Pemphigus Vulgaris: A Retrospective Cohort Study of Clinical Features, Treatments, and Outcomes. / Pénfigo vulgar: estudio de cohorte retrospectivo de sus características clínicas, tratamientos empleados y evolución.

BACKGROUND: Pemphigus vulgaris (PV) is an uncommon, serious disease that is treated with systemic corticosteroids and corticosteroid-sparing agents. OBJECTIVES: To describe and analyze the demographic and clinical characteristics of patients with PV. MATERIAL AND METHODS: Retrospective cohort study of adults diagnosed with PV over a period of 12years. RESULTS: PV presented with mucosal lesions in 20 of the 32 patients studied (63%); the most common site was the oral mucosa followed by the vulva. Mucosal involvement was more common in women (P=.03). Lesions were found at more than 1 mucosal site in patients whose disease began in the mucosa, independently of age or sex (P=.003). Disease onset before the age of 40years was associated with generalized skin lesions (P=.003), a need for corticosteroid-sparing therapy (P=.05), and refractory PV (P=.02). Azathioprine was the most widely prescribed corticosteroid-sparing agent (in 22 patients). Eight patients (25%) were dependent on corticosteroids and disease recurred in 26 (81%). Complete remission, with or without treatment, was achieved in 15 patients (47%). Patients remained disease-free for a median of 14months, and 2 patients died (6%). CONCLUSION: Onset before the age of 40 years could be a sign of poor prognosis in patients with PV, as it was significantly associated with a higher risk of generalized skin involvement, a need for corticosteroid-sparing therapy, and refractory disease.

Cutaneous Metastases From Breast Cancer: An 8-Year Review of Cases at a Tertiary Care Hospital. / Metástasis cutáneas de cáncer de mama: 8 años de revisión en un centro de tercera complejidad.

BACKGROUND AND OBJECTIVES: Breast cancer is the most common cause of cutaneous metastases. In our review of the literature, we found no studies that have investigated the prevalence of cutaneous metastases from breast cancer in Latin America or compared survival in relation to the site of cutaneous involvement or the presence of visceral metastases. The aims of this study were to characterize the prevalence and clinical features of cutaneous metastases from breast cancer and analyze survival in relation to site of involvement and the concomitant presence of visceral metastases. MATERIALS AND METHODS: Retrospective cohort study. We evaluated patients with breast cancer and histologically confirmed cutaneous metastases. RESULTS: Data from 914 patients with breast cancer seen between 2007 and 2014 were analyzed. Thirty-one of the patients, all women, had cutaneous metastases (prevalence, 3.4%; 95% CI, 2.3%-4.7%). The most common form of metástasis was nodular, metachronous, and asymptomatic. There were discrepancies between the immunohistochemical findings for the primary tumor and the metastases in 5 of 21 women. The metastases were locorregional in 23 patients and distant in 8. No differences were observed between patients with locorregional and distant metastases for survival after diagnosis of the primary tumor (median of 4.7 vs. 4.8 years; P=.085) or the cutaneous metastases (median of 2.9 vs. 1.1 years, P=.06). Women with a simultaneous diagnosis of cutaneous and visceral metastases had the shortest survival. CONCLUSIONS: This is the first study in Latin America to estimate the prevalence of cutaneous metastases from breast cancer and we found it to be lower than rates reported for other parts of the world.

Clinical Characteristics and Outcomes in a Population With Disseminated Herpes Zoster: A Retrospective Cohort Study. / Características clínicas y evolutivas de una población con herpes zoster diseminado: un estudio de cohorte retrospectiva.

INTRODUCTