Weight gain prevention buffers the impact of CETP rs3764261 on high density lipoprotein cholesterol in young adulthood: The Study of Novel Approaches to Weight Gain Prevention (SNAP).

BACKGROUND AND AIMS: Two weight gain prevention strategies, one targeting small changes to diet and physical activity and a second targeting large changes, significantly reduced weight gain in young adulthood. We examined whether weight gain prevention blunts genetic risk for body weight increase and/or high density lipoprotein cholesterol (HDL-C) lowering over two years. METHODS AND RESULTS: Participants were 524 male and female young adults (mean age = 28.2, SD = 4.3; mean BMI = 25.5, SD = 2.6). Obesity-related SNPs accounting for ≥ 0.04% of the variance were genotyped and combined into a genetic risk score. For HDL-C, SNPs within CETP, LIPC and FADS2 were genotyped. The obesity-related genetic risk score did not predict change in BMI independently or in interaction with treatment arm. However, consistent with the prior literature, each copy of the HDL-C risk, C, allele at CETP rs3764261 was associated with lower HDL-C at baseline. Moreover, significant interaction between SNP and treatment arm for change in HDL-C was observed (p = 0.02). In the control group, HDL-C change was dependent upon rs3764261 (p = 0.004) with C allele carriers showing a continued reduction in HDL-C. In contrast, within the two intervention groups, HDL-C increased on average with no differential effect of rs3764261 (p > 0.24). Notably, even among carriers of the CC genotype, small and large change arms were associated with increased HDL-C and the control arm a reduction (p = 0.013). CONCLUSIONS: The C allele at CETP rs3764261 is a strong risk factor for low HDL-C in young adulthood but weight gain prevention may mitigate this risk. CLINICAL TRIAL REGISTRY NUMBER AND WEBSITE: clinicaltrials.gov Identifier: NCT01183689, https://clinicaltrials.gov/.

CYP3A4 genotype is associated with sildenafil concentrations in patients with heart failure with preserved ejection fraction.

Despite its established inter-individual variability, sildenafil has been the subject of only a few pharmacogenetic investigations, with limited data regarding the genetic modulators of its pharmacokinetics. We conducted a pharmacogenetic sub-study of patients randomized to sildenafil (n=85) in the RELAX trial, which investigated the impact of high-dose sildenafil in patients with heart failure with preserved left ventricular ejection fraction (HFpEF). In the overall population, the CYP3A4 inferred phenotype appeared associated with the dose-adjusted peak concentrations of sildenafil at week 12 and week 24 (adjusted P=0.045 for repeated measures analysis), although this P-value did not meet our corrected significance threshold of 0.0167. In the more homogeneous Caucasian subgroup, this association was significant (adjusted P=0.0165 for repeated measures). Hence, CYP3A4 inferred phenotype is associated with peak sildenafil dose-adjusted concentrations in patients with HFpEF receiving high doses of sildenafil. The clinical impact of this association requires further investigation.

A pharmacogenetic investigation of intravenous furosemide in decompensated heart failure: a meta-analysis of three clinical trials.

We conducted a meta-analysis of pharmacogenomic substudies of three randomized trials conducted in patients with decompensated heart failure (HF) that were led by National Heart Lung and Blood Institute (NHLBI)-funded HF Network to test the hypothesis that candidate genes modulate net fluid loss and weight change in patients with decompensated HF treated with a furosemide-based diuretic regimen. Although none of the genetic variants previously shown to modulate the effects of loop diuretics in healthy individuals were associated with net fluid loss after 72 h of treatment, a set of rare variants in the APOL1 gene, which codes for apolipoprotein L1 (P=0.0005 in the random effects model), was associated with this end point. Moreover, a common variant in the multidrug resistance protein-4 coding gene (ABCC4, rs17268282) was associated with weight loss with furosemide use (P=0.0001). Our results suggest that both common and rare genetic variants modulate the response to a furosemide-based diuretic regimen in patients with decompensated HF.

Common variants associated with changes in levels of circulating free fatty acids after administration of glucose-insulin-potassium (GIK) therapy in the IMMEDIATE trial.

Glucose-insulin-potassium (GIK) therapy may promote a shift from oxygen-wasteful free fatty acid (FFA) metabolism to glycolysis, potentially reducing myocardial damage during ischemia. Genetic variation associated with FFA response to GIK was investigated in an IMMEDIATE (Immediate Myocardial Metabolic Enhancement During Initial Assessment and Treatment in Emergency care) sub-study (n=117). In patients with confirmed acute coronary syndromes, associations between 132 634 variants and 12-h circulating FFA response were assessed. Between initial and 6-h measurements, three LINGO2 variants were associated with increased levels of total FFA (P-value for 2 degree of freedom test, P2df ⩽5.51 × 10-7). Lead LINGO2 single-nucleotide polymorphism, rs12003487, was nominally associated with reduced 30-day ejection fraction (P2df=0.03). Several LINGO2 signals were linked to alterations in epigenetic profile and gene expression levels. Between 6 and 12 h, rs7017336 nearest to IMPA1/FABP12 showed an association with decreased saturated FFAs (P2df=5.47 × 10-7). Nearest to DUSP26, rs7464104 was associated with a decrease in unsaturated FFAs (P2df=5.51 × 10-7). Genetic variation may modify FFA response to GIK, potentially conferring less beneficial outcomes.

Genetic modifiers of response to glucose-insulin-potassium (GIK) infusion in acute coronary syndromes and associations with clinical outcomes in the IMMEDIATE trial.

Modifiers of response to glucose, insulin and potassium (GIK) infusion may affect clinical outcomes in acute coronary syndromes (ACS). In an Immediate Myocardial Metabolic Enhancement During Initial Assessment And Treatment In Emergency Care (IMMEDIATE) trial's sub-study (n = 318), we explored effects of 132,634 genetic variants on plasma glucose and potassium response to 12-h GIK infusion. Associations between metabolite-associated variants and infarct size (n = 84) were assessed. The 'G' allele of rs12641551, near ACSL1, as well as the 'A' allele of XPO4 rs2585897 were associated with a differential glucose response (P for 2 degrees of freedom test, P2df ⩽ 4.75 × 10(-7)) and infarct size with GIK (P2df < 0.05). Variants within or near TAS1R3, LCA5, DNAH5, PTPRG, MAGI1, PTCSC3, STRADA, AKAP12, ARFGEF2, ADCYAP1, SETX, NDRG4 and ABCB11 modified glucose response, and near CSF1/AHCYL1 potassium response (P2df ⩽ 4.26 × 10(-7)), but not outcomes. Gene variants may modify glucose and potassium response to GIK infusion, contributing to cardiovascular outcomes in ACS.

Genetic variation at glucose and insulin trait loci and response to glucose-insulin-potassium (GIK) therapy: the IMMEDIATE trial.

The mechanistic effects of intravenous glucose, insulin and potassium (GIK) in cardiac ischemia are not well understood. We conducted a genetic sub-study of the Immediate Myocardial Metabolic Enhancement During Initial Assessment and Treatment in Emergency care (IMMEDIATE) Trial to explore effects of common and rare glucose and insulin-related genetic loci on initial to 6-h and 6- to 12-h change in plasma glucose and potassium. We identified 27 NOTCH2/ADAM30 and 8 C2CD4B variants conferring a 40-57% increase in glucose during the first 6 h of infusion (P<5.96 × 10(-6)). Significant associations were also found for ABCB11 and SLC30A8 single-nucleotide polymorphisms (SNPs) and glucose responses, and an SEC61A2 SNP with a potassium response to GIK. These studies identify genetic factors that may impact the metabolic response to GIK, which could influence treatment benefits in the setting of acute coronary syndromes (ACS).

FTO predicts weight regain in the Look AHEAD clinical trial.

BACKGROUND: Genome-wide association studies have provided new insights into the genetic factors that contribute to the development of obesity. We hypothesized that these genetic markers would also predict magnitude of weight loss and weight regain after initial weight loss. METHODS: Established obesity risk alleles available on the Illumina CARe iSelect (IBC) chip were characterized in 3899 overweight or obese participants with type 2 diabetes from the Look AHEAD (Action for Health in Diabetes), a randomized trial to determine the effects of intensive lifestyle intervention (ILI) and diabetes support and education (DSE) on cardiovascular morbidity and mortality. Primary analyses examined the interaction between 13 obesity risk polymorphisms in eight genes and randomized treatment arm in predicting weight change at year 1, and weight regain at year 4 among individuals who lost 3% or more of their baseline weight by year 1. RESULTS: No single-nucleotide polymorphisms (SNPs) were significantly associated with magnitude of weight loss or interacted with treatment arm at year 1. However, fat mass and obesity associated gene (FTO) rs3751812 predicted weight regain within DSE (1.56 kg per risk allele, P=0.005), but not ILI (P=0.761), resulting in SNP × treatment arm interaction (P=0.009). In a partial replication of prior research, the obesity risk (G) allele at BDNF rs6265 was associated with greater weight regain across treatment arms (0.773 kg per risk allele), although results were of borderline statistical significance (P=0.051). CONCLUSIONS: Variations in the FTO and BDNF loci may contribute risk of weight regain after weight loss.

Association between arterial stiffness and variations in oestrogen-related genes.

Increased arterial stiffness and wave reflection have been identified as cardiovascular disease risk factors. In light of significant sex differences and the moderate heritability of vascular function measures, we hypothesized that variation in the genes coding for oestrogen receptors alpha (ESR1) and beta (ESR2) and aromatase (CYP19A1) is associated with aortic stiffness and pressure wave reflection as measured by non-invasive arterial tonometry. In all, 1261 unrelated Framingham Offspring Study participants who attended the seventh examination cycle (mean age 62+/-10 years, 52% women) and had arterial tonometry and genotyping data were included in the study. Analysis of covariance was used to assess the association of polymorphisms with forward wave amplitude, augmented pressure, augmentation index (AI), carotid-femoral pulse wave velocity and mean arterial pressure with adjustment for potential confounders. In the sex-pooled analysis, those homozygous for the minor allele at any of four ESR1 variants that were in strong linkage disequilibrium ((TA)(n), rs2077647, rs2234693 and rs9340799) had on an average 18% higher augmented pressure and 16% greater AI compared with carriers of one or two major alleles (P=0.0002-0.01). A similar magnitude of association was detected in those homozygous for the common allele at two ESR2 single-nucleotide polymorphisms (P=0.007-0.02). Our results are consistent with the hypothesis that variation in ESR1 and ESR2, but not CYP19A1, is associated with an increased wave reflection that may contribute to associations between these variants and adverse clinical events demonstrated earlier. Our findings will need to be replicated in additional cohorts.

The Filshie clip for laparoscopic adnexal surgery.

BACKGROUND: Gynecologic endoscopic procedures are increasingly common and require the ability to control large vascular structures. METHOD: The Filshie clip is a silicone-lined, titanium occlusive device, originally designed and Food and Drug Administration (FDA) approved for surgical contraception. This device also has the potential for occluding vascular structures during laparoscopic surgery. EXPERIENCE AND RESULTS: We describe a salpingectomy, an excision of bilateral hydrosalpinges, and a salpingo-oopherectomy. We performed all procedures laparoscopically using this device as the primary modality for assuring hemostasis. CONCLUSION: The Filshie clip is a useful and economical device for assuring hemostasis during gynecologic endoscopic surgery.

Friend of GATA 2 physically interacts with chicken ovalbumin upstream promoter-TF2 (COUP-TF2) and COUP-TF3 and represses COUP-TF2-dependent activation of the atrial natriuretic factor promoter.

