This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musculoskeletal cartilages –for example articular cartilage, meniscus, temporomandibular joint disc, and intervertebral disc – are characterized by low vascularity and cellularity, and are amenable to scaffold-free tissue engineering approaches. Scaffold-free approaches, particularly the self-assembling process, mimic elements of developmental processes underlying these tissues. Discussed are various scaffold-free approaches for musculoskeletal cartilage tissue engineering, such as cell sheet engineering, aggregation, and the self-assembling process, as well as the availability and variety of cells used. Immunological considerations are of particular importance as engineered tissues are frequently of allogeneic, if not xenogeneic, origin. Factors that enhance the matrix production and mechanical properties of these engineered cartilages are also reviewed, as the fabrication of biomimetically suitable tissues is necessary to replicate function and ensure graft survival in vivo. The concept of combining scaffold-free and scaffold-based tissue engineering methods to address clinical needs is also discussed. Inasmuch as scaffold-based musculoskeletal tissue engineering approaches have been employed as a paradigm to generate engineered cartilages with appropriate functional properties, scaffold-free approaches are emerging as promising elements of a translational pathway not only for musculoskeletal cartilages but for other tissues as well.
Scaffoldless; Cell Sheet Engineering; Aggregate; Self-assembling Process; Self-assembly; Self-organization; Tissue Engineering Paradigm
The down regulation of glutamic acid decarboxylase67 (GAD1), reelin (RELN), and BDNF expression in brain of schizophrenia (SZ) and bipolar (BP) disorder patients is associated with overexpression of DNA methyltransferase1 (DNMT1) and ten-eleven translocase methylcytosine dioxygenase1 (TET1). DNMT1 and TET1 belong to families of enzymes that methylate and hydroxymethylate cytosines located proximal to and within cytosine phosphodiester guanine (CpG) islands of many gene promoters, respectively. Altered promoter methylation may be one mechanism underlying the down-regulation of GABAergic and glutamatergic gene expression. However, recent reports suggest that both DNMT1 and TET1 directly bind to unmethylated CpG rich promoters through their respective Zinc Finger (ZF-CXXC) domains. We report here, that the binding of DNMT1 to GABAergic (GAD1, RELN) and glutamatergic (BDNF-IX) promoters is increased in SZ and BP disorder patients and this increase does not necessarily correlate with enrichment in promoter methylation. The increased DNMT1 binding to these promoter regions is detected in the cortex but not in the cerebellum of SZ and BP disorder patients, suggesting a brain region and neuron specific dependent mechanism. Increased binding of DNMT1 positively correlates with increased expression of DNMT1 and with increased binding of MBD2. In contrast, the binding of TET1 to RELN, GAD1 and BDNF-IX promoters failed to change. These data are consistent with the hypothesis that the down-regulation of specific GABAergic and glutamatergic genes in SZ and BP disorder patients may be mediated, at least in part, by a brain region specific and neuronal-activity dependent DNMT1 action that is likely independent of its DNA methylation activity.
epigenetics; methylation; schizophrenia; bipolar disorder; glutamate decarboxylase; DNMT1; TET1
Based on postmortem brain studies, our overarching epigenetic hypothesis is that chronic schizophrenia (SZ) is a psychopathological condition involving dysregulation of the dynamic equilibrium among DNA-methylation/demethylation network components and the expression of SZ target genes, including GABAergic and glutamatergic genes.
SZ has a natural course, starting with a prodrome, a first episode that occurs in adolescence or in young adults, and later deterioration over the adult years. Hence, the epigenetic status at each neurodevelopmental stage of the disease cannot be studied just in postmortem brain of chronic SZ patients, but requires the use of a neurodevelopmental animal models. We have directed the focus of our research toward studying the epigenetic signature of SZ brain in the offspring of dams stressed during pregnancy (PRS mice). Adult PRS-mice have behavioral deficits reminiscent of behaviors observed in psychotic patients. The adult PRS brain, like that of postmortem chronic SZ patients, is characterized by a significant increase in DNA-methyltransferase 1 (DNMT1), Tet methylcytosine dioxygenase 1 (TET1), 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at SZ candidate gene promoters, and a reduction in the expression of glutamatergic and GABAergic genes. In PRS mice, measurements of epigenetic biomarkers for SZ can be assessed at different stages of development with the goal of further elucidating the pathophysiology of this disease and predicting treatment responses at specific stages of the illness, with particular attention to early detection and possibly early intervention.
DNA-methyltransferase; DNA-demethylase; GAD1; RELN; BDNF; Prenatally stressed mice; schizophrenia; Sadenosyl-methionine; valproic acid; clozapine
It is becoming increasingly clear that a dysfunction of the GABAergic/glutamatergic network in telencephalic brain structures may be the pathogenetic mechanism underlying psychotic symptoms in schizophrenia (SZ) and bipolar (BP) disorder patients. Data obtained in Costa’s laboratory (1996–2009) suggest that this dysfunction may be mediated primarily by a downregulation in the expression of GABAergic genes (e.g., glutamic acid decarboxylase67 [GAD67] and reelin) associated with DNA-methyltransferase (DNMT)-dependent hypermethylation of their promoters.
A pharmacological strategy to reduce the hypermethylation of GABAergic promoters is to administer drugs, such as the histone deacetylase (HDAC) inhibitor valproate (VPA), that induce DNA-demethylation when administered at doses that facilitate chromatin remodeling. The benefits elicited by combining VPA with antipsychotics in the treatment of BP disorder suggest that an investigation of the epigenetic interaction of these drugs is warranted.
Our studies in mice suggest that when associated with VPA, clinically relevant doses of clozapine elicit a synergistic potentiation of VPA-induced GABAergic promoter demethylation. Olanzapine and quetiapine (two clozapine congeners) also facilitate chromatin remodeling but at doses higher than used clinically, whereas haloperidol and risperidone are inactive. Hence, the synergistic potentiation of VPA’s action on chromatin remodeling by clozapine appears to be a unique property of the dibenzepines and is independent of their action on catecholamine or serotonin receptors.
By activating DNA-demethylation, the association of clozapine or its derivatives with VPA or other more potent and selective HDAC inhibitors may be considered a promising treatment strategy for normalizing GABAergic promoter hypermethylation and the GABAergic gene expression downregulation detected in the postmortem brain of SZ and BP disorder patients.
To describe CT findings in dogs and cats with temporomandibular joint (TMJ) disorders.
41 dogs and 17 cats.
Medical records and CT images of the skull were reviewed for dogs and cats that were examined at a dentistry and oral surgery specialty practice between 2006 and 2011.
Of 142 dogs and 42 cats evaluated, 41 dogs and 17 cats had CT findings consistent with a TMJ disorder. In dogs, the most common TMJ disorder was osteoarthritis; however, in most cases, there were other TMJ disorders present in addition to osteoarthritis. Osteoarthritis was more frequently identified at the medial aspect rather than the lateral aspect of the TMJ, whereas the frequency of osteoarthritic involvement of the dorsal and ventral compartments did not differ significantly. In cats, fractures were the most common TMJ disorder, followed by osteoarthritis. Clinical signs were observed in all dogs and cats with TMJ fractures, dysplasia, ankylosis, luxation, and tumors; however, only 4 of 15 dogs and 2 of 4 cats with osteoarthritis alone had clinical signs.
Conclusions and Clinical Relevance
Results indicated that TMJ disorders are frequently present in combination. Osteoarthritis was the most common TMJ disorder in dogs and the second most common TMJ disorder in cats. Computed tomography should be considered as a tool for the diagnosis of TMJ disorders in dogs and cats with suspected orofacial disorders and pain.
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms related to altered social interactions/communication and restricted and repetitive behaviors. In addition to genetic risk, epigenetic mechanisms (which include DNA methylation/demethylation) are thought to be important in the etiopathogenesis of ASD. We studied epigenetic mechanisms underlying the transcriptional regulation of candidate genes in cerebella of ASD patients, including the binding of MeCP2 (methyl CpG binding protein-2) to the glutamic acid decarboxylase 67 (GAD1), glutamic acid decarboxylase 65 (GAD2), and Reelin (RELN) promoters and gene bodies. Moreover, we performed methyl DNA immunoprecipitation (MeDIP) and hydroxymethyl DNA immunoprecipitation (hMeDIP) to measure total 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in the same regions of these genes. The enrichment of 5-hmC and decrease in 5-mC at the GAD1 or RELN promoters detected by 5-hmC and 5-mC antibodies was confirmed by Tet-assisted bisulfite (TAB) pyrosequencing. The results showed a marked and significant increase in MeCP2 binding to the promoter regions of GAD1 and RELN, but not to the corresponding gene body regions in cerebellar cortex of ASD patients. Moreover, we detected a significant increase in TET1 expression and an enrichment in the level of 5-hmC, but not 5-mC, at the promoters of GAD1 and RELN in ASD when compared with CON. Moreover, there was increased TET1 binding to these promoter regions. These data are consistent with the hypothesis that an increase of 5-hmC (relative to 5-mC) at specific gene domains enhances the binding of MeCP2 to 5-hmC and reduces expression of the corresponding target genes in ASD cerebella.
autism; cerebellum; epigenetics; gene expression; postmortem
Costochondral cells may be isolated with minimal donor site morbidity and are unaffected by pathologies of the diarthrodial joints. Identification of optimal exogenous stimuli will allow abundant and robust hyaline articular cartilage to be formed from this cell source.
In a three factor, two level full factorial design, the effects of hydrostatic pressure (HP), transforming growth factor β1 (TGF-β1), and chondroitinase ABC (C-ABC), and all resulting combinations, were assessed in third passage expanded, redifferentiated costochondral cells. After 4 wks, the new cartilage was assessed for matrix content, superficial zone protein (SZP), and mechanical properties.
Hyaline articular cartilage was generated, demonstrating the presence of type II collagen and SZP, and the absence of type I collagen. TGF-β1 upregulated collagen synthesis by 175% and glycosaminoglycan synthesis by 75%, resulting in a nearly 200% increase in tensile and compressive moduli. C-ABC significantly increased collagen content, and fibril density and diameter, leading to a 125% increase in tensile modulus. Hydrostatic pressure increased fibril diameter by 30% and tensile modulus by 45%. Combining TGF-β1 with C-ABC synergistically increased collagen content by 300% and tensile strength by 320%, over control. No significant differences were observed between C-ABC/TGF-β1 dual treatment and HP/C-ABC/TGF-β1.
Employing biochemical, biophysical, and mechanical stimuli generated robust hyaline articular cartilage with a tensile modulus of 2 MPa and a compressive instantaneous modulus of 650 kPa. Using expanded, redifferentiated costochondral cells in the self-assembling process allows for recapitulation of robust mechanical properties, and induced SZP expression, key characteristics of functional articular cartilage.
One of the greatest challenges of nanoparticle cancer therapy is the delivery of adequate numbers of nanoparticles to the tumor site. Iron oxide nanoparticles (IONPs) have many favorable qualities, including their nontoxic composition, the wide range of diameters in which they can be produced, the cell-specific cytotoxic heating that results from their absorption of energy from a nontoxic, external alternating magnetic field (AMF), and the wide variety of functional coatings that can be applied. Although IONPs can be delivered via an intra-tumoral injection to some tumors, the resulting tumor IONP distribution is generally inadequate; additionally, local tumor injections do not allow for the treatment of systemic or multifocal disease. Consequently, the ultimate success of nanoparticle based cancer therapy likely rests with successful systemic, tumor-targeted IONP delivery.
In this study, we used a surface-based, bilateral, noninvasive static magnetic field gradient produced by neodymium-boron-iron magnets (80 T/m to 130 T/m in central plane between magnets), a rabbit ear model, and systemically-delivered starch-coated 100 nm magnetic (iron oxide) nanoparticles to demonstrate a spatially-defined increase in the local tissue accumulation of IONPs. In this non-tumor model, the IONPs remained within the local vascular space. It is anticipated that this technique can be used to enhance IONP delivery significantly to the tumor parenchyma/cells.
iron oxide nanoparticles; magnetic capture; in vivo; rabbit ear model; hyperthermia
To study the ability of the logistic EuroSCORE to predict operative risk in contemporary cardiac surgery.
Retrospective analysis of prospectively collected data.
All National Health Service centres undertaking adult cardiac surgery in northwest England.
All patients undergoing cardiac surgery between April 2002 and March 2004.
Main outcome measures
The predictive ability of the logistic EuroSCORE was assessed by analysing how well it discriminates between patients with differing observed risk by using the area under the receiver operating characteristic (ROC) curve and studying how well it is calibrated against observed in‐hospital mortality. The performance of the EuroSCORE was examined in the following surgical subgroups: all cardiac surgery, isolated coronary artery surgery, isolated valve surgery, combined valve and coronary surgery, mitral valve surgery, aortic valve surgery and other surgery.
9995 patients underwent surgery. The discrimination of the logistic EuroSCORE was good with a ROC curve area of 0.79 for all cardiac surgery (range 0.71–0.79 in the subgroups). For all operations, the predicted mortality was 5.7% and observed mortality was 3.3%. The logistic EuroSCORE overpredicted observed mortality for all subgroups but by differing degrees (p = 0.02)
The logistic EuroSCORE is a reasonable overall predictor for contemporary cardiac surgery but overestimates observed mortality. Its accuracy at predicting risk in different surgical subgroups varies. The logistic EuroSCORE should be recalibrated before it is used to gain reassurance about outcomes. Caution should be exercised when using it to compare hospitals or surgeons with a different operative case mix.
To develop a multivariate prediction model for major adverse cardiac events (MACE) after percutaneous coronary interventions (PCIs) by using the North West Quality Improvement Programme in Cardiac Interventions (NWQIP) PCI Registry.
All NHS centres undertaking adult PCIs in north west England.
Retrospective analysis of prospectively collected data on 9914 consecutive patients undergoing adult PCI between 1 August 2001 and 31 December 2003. A multivariate logistic regression analysis was undertaken, with the forward stepwise technique, to identify independent risk factors for MACE. The area under the receiver operating characteristic (ROC) curve and the Hosmer‐Lemeshow goodness of fit statistic were calculated to assess the performance and calibration of the model, respectively. The statistical model was internally validated by using the technique of bootstrap resampling.
Main outcome measures
MACE, which were in‐hospital mortality, Q wave myocardial infarction, emergency coronary artery bypass graft surgery, and cerebrovascular accidents.
Independent variables identified with an increased risk of developing MACE were advanced age, female sex, cerebrovascular disease, cardiogenic shock, priority, and treatment of the left main stem or graft lesions during PCI. The ROC curve for the predicted probability of MACE was 0.76, indicating a good discrimination power. The prediction equation was well calibrated, predicting well at all levels of risk. Bootstrapping showed that estimates were stable.
A contemporaneous multivariate prediction model for MACE after PCI was developed. The NWQIP tool allows calculation of the risk of MACE permitting meaningful risk adjusted comparisons of performance between hospitals and operators.
major adverse cardiac events; percutaneous coronary interventions; risk prediction
Little is known about the effect of surgical training on outcomes in thoracic surgery. The impact of surgeon training on outcomes following lung resection was examined, focusing on lobectomy as a marker operation.
328 consecutive patients who underwent lobectomy at our institution between 1 October 2001 and 30 June 2003 were studied. Data were collected prospectively during the patient's admission as part of routine clinical practice and validated by a designated audit officer. Patient characteristics and postoperative outcomes were compared between trainee led and consultant led operations.
In 115 cases (35.1%) the operation was performed by a trainee thoracic surgeon as the first operator. There were no significant differences in patient characteristics between the two groups. In‐hospital mortality was similar for operations led by trainees and consultants (3.5% and 2.8%, respectively; p>0.99). Outcomes in the two groups did not differ significantly with respect to respiratory, cardiovascular, renal, neurological, chest infection, bleeding, and gastrointestinal complications. Survival rates at 1 year were 82.6% for procedures led by trainees compared with 81.7% for procedures led by consultants (p = 0.83).
With appropriate supervision, trainee thoracic surgeons can perform lobectomies safely without compromising short or intermediate term patient outcome.
training; lobectomy; mortality; morbidity; thoracic surgery
To assess the cost effectiveness of drug eluting stents (DES) compared with conventional stents for treatment of symptomatic coronary artery disease in the UK.
Cost–utility analysis of audit based patient subgroups by means of a simple economic model.
12 month audit data for 2884 patients receiving percutaneous coronary intervention with stenting at the Cardiothoracic Centre Liverpool between January 2000 and December 2002.
Main outcome measures
Risk of repeat revascularisation within 12 months of index procedure and reduction in risk from use of DES. Economic modelling was used to estimate the cost–utility ratio and threshold price premium.
Four factors were identified for patients undergoing elective surgery (n = 1951) and two for non‐elective surgery (n = 933) to predict risk of repeat revascularisation within 12 months. Most patients fell within the subgroup with lowest risk (57% of the elective surgery group with 5.6% risk and 91% of the non‐elective surgery group with 9.9% risk). Modelled cost–utility ratios were acceptable for only one group of high risk patients undergoing non‐elective surgery (only one patient in audit data). Restricting the number of DES for each patient improved results marginally: 4% of stents could then be drug eluting on economic grounds. The threshold price premium justifying 90% substitution of conventional stents was estimated to be £112 (US$212, [euro ]162) (sirolimus stents) or £89 (US$167, [euro ]130) (paclitaxel stents).
At current UK prices, DES are not cost effective compared with conventional stents except for a small minority of patients. Although the technology is clearly effective, general substitution is not justified unless the price premium falls substantially.
drug eluting stents; cost–benefit analysis; percutaneous coronary intervention
Objectives: The purpose of this study was to define magnetic resonance imaging (MRI) correlates of normal brain ageing, with the specific objective of investigating whether the size of the hippocampus is selectively correlated with age related memory performance in non-demented individuals in their ninth and tenth decades of life.
Methods: Hippocampal size was estimated using MRI based volumetry and qualitative visual assessment in 102 community dwelling individuals aged between 81 and 94 years. Participants were evaluated on a variety of clinical and experimental instruments, including a comprehensive neuropsychological test battery. All participants underwent neurological examination, an extensive medical history was obtained, and an informant confirmed details of each participant's functional ability.
Results: Both visual and volumetric hippocampal measures were identified as robust predictors of memory performance, even when the influence of age related illnesses and sociodemographic variables was accounted for. When the sample was reduced to include the most cognitively healthy participants who were rated by an informant as showing no evidence of cognitive decline, the left hippocampal measures remained significant predictors of delayed retention of verbal material.
Conclusions: These findings suggest that hippocampal volumes are selectively correlated with memory functioning in both normal and successful ageing.
OBJECTIVE: To identify current myocardial protection strategies for coronary artery bypass grafting (CABG) across the UK and Ireland. METHODS: A questionnaire survey of 15 questions was sent to practising cardiac surgeons between June and October 2002. The list of surgeons was obtained from the Society of Cardiothoracic Surgeons of Great Britain and Ireland database and they were contacted by postal and electronic mail. RESULTS: 118 (73.7%) out of 160 surgeons responded to the survey. 61 (51.7%) perform CABG on-pump (ONCAB) while 10 (8.5%) practice off-pump CABG (OPCAB). 47 (39.8%) perform either depending on individual cases. Of the 108 surgeons performing ONCAB, 91 (84.3%) use cardioplegia while 17 (15.7%) use cross-clamp and fibrillation techniques. Of those using cardioplegia, 76 (83.5%) use blood cardioplegia, 15 (19.7%) use warm-blood and 60 (78.9%) use cold-blood cardioplegia. 15(16.5%) use crystalloid cardioplegia. Retrograde cardioplegia is used by 23 (25.2%). We find an interesting variation of practice in relation to specifics like warm induction, graft cardioplegia, hot-shot, single cross-clamp, hypothermia and venting procedures. An overwhelming majority of surgeons performing OPCAB use the Octopus stabiliser (n=44, 77.2%) with some others preferring the Genzyme system. Supplementary stabilisation is not commonly used. While most OPCAB surgeons use intracoronary shunts (n=51), some prefer blockers (n=9) and others use coronary sloops (n=36). Ischaemic preconditioning is not commonly practised. Several surgeons have changed their practice of myocardial protection in the last 5 years (n=45). CONCLUSIONS: This survey gives us an interesting insight into current myocardial protection practices in the UK and Ireland and may be useful for future reference.
if preclinical syndromes for Alzheimer's disease, vascular dementia,
and Parkinson's disease and related dementias exist. Identification of
dementia at early or even preclinical stages has important implications
dwelling sample of 647 subjects aged 75 and over at recruitment were
followed up for a mean period of 3.19 years (range 2.61 to 4.51 years).
Each subject was asked to participate in a medical assessment which
included a standardised medical history examining both past and current
health and medication usage; a neuropsychological battery (mini mental
state examination, Reid memory test, verbal fluency, subsets of the
Boston naming test and similarities, clock drawing and copied drawings)
and physical examination. Preclinical syndromes for the three
predominant dementias (Alzheimer's disease, vascular dementia and
Parkinson's disease, and related dementias) and their combinations
were defined using cognitive, motor, and vascular features. Their
longitudinal outcome as defined by death and dementia incidence was examined.
syndromes affected 55.7% (n=299) of subjects. Preclinical syndromes
showed a trend for an increased odds of death (odds ratio 1.72, p=0.056) and a significantly increased odds of developing dementia
(odds ratio 4.81, p<0.001). Preclinical syndromes were highly
sensitive, detecting 52 of 58 (89.7%) incident dementias. Two hundred
and sixteen of 268 (80.6%) preclinical subjects did not show dementia
over the 3 year period (positive predictive value 19.4%). Subjects
defined as having a combination of cognitive, extrapyramidal, and
vascular features were at greatest risk of progressing to dementia.
syndromes were sensitive and significant predictors of dementia. In
view of their poor positive predictive value, the preclinical syndromes
as defined in this study remain a research tool needing both
definitional refinement and greater periods of observation. Multiple
coexistent preclinical disorders resulted in a greater incidence of
dementia, providing evidence for an additive role between multiple disorders.
Transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT) are expressed at high levels in the liver and also in at least one other cell type. We report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the alpha 1-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. We had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and alpha 1-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the result of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and alpha 1-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.
To aid general practitioners and other non-psychiatrists in the better recognition of mental illness short scales measuring anxiety and depression were derived by latent trait analysis from a standardised psychiatric research interview. Designed to be used by non-psychiatrists, they provide dimensional measures of the severity of each disorder. The full set of nine questions need to be administered only if there are positive answers to the first four. When assessed against the full set of 60 questions contained in the psychiatric assessment schedule they had a specificity of 91% and a sensitivity of 86%. The scales would be used by non-psychiatrists in clinical investigations and possibly also by medical students to familiarise them with the common forms of psychiatric illness, which are often unrecognised in general medical settings.
We have previously described the isolation and characterization of genomic clones corresponding to the mouse alpha 1-antitrypsin gene (Krauter et al., DNA 5:29-36, 1986). In this report, we have analyzed the DNA sequences upstream of the RNA start site that direct hepatoma cell-specific expression of this gene when incorporated into recombinant plasmids. The 160 nucleotides 5' to the cap site direct low-level expression in hepatoma cells, and sequences between -520 and -160 bp upstream of the RNA start site functioned as a cell-specific enhancer of expression both with the alpha 1-antitrypsin promoter and when combined with a functional beta-globin promoter. Within the enhancer region, three binding sites for proteins present in hepatoma nuclear extracts were identified. The location of each site was positioned, using both methylation protection and methylation interference experiments. Each protein-binding site correlated with a functionally important region necessary for full enhancer activity. These experiments demonstrated a complex arrangement of regulatory elements comprising the alpha 1-antitrypsin enhancer. Significant qualitative differences exist between the findings presented here and the cis-acting elements operative in regulating expression of the human alpha 1-antitrypsin gene (Ciliberto et al., Cell 41:531-540, 1985; De Simone et al., EMBO J. 6:2759-2766, 1987).
We previously defined two distinct cell-specific DNA elements controlling the transient expression of the transthyretin gene in Hep G2 (human hepatoma) cells: a proximal promoter region (-202 base pairs [bp] to the cap site), and a far-upstream cell-specific enhancer located between 1.6 and 2.15 kilobases (kb) 5' of the cap site (R. H. Costa, E. Lai, and J. E. Darnell, Jr., Mol. Cell. Biol. 6:4697-4708, 1986). In this report, we located the effective transthyretin enhancer element within a 100-bp region between 1.96 and 1.86 kb 5' to the mRNA cap site. In Hep G2 nuclear extracts, three protein-binding sites within this minimal enhancer element were identified by gel mobility and methylation protection experiments. Each binding site was required for full enhancer activity in Hep G2 transient expression assays. Competition experiments in protein-binding assays suggested that two of the three sites were recognized by a similar factor and that the protein interaction with the third site was different. The nuclear protein(s) which bound to the two homologous sites was found mainly or only in cells of hepatic origin, suggesting an involvement of this region in the cell-specific function of this enhancer. The nuclear protein(s) recognizing the third enhancer region was also found in HeLa and spleen cells.
The transthyretin (TTR) gene is regulated by two DNA regions which elicit hepatocyte-specific expression: a proximal promoter and distal enhancer. The TTR promoter and enhancer are composed of at least eight DNA binding sites for three different hepatocyte nuclear factors (HNF), CCAAT/enhancer binding protein (C/EBP), and AP-1/cJun. Site directed mutations within each of the HNF binding sites in the TTR promoter were introduced to evaluate their contribution to transcriptional activity in hepatoma cells. The data indicate that the strong affinity HNF-3-S binding site (-106 to -94) is absolutely required for TTR promoter activity since several mutations in this site eliminate TTR expression in the context of its enhancer. Conversion of a second weak affinity HNF3-W site (-140 to -131) in the TTR promoter to a high affinity site resulted in higher levels of expression. TTR mutations that disrupted several weak affinity sites (HNF1, HNF3-W, and HNF4) only slightly diminished expression levels in the presence of the TTR enhancer. In contrast, when we deleted the TTR enhancer from these HNF mutant constructs, TTR expression decreased to undetectable levels. This result suggests cooperation between the factors binding to the TTR promoter and enhancer regions. These results also demonstrate that the HNF3-S site alone is not sufficient to activate TTR transcription, but rather requires the participation of three cell-specific factors to elicit minimal promoter activity. The complexity of this promoter design and the requirement for a minimal number of cell-specific factors to achieve transcription allows us to propose a model which may explain the maintenance of tissue-specific expression of TTR.