), are found in ∼75% of patients with familial PAH, but a possible nexus between increased inflammation and diminished BMP signaling has hitherto remained elusive. We previously showed that BMP4 triggers nuclear localization of the Myocardin-related transcription factor A (MRTF-A) in human pulmonary artery smooth muscle cells (PASMC), resulting in the induction of contractile proteins. Here we report the BMPR2-dependent repression of a set of inflammatory mediators in response to BMP4 stimulation of PASMC. Forced expression of MRTF-A precisely emulates the anti-inflammatory effect of BMP4, while MRTF-A depletion precludes BMP4-mediated cytokine inhibition. BMP4 and MRTF-A block signaling through NF-κB, the keystone of most pathways leading to inflammatory responses, at the level of chromatin recruitment and promoter activation. Moreover, MRTF-A physically interacts with RelA/p65, the NF-κB subunit endowed with a transcription activation domain. Interestingly, the MRTF-A-NF-κB interaction is mutually antagonistic: stimulation of NF-κB signaling by TNFα, as well as p65 overexpression, hinders MRTF-A activity and the expression of contractile genes. Thus, a molecular inhibitory pathway linking BMP4 signaling, activation of MRTF-A, and inhibition of NF-κB provides insights into the etiology of PAH and a potential focus of therapeutic intervention.

Pulmonary fibrosis is characterized by the excessive deposition of a collagen-rich extracellular matrix. The accumulation of collagen within the lung interstitium leads to impaired respiratory function. Furthermore, smooth muscle actin-positive myofibroblasts within the fibrotic lung contribute to disease progression. Because collagen and smooth muscle cell α-actin are coordinately expressed in the setting of fibrosis, the hypothesis was tested that specific transcriptional regulators of the myocardin family might also regulate collagen gene expression in myofibroblasts. Myocardin-related transcription factors (MRTFs), through their interaction with the serum-response factor (SRF) on CArG box regulatory elements (CC(A/T)6GG), are important regulators of myofibroblast differentiation. MRTF-A transactivated type I collagen gene reporters as much as 100-fold in lung myofibroblasts. Loss of functional MRTF-A using either a dominant negative MRTF-A isoform, shRNA targeting MRTF-A, or genetic deletion of MRTF-A in lung fibroblasts significantly disrupted type I collagen synthesis relative to controls. Analysis of the COL1A2 proximal promoter revealed a noncanonical CArG box (CCAAACTTGG), flanked by several Sp1 sites important for MRTF-A activation. Chromatin immunoprecipitation experiments confirmed the co-localization of MRTF-A, SRF, and Sp1 bound to the same region of the COL1A2 promoter. Mutagenesis of either the noncanonical CArG box or the Sp1 sites significantly disrupted MRTF-A activation of COL1A2. Together, our findings show that MRTF-A is an important regulator of collagen synthesis in lung fibroblasts and exhibits a dependence on both SRF and Sp1 function to enhance collagen expression.

In the postnatal vasculature, fully differentiated and quiescent vascular smooth muscle cells (VSMCs) in a "contractile" phenotype are required for the normal regulation of vascular tone. The transforming growth factor-β (TGF-β) superfamily of growth factors (TGF-βs and bone morphogenetic proteins (BMPs)) are potent inducers of contractile phenotype and mediate (i) induction of contractile genes, and (ii) inhibition of VSMC growth and migration. Transcription of contractile genes is positively regulated by a regulatory DNA element called a CArG box. The CArG box is activated by the binding of serum response factor and its coactivators, myocardin (Myocd) or Myocd-related transcription factors (MRTFs). Krüppel-like factor-4 (KLF4) is known to inhibit activation of the CArG box. However, the potential role of KLF4 in the contractile activities of TGF-β or BMP has not been explored. Here, we demonstrate that TGF-β and BMP4 rapidly down-regulate KLF4 through induction of microRNA-143 (miR-143) and miR-145, which leads to a reduction of KLF4 transcripts and decreased KLF4 protein expression. Inhibition of miR-145 prevents down-regulation of KLF4 and activation of contractile genes by TGF-β or BMP4, suggesting that modulation of KLF4 is a prerequisite for induction of contractile genes by TGF-β and BMP4. Interestingly, both TGF-β and BMP4 activate transcription of the miR-143/145 gene cluster through the CArG box, however, TGF-β mediates this effect through induction of Myocd expression, whereas BMP4 utilizes nuclear translocation of MRTF-A. Thus, this study sheds light on both the similarities and the differences of TGF-β and BMP4 signaling in the regulation of KLF4 and contractile genes.

Mice lacking aortic carboxypeptidase-like protein (ACLP) exhibit a gastroschisis (GS) like abdominal wall defect. The objectives of this study were to evaluate the pathophysiological features of GS in ACLP mice and to characterize the neuromuscular development of the eviscerated intestine (EI). ACLP mice were created by heterozygous mating from previously generated mice with targeted disruption of ACLP. Specimens were processed for H&E, and immunohistochemistry for smooth muscle cells [SMC, alpha-smooth muscle actin (alpha-SMA) antibody], interstitial cells of Cajal (ICC, c-kit-antibody), neural crest cells (NCC, Hox-b5-antibody), and enteric neurons (EN, PGP9.5-, alpha-internexin, and synaptophysin antibody). From 47 fetuses genotyped, 13 (27.7%) were wild type, 20 (42.5%) were heterozygous, and 14 (29.8%) were ACLP homozygous. In GS mice, expression of c-kit, Hox-b5, PGP-9.5, alpha-internexin, and synaptophysin were almost completely absent and only faint alpha-SMA expression was seen in the EI. In contrast, c-kit, Hox-b5, PGP9.5, alpha-internexin, synaptophysin, and alpha-SMA expression in intra-abdominal intestine in GS fetuses was the same as control intestine. The defect observed in ACLP mice closely resembles GS. Absence of ICC, NCC, EN, and immature differentiation of SMC supports an associated defect in neuromuscular development that is restricted to the EI.

Objective— An absence of cysteine-rich protein 2 (CRP2) enhances vascular smooth muscle cell (VSMC) migration and increases neointima formation after arterial injury; therefore, CRP2 plays an important role in the response to vascular injury. The goal of the present study was to elucidate the molecular mechanisms that preserve CRP2 expression in the adult vasculature and thus might serve to inhibit the response to injury. Methods and Results— We generated a series of transgenic mice harboring potential Csrp2 regulatory regions with a lacZ reporter. We determined that the 12-kb first intron was necessary for transgene activity in adult but not in developing vasculature. Within the intron we identified a 6.3-kb region that contains 2 CArG boxes. Serum response factor preferentially bound to CArG2 box in gel mobility shift and chromatin immunoprecipitation assays; additionally, serum response factor coactivator myocardin factors activated CRP2 expression via the CArG2 box. Mutational analysis revealed that CArG2 box was important in directing lacZ expression in VSMC of adult vessels. Conclusion— Although CRP2 expression during development is independent of CArG box regulatory sites, CRP2 expression in adult VSMC requires CArG2 element within the first intron. Our results suggest that distinct mechanisms regulate CRP2 expression in VSMC that are controlled by separate embryonic and adult regulatory modules.

The inducible form of nitric oxide synthase (NOS2) plays an important role in sepsis incurred as a result of infection with Gram-negative bacteria that elaborate endotoxin. The HMGA1 (high-mobility group A1) architectural transcription factor facilitates NOS2 induction by binding a specific AT-rich Oct (octamer) sequence in the core NOS2 promoter via AT-hook motifs. The small-molecule MGB (minor-groove binder) netropsin selectively targets AT-rich DNA sequences and can interfere with transcription factor binding. We therefore hypothesized that netropsin would improve survival from murine endotoxaemia by attenuating NOS2 induction through interference with HMGA1 DNA binding to the core NOS2 promoter. Netropsin improved survival from endotoxaemia in wild-type mice, yet not in NOS2-deficient mice, supporting an important role for NOS2 in the beneficial effects of MGB administration. Netropsin significantly attenuated NOS2 promoter activity in macrophage transient transfection studies and the AT-rich HMGA1 DNA-binding site was critical for this effect. EMSAs (electrophoretic mobility-shift assays) demonstrated that netropsin interferes with HMGA1 NOS2 promoter binding and NMR spectroscopy was undertaken to characterize this disruption. Chemical shift perturbation analysis identified that netropsin effectively competes both HMGA1 DNA-binding AT-hooks from the AT-rich NOS2 promoter sequence. Furthermore, NOESY data identified direct molecular interactions between netropsin and A/T base pairs within the NOS2 promoter HMGA1-binding site. Finally, we determined a structure of the netropsin/NOS2 promoter Oct site complex from molecular modelling and dynamics calculations. These findings represent important steps toward refined structure-based ligand design of novel compounds for therapeutic benefit that can selectively target key regulatory regions within genes that are important for the development of critical illness.