Friend of GATA (FOG)-2 is a multi-zinc finger transcriptional corepressor protein that binds specifically to GATA4. Gene targeting studies have demonstrated that FOG-2 is required for normal cardiac morphogenesis, including the development of the coronary vasculature, left ventricular compact zone, and heart valves. To better understand the molecular mechanisms by which FOG-2 regulates these cardiac developmental programs, we screened a mouse day 11 embryo library using a yeast two-hybrid interaction trap with the fifth and sixth zinc fingers of FOG-2 as bait. Using this approach, we isolated clones encoding the orphan nuclear receptors chicken ovalbumin upstream promoter-transcription factor (COUP-TF) 2 and COUP-TF3. COUP-TF2-null embryos die during embryonic development with defective angiogenesis and cardiac defects, a pattern that partly resembles the FOG-2-null phenotype. The interaction between COUP-TF2 and FOG-2 in mammalian cells was confirmed by co-immunoprecipitation of these proteins from transfected COS-7 cells. The sites of binding interaction between COUP-TF2 and FOG-2 were mapped to zinc fingers 5 and 6 and fingers 7 and 8 of FOG-2 and to the carboxyl terminus of the COUP-TF proteins. Binding to COUP-TF2 was specific because FOG-2 did not interact with the ligand-binding domains of retinoid X receptor alpha, glucocorticoid receptor, and peroxisome proliferating antigen receptor gamma, which are related to the COUP-TF proteins. Full-length FOG-2 markedly enhanced transcriptional repression by GAL4-COUP-TF2(117-414), but not by a COUP-TF2 repression domain mutant. Moreover, FOG-2 repressed COUP-TF2dependent synergistic activation of the atrial natriuretic factor promoter by both GATA4 and the FOG-2-independent mutant GATA4-E215K. Taken together, these findings suggest that FOG-2 functions as a corepressor for both GATA and COUP-TF proteins.

Buprenorphine treatment of pregnant opioid--dependent women: maternal and neonatal outcomes.

This open-label prospective study examined maternal and neonatal safety and efficacy outcome measures during and following prenatal buprenorphine exposure. Three opioid-dependent pregnant women received 8 or 12 mg sublingual buprenorphine tablets daily for 15-16 weeks prior to delivery. Results showed that buprenorphine in combination with comprehensive prenatal care was safe and effective in these women. Prenatal exposure to buprenorphine resulted in normal birth outcomes, a mean of 4.33 days (minimum possible=4) hospitalization, and a 'relatively mild' neonatal abstinence syndrome comprised primarily of tremors (disturbed), hyperactive moro and shortened sleep after feeding. The infants required no pharmacological treatment. Onset of neonatal abstinence signs occurred within the first 12 h after birth, peaked by 72 h and returned to below pre-12 h levels by 120 h. It is concluded that buprenorphine has potential utility for the treatment of pregnant opioid-dependent women.

Effect of academic affiliation and obstetric volume on clinical outcome and cost of childbirth.

OBJECTIVE: To determine whether the academic affiliation and obstetric volume of the delivering hospital has an impact on clinical and economic outcomes. METHODS: We performed a cross-sectional analysis of data for all births in the State of Maryland during 1996. Acute hospital discharge data were obtained from the publicly available Maryland Health Services Cost Review Commission database. Institutions were classified as community hospitals, community teaching hospitals, and academic medical centers. Principal outcome variables included cesarean birth and complication rates, total hospital charges, and length of stay. RESULTS: A total of 63,143 cases were identified for analysis. The cesarean delivery rate was lower among academic medical centers, compared with community teaching hospitals and community hospitals (18.4% compared with 24.3% and 21.2%, respectively). After adjustment for patient case-mix, the adjusted odds ratio (OR) for cesarean birth was 0.66 at academic medical centers and 1.23 at community teaching hospitals compared with community hospitals (P <.01). Rates of episiotomy and serious complications were lower at academic medical centers compared with community hospitals. Adjusted total hospital charges were lower and length of stay was shorter for community hospitals compared with academic medical centers ($2937 compared with $3564 and 2.2 days compared with 2.5 days, respectively). CONCLUSION: Hospital academic affiliation was an important predictor of clinical outcomes. Better clinical outcomes were found primarily among patients at academic medical centers, although these institutions demonstrated moderately higher resource utilization, compared with community hospitals.

Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro.

Human pluripotent stem cells (hPSCs) have been derived from the inner cell mass cells of blastocysts (embryonic stem cells) and primordial germ cells of the developing gonadal ridge (embryonic germ cells). Like their mouse counterparts, hPSCs can be maintained in culture in an undifferentiated state and, upon differentiation, generate a wide variety of cell types. Embryoid body (EB) formation is a requisite step in the process of in vitro differentiation of these stem cells and has been used to derive neurons and glia, vascular endothelium, hematopoietic cells, cardiomyocytes, and glucose-responsive insulin-producing cells from mouse PSCs. EBs generated from human embryonic germ cell cultures have also been found to contain a wide variety of cell types, including neural cells, vascular endothelium, muscle cells, and endodermal derivatives. Here, we report the isolation and culture of cells from human EBs as well as a characterization of their gene expression during growth in several different culture environments. These heterogeneous cell cultures are capable of robust and long-term [>70 population doublings (PD)] proliferation in culture, have normal karyotypes, and can be cryopreserved, clonally isolated, and stably transfected. Cell cultures and clonal lines retain a broad pattern of gene expression including simultaneous expression of markers normally associated with cells of neural, vascular/hematopoietic, muscle, and endoderm lineages. The growth and expression characteristics of these EB-derived cells suggest that they are relatively uncommitted precursor or progenitor cells. EB-derived cells may be suited to studies of human cell differentiation and may play a role in future transplantation therapies.

A syndrome of tricuspid atresia in mice with a targeted mutation of the gene encoding Fog-2.

Tricuspid atresia (TA) is a common form of congenital heart disease, accounting for 1-3% of congenital cardiac disorders. TA is characterized by the congenital agenesis of the tricuspid valve connecting the right atrium to the right ventricle and both an atrial septal defect (ASD) and a ventricular septal defect (VSD). Some patients also have pulmonic stenosis, persistence of a left-sided superior vena cava or transposition of the great arteries. Most cases of TA are sporadic, but familial occurrences with disease in multiple siblings have been reported. Gata4 is a zinc-finger transcription factor with a role in early cardiac development. Gata4-deficient mice fail to form a ventral heart tube and die of circulatory failure at embryonic day (E) 8.5 (refs 6,7). Zfpm2 (also known as Fog-2) is a multi-zinc-finger protein that is co-expressed with Gata4 in the developing heart beginning at E8.5 (refs 8-10). Zfpm2 interacts specifically with the N-terminal zinc finger of Gata4 and represses Gata4-dependent transcription. Here we use targeted mutagenesis to explore the role of Zfpm2 in normal cardiac development. Zfpm2-deficient mice died of congestive heart failure at E13 with a syndrome of tricuspid atresia that includes an absent tricuspid valve, a large ASD, a VSD, an elongated left ventricular outflow tract, rightward displacement of the aortic valve and pulmonic stenosis. These mice also display hypoplasia of the compact zone of the left ventricle. Our findings indicate the importance of Zfpm2 in the normal looping and septation of the heart and suggest a genetic basis for the syndrome of tricuspid atresia.

Economic and clinical outcomes of microlaparoscopic and standard laparoscopic sterilization. A comparison.

OBJECTIVE: To compare micro-laparoscopic surgical sterilization and standard laparoscopic sterilization with respect to cost effectiveness and patient preferences. STUDY DESIGN: A retrospective study of all laparoscopic surgical sterilizations performed under general anesthesia at Johns Hopkins Bayview Medical Center--16 micro-laparoscopies and 34 standard laparoscopies. Cases selected for review were limited to patients undergoing surgical contraception and not requiring additional, concurrent procedures. Laparoscopic surgical sterilization was performed using a double-puncture technique with silicone band application. In each case either a standard, 10-mm laparoscope or a 2-mm micro-laparoscope was used, and the procedure was performed under general anesthesia. Postoperative pain management was achieved by nonsteroidal antiinflammatory drugs and/or narcotic analgesia. All cases were performed by residents under faculty supervision. Medical records and hospital billing records were reviewed, and a standardized telephone interview was conducted to assess postoperative quality of life and patient satisfaction. RESULTS: Both techniques were comparable in cost effectiveness. There was no significant difference in operating room time, average operating room costs, average ancillary department costs, instrument and supply costs, or length of stay. Postoperative discomfort was significantly less with microlaparoscopy (P = .05), and patient satisfaction was higher in the microlaparoscopy group. CONCLUSION: Microlaparoscopy and the standard laparoscopic approach for surgical sterilization are associated with similar hospital charges. Postoperative pain and overall patient satisfaction were significantly better with microlaparoscopy than standard laparoscopy.

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.

A functionally conserved N-terminal domain of the friend of GATA-2 (FOG-2) protein represses GATA4-dependent transcription.

GATA4 is a transcriptional activator of cardiac-restricted promoters and is required for normal cardiac morphogenesis. Friend of GATA-2 (FOG-2) is a multizinc finger protein that associates with GATA4 and represses GATA4-dependent transcription. To better understand the transcriptional repressor activity of FOG-2 we performed a functional analysis of the FOG-2 protein. The results demonstrated that 1) zinc fingers 1 and 6 of FOG-2 are each capable of interacting with evolutionarily conserved motifs within the N-terminal zinc finger of mammalian GATA proteins, 2) a nuclear localization signal (RKRRK) (amino acids 736-740) is required to program nuclear targeting of FOG-2, and 3) FOG-2 can interact with the transcriptional co-repressor, C-terminal-binding protein-2 via a conserved sequence motif in FOG-2 (PIDLS). Surprisingly, however, this interaction with C-terminal-binding protein-2 is not required for FOG-2-mediated repression of GATA4-dependent transcription. Instead, we have identified a novel N-terminal domain of FOG-2 (amino acids 1-247) that is both necessary and sufficient to repress GATA4-dependent transcription. This N-terminal repressor domain is functionally conserved in the related protein, Friend of GATA1. Taken together, these results define a set of evolutionarily conserved mechanisms by which FOG proteins repress GATA-dependent transcription and thereby form the foundation for genetic studies designed to elucidate the role of FOG-2 in cardiac development.

Partner effects on a woman's intention to conceive: 'not with this partner'.

CONTEXT: Current definitions of pregnancy intention that are useful at aggregate levels are weak at the individual level. This is especially true in social contexts where childbearing and pregnancy often occur within casual or transient relationships. METHODS: Extensive data on lifetime partnerships and sexual behaviors, including pregnancies and births, from 250 low-income women who had experienced a total of 839 pregnancies are used to explore correlates of intention to conceive, as well as the extent to which women attribute their intentions to a current partnership. RESULTS: Some 57% of reported pregnancies were unintended. Overall, 21% of the women had not wished to conceive at least one of their pregnancies with the partner who impregnated them; that proportion rose to 33% among women who had had only unintended pregnancies. Even among women who had had no unintended pregnancies, 18% had had at least one conception that they had not wanted with their partner at the time of conception. Women were less likely to say they had not wanted to conceive with a particular partner if they were living with that partner than if they were not. The likelihood of not having wanted a pregnancy with a given partner rose with the lifetime number of serious partners. Pregnancies that were not wanted with a particular partner were more than twice as likely to end in abortion as were those that were (33% vs. 14%). CONCLUSIONS: Among these women, the desire to avoid childbearing relates more to the couple involved in the conception than to abstract notions of completed family size. It would therefore be useful to include items pertaining to partner relationships in future studies of pregnancy intention.

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.

Characterization of the mUBC9-binding sites required for E2A protein degradation.

Mammalian Ubc9 (mUbc9) is required for rapid degradation of the E2A proteins E12 and E47 by the ubiquitin-proteasome system. We have shown elsewhere that mUbc9 interacts with amino acids 477-530 of E12/E47. Here we test the hypothesis that this region, rich in proline, glutamic acid, serine, and threonine (PEST) residues, serves as the E2A protein degradation domain (DD). An E2A protein lacking this region, E47Delta(478-531), was significantly more stable than wild-type E47(half-life of more than 6 h versus 55 min). Deletion of the E2A DD had no effect on the E-box-binding and transcriptional activity of E47. We mapped two discreet mUbc9-interacting regions within the E2A DD: amino acids 476-494 and 505-513. E2A(505-513) interacted with mUbc9 but not with human Ubc5, MyoD, Id3, or the polymyositis-scleroderma autoantigen. Substitution of the E2A(505-513) central hydrophobic residues with basic residues abolished interaction with mUbc9. Also, full-length E47 lacking the second mUbc9-interacting region was significantly more stable than wild-type E47. Reintroduction of the E2A DD into the long-lived, naturally occurring chimeric oncoprotein E2A-HLF (hepatic leukemic factor) destabilized it, suggesting that this domain can transfer a degradation signal to a heterologous protein. E2A-HLF-DD chimeric protein was stabilized by the proteasome inhibitor LLNL, indicating the role of the ubiquitin-proteasome system mediating degradation through the E2A degradation domain. Our experiments indicate that the E2A DD mediates E2A protein interactions with the ubiquitin-proteasome system and that the E2A DD is required for metabolism of these widely expressed proteins.