Background— The striated preferentially expressed gene (Speg) generates 4 different isoforms through alternative promoter use and tissue-specific splicing. Depending on the cell type, Speg isoforms may serve as markers of striated or smooth muscle differentiation. Methods and Results— To elucidate function of Speg gene isoforms, we disrupted the Speg gene locus in mice by replacing common exons 8, 9, and 10 with a lacZ gene. β-Galactosidase activity was detected in cardiomyocytes of the developing heart starting at day 11.5 days post coitum (dpc). β-Galactosidase activity in other cell types, including vascular smooth muscle cells, did not begin until 18.5 dpc. In the developing heart, protein expression of only Spegα and Spegβ isoforms was present in cardiomyocytes. Homozygous Speg mutant hearts began to enlarge by 16.5 dpc, and by 18.5 dpc, they demonstrated dilation of right and left atria and ventricles. These cardiac abnormalities in the absence of Speg were associated with a cellular hypertrophic response, myofibril degeneration, and a marked decrease in cardiac function. Moreover, Speg mutant mice exhibited significant neonatal mortality, with increased death occurring by 2 days after birth. Conclusions— These findings demonstrate that mutation of the Speg locus leads to cardiac dysfunction and a phenotype consistent with a dilated cardiomyopathy.

CRP2 (cysteine-rich protein) is a vascular smooth muscle cell (VSMC)-expressed LIM-only protein. CRP2 associates with the actin cytoskeleton and interacts with transcription factors in the nucleus to mediate smooth muscle cell gene expression. Using Csrp2 (gene symbol of the mouse CRP2 gene)-deficient mice, we previously demonstrated that an absence of CRP2 enhances VSMC migration and increases neointima formation following arterial injury. Despite its importance in vascular injury, the molecular mechanisms controlling CRP2 expression in VSMC are largely unknown. Transforming growth factor beta (TGFbeta), a key factor present in the vessel wall in the early phases of arterial response to injury, plays an important role in modulating lesion formation. Because both CRP2 and TGFbeta are mediators of VSMC responses, we examined the possibility that TGFbeta might regulate CRP2 expression. TGFbeta significantly induced CRP2 mRNA and protein expression in VSMCs. Promoter analysis identified a conserved cAMP-responsive element (CRE)-like site (TAACGTCA) in the Csrp2 promoter that was critical for basal promoter activity and response to TGFbeta. Gel mobility shift assays revealed that mainly ATF2 bound to this CRE-like element, and mutation of the CRE sequences abolished binding. TGFbeta enhanced the activation of ATF2, leading to increased phospho-ATF2 levels within the DNA-protein complexes. Furthermore, ATF2-transactivated Csrp2 promoter activity and TGFbeta enhanced this activation. In addition, a phosphorylation-negative ATF2 mutant construct decreased basal and TGFbeta-mediated Csrp2 promoter activity. Our results show for the first time in VSMC that TGFbeta activates ATF2 phosphorylation and Csrp2 gene expression via a CRE promoter element.

Nitric oxide synthase (NOS)2, an inducible enzyme that produces NO during inflammation, is transcriptionally regulated. Our goal was to determine whether high mobility group (HMG)A1 contributes to human (h)NOS2 gene regulation. Using a small molecule inhibitor of HMGA1 binding to DNA, or a dominant-negative form of HMGA1, we blunted the induction of hNOS2 by pro-inflammatory stimuli. Binding of HMGA1 in the region −3506 to −3375 of the hNOS2 promoter, a region not previously known to be involved in hNOS2 regulation, contributed to the induction of hNOS2 promoter in conjunction with upstream enhancer regions. We demonstrate a previously unknown role for HMGA1 in the regulation of hNOS2.

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that is acutely induced by inflammatory stimuli, and the products of HO-1-mediated heme degradation have anti-inflammatory properties. In many different pathophysiologic states, the up-regulation of HO-1 has been shown to be beneficial in combating the detrimental consequences of increased inflammation. Ets transcription factors are known to be important mediators of inflammatory responses, and the ternary complex factor subfamily of Ets proteins has both transcriptional activation and repression activity. The present study demonstrates that of several ternary complex factor subfamily members, only Elk-3 represses HO-1 promoter activity in macrophages. Endotoxin administration to macrophages led to a dose-dependent decrease in endogenous Elk-3 mRNA levels, and this reduction in Elk-3 preceded the LPS-mediated up-regulation of HO-1 message. Analogous results also occurred in lung tissue of mice exposed to endotoxin. Two putative Ets binding sites (EBS1 and EBS2) are present in the downstream region of the murine HO-1 promoter (bp −125 and −93, respectively), and we recently showed that the EBS2 site is essential for HO-1 induction by endotoxin. In contrast, the present study demonstrates that the repressive effect of Elk-3 on HO-1 promoter activity is dependent on the EBS1 site. Taken together, our data reveal that Elk-3 serves as an important repressor of HO-1 gene transcription and contributes to the tight control of HO-1 gene regulation in the setting of inflammatory stimuli.

We recently isolated a novel bHLH/PAS protein, CLIF (cycle like factor), by yeast two-hybrid screening of human umbilical endothelial cell cDNA library. CLIF is preferentially expressed in endothelial and neuronal cells. Because CLIF is expressed in vascular endothelial cells and forms a heterodimer with CLOCK, the key transcription factor controlling the circadian rhythm, we hypothesized that CLIF regulates the circadian oscillation of PAI-1 gene expression in endothelial cells. Northern blot analysis of mouse organs showed circadian oscillations of PAI-I mRNA levels. In addition, the clock-related genes also showed circadian oscillation in peripheral tissues. In endothelial cells, the heterodimer of CLIF and CLOCK upregulated the PAI-1 gene expression through E-box sites. Furthermore, Period and Cryptochrome, which are negative regulators in the feedback loop of the biological clock, inhibited PAI-1 promoter activation by the CLOCK:CLIF heterodimer. These results suggest that the peripheral tissues have their own biological clock and CLIF regulates the circadian oscillation of PAI-1 gene expression in endothelial cells. This study suggests a novel molecular mechanism of the morning onset of myocardial infarction. Here we review our recent work and literature.

In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix.

In response to arterial injury, medial vascular smooth muscle cells (VSMCs) proliferate and migrate into the intima, contributing to the development of occlusive vascular disease. The LIM protein cysteine-rich protein (CRP) 2 associates with the actin cytoskeleton and may maintain the cytoarchitecture. CRP2 also interacts with transcription factors in the nucleus to mediate SMC gene expression. To test the hypothesis that CRP2 may be an important regulator of vascular development or function we generated Csrp2 (gene symbol of the mouse CRP2 gene)-deficient ( Csrp2 −/− ) mice by targeted mutation. Csrp2 −/− mice did not have any gross vascular defects or altered expression levels of SM α-actin, SM22α, or calponin. Following femoral artery injury, CRP2 expression persisted in the vessel wall at 4 days and then decreased by 14 days. Intimal thickening was enhanced 3.4-fold in Csrp2 −/− compared with wild-type (WT) mice 14 days following injury. Cellular proliferation was similar between WT and Csrp2 −/− VSMC both in vivo and in vitro. Interestingly, Csrp2 −/− VSMC migrated more rapidly in response to PDGF-BB and had increased Rac1 activation. Our data demonstrate that CRP2 is not required for vascular development. However, an absence of CRP2 enhanced VSMC migration and increased neointima formation following arterial injury.

Adipogenesis is characterized by early remodeling of the extracellular matrix, allowing preadipocytes to adopt a more spherical shape and optimize lipid accumulation as they mature. Aortic carboxypeptidase-like protein (ACLP), found in collagen-rich tissues including adipose tissue, is expressed in 3T3-L1 and 3T3-F442A preadipocytes, and is downregulated during adipogenesis. We now report that ACLP is found in medium conditioned by 3T3-L1 preadipocytes. Transforming growth factor (TGF) β, a known modulator of fibrillar matrix protein production, increased ACLP expression by 2.4 ± 0.4-fold (mean ± SE; n = 3) in 3T3-L1 preadipocytes, through a mechanism that requires p42/44 MAPK activity. Addition of TGFβ to differentiation medium, which inhibits adipogenesis, raised ACLP levels in 3T3-L1 cells. However, sustained expression of ACLP in stable clones of 3T3-L1 or 3T3-F442A preadipocytes did not interfere with adipogenesis.

Increased production of reactive oxygen species contributes to the etiology of diabetes complications. Pathophysiological stimuli that increase oxidative stress upregulate heme oxygenase (HO)-1, a cytoprotective heme-degrading enzyme. We hypothesized that HO-1 may be important in myocardial injury that is exacerbated by diabetes. To test this hypothesis, the left anterior descending coronary arteries of nondiabetic and diabetic wild-type (HO-1(+/+)) and HO-1 null (HO-1(-/-)) mice were ligated for 1 h followed by 24 h reperfusion. The absence of HO-1 significantly increased myocardial infarct size (36.4 +/- 2.0 vs. 21.4 +/- 1.8% in HO-1(+/+) mice), while cardiac-specific overexpression of HO-1 protected against myocardial ischemic injury in diabetic mice. Despite similar high blood glucose levels, diabetic HO-1(-/-) mice had fourfold higher oxidative stress and larger infarcts (56.0 +/- 2.8%) than diabetic HO-1(+/+) mice (30.8 +/- 6.1%). Moreover, hyperglycemia increased the mortality of HO-1(-/-) mice (31.3%) after ischemia/reperfusion injury, and 55% of diabetic HO-1(-/-) mice had mural thrombi in the left ventricles. The increased mortality of diabetic HO-1(-/-) mice may be in part due to formation of left ventricular mural thrombi. Our data demonstrate that the absence of HO-1 renders animals more susceptible to myocardial ischemia/reperfusion damage and diabetes worsens the injury.

Heme oxygenase (HO) enzymes catalyze the initial reaction in heme catabolism. HO-1 is an inducible isoform that is up-regulated by diverse stimuli, including inflammatory cytokines and factors that promote oxidative stress. HO-1 is a cytoprotective enzyme that degrades heme, a potent oxidant, to generate carbon monoxide, biliverdin (subsequently reduced to bilirubin), and iron. Recently, we found that thioredoxin (TRX), a disulfide reductase enzyme known to be important for the binding of transcription factors to DNA, contributes to the induction of HO-1 by inflammatory mediators. In the present study, we extended this observation and determined that, similar to HO-1, TRX and TRX reductase (TR) are induced by bacterial lipopolysaccharide in macrophages at the level of mRNA and protein. However, maximal induction of TRX and TR precedes that of HO-1. Increased expression of HO-1 in the cytoplasm of inflammatory cells corresponds to a translocation of TRX into the nucleus of these cells. Finally, transfection of TRX into macrophages promoted an increase in HO-1 protein. Taken together, these data support the concept that the TRX system contributes to the up-regulation of HO-1 under conditions associated with increased oxidative stress.

Cathepsin S is a cysteine protease with potent endoproteolytic activity and a broad pH profile. Cathepsin S activity is essential for complete processing of the MHC class II-associated invariant chain within B cells and dendritic cells, and may also be important in extracellular matrix degradation in atherosclerosis and emphysema. Unique among cysteine proteases, cathepsin S activity is up-regulated by IFN-γ. Given its importance, we sought to elucidate the pathway by which IFN-γ increases cathepsin S expression. Our data demonstrate that the cathepsin S promoter contains an IFN-stimulated response element (ISRE) that is critical for IFN-γ-induced gene transcription in a cell line derived from type II alveolar epithelial (A549) cells. IFN response factor (IRF)-2 derived from A549 nuclear extracts associates with the ISRE oligonucleotide in gel shift assays, but is quickly replaced by IRF-1 following stimulation with IFN-γ. The time course of IRF-1/ISRE complex formation correlates with increased levels of IRF-1 protein and cathepsin S mRNA. Overexpression of IRF-1, but not IRF-2, markedly augments cathepsin S promoter activity in A549 cells. Furthermore, overexpression of IRF-1 increases endogenous cathepsin S mRNA levels in 293T epithelial cells. Finally, freshly isolated bone marrow cells from IRF-1−/− mice fail to up-regulate cathepsin S activity in response to IFN-γ. Thus, IRF-1 is the critical transcriptional mediator of IFN-γ-dependent cathepsin S activation. These data elucidate a new pathway by which IRF-1 may affect MHC class II processing and presentation.

Background — Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. Methods and Results — Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1β protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. Conclusions — This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel “pre-event” gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.

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.

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.

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.

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.

Aortic preferentially expressed gene-1 (APEG-1) was originally identified as a 1.4-kilobase (kb) transcript preferentially expressed in differentiated vascular smooth muscle cells (VSMC). Its expression is markedly down-regulated in de-differentiated VSMC, suggesting a role for APEG-1 in VSMC differentiation. We have now determined that APEG-1 is a single-copy gene in the human, rat, and mouse genomes and have mapped human APEG-1 to chromosome 2q34. To study the molecular mechanisms regulating its expression, we characterized the genomic organization and promoter of mouse APEG-1. APEG-1 spans 4.5 kb in the mouse genome and is composed of five exons. Using reporter gene transfection analysis, we found that a 2.7-kb APEG-15′-flanking sequence directed a high level of promoter activity only in VSMC. Its activity was minimal in five other cell types. A repressor region located within an upstream 685-base pair sequence suppressed the activity of this 2.7-kb promoter. Further deletion and mutation analyses identified an E box motif as a positive regulatory element, which was bound by nuclear protein prepared from VSMC. In conjunction with its flanking sequence, this E box motif confers VSMC-specific enhancer activity to a heterologous SV40 promoter. To our knowledge, this is the first demonstration of an E box motif that mediates gene expression restricted to VSMC.

Phenotypic modulation of vascular smooth muscle cells plays an important role in the pathogenesis of arteriosclerosis. In a screen of proteins expressed in human aortic smooth muscle cells, we identified a novel gene product designated aortic carboxypeptidase-like protein (ACLP). The approximately 4-kilobase human cDNA and its mouse homologue encode 1158 and 1128 amino acid proteins, respectively, that are 85% identical. ACLP is a nonnuclear protein that contains a signal peptide, a lysine- and proline-rich 11-amino acid repeating motif, a discoidin-like domain, and a C-terminal domain with 39% identity to carboxypeptidase E. By Western blot analysis and in situ hybridization, we detected abundant ACLP expression in the adult aorta. ACLP was expressed predominantly in the smooth muscle cells of the adult mouse aorta but not in the adventitia or in several other tissues. In cultured mouse aortic smooth muscle cells, ACLP mRNA and protein were up-regulated 2-3-fold after serum starvation. Using a recently developed neural crest cell to smooth muscle cell in vitro differentiation system, we found that ACLP mRNA and protein were not expressed in neural crest cells but were up-regulated dramatically with the differentiation of these cells. These results indicate that ACLP may play a role in differentiated vascular smooth muscle cells.

Several members of the LIM protein family have important roles in development and differentiation. We recently isolated a rat cDNA encoding a new member of this family, CRP2/SmLIM, that contains two LIM domains and is expressed preferentially in vascular smooth muscle cells (VSMC). To study the molecular mechanisms that regulate VSMC-specific transcription of the Crp2/SmLim gene, we cloned the cDNA and gene of mouse Crp2/SmLim. Mouse Crp2/SmLim is a single copy gene of six exons and five introns spanning approximately 20 kilobases of genomic DNA. By 5'-rapid amplification of cDNA ends and S1 nuclease protection assay, we determined that the transcription start site is an A residue 80 base pairs 5' of the translation initiation codon. A TATA-like sequence is located 27 base pairs 5' of the transcription start site, and there are potential cis-acting elements (GATA, Sp1, AP-2, E box, CCAC box, and GArC motif) in the 5'-flanking sequence. In transient transfection assays in rat aortic smooth muscle cells in primary culture, 5 kilobases of the Crp2/SmLim 5'-flanking sequence generated a high level of luciferase reporter gene activity. By deletion analysis and gel mobility shift assay, we found that the region between bases -74 and -39 of this 5 kilobase DNA fragment binds Sp1 and confers basal promoter activity in the Crp2/SmLim gene. In vitro, the 5-kilobase fragment was active in multiple cell types. In vivo, however, the 5-kilobase fragment directed high level expression of the lacZ reporter gene preferentially in the VSMC of transgenic mice, indicating the presence of VSMC-specific element(s) in this fragment.

Members of the erythroid Krüppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the beta-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the beta-globin gene as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell cDNA library. Sequence analysis indicated that our clone, hEZF, is the human homologue of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein bound specifically to a probe containing the CACCC core sequence. In co-transfection experiments, we found that sense but not antisense hEZF decreased the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, we mapped a repression domain in hEZF to amino acids 181-388. We also found that amino acids 91-117 of hEZF confer an activation function on the GAL4 DNA-binding domain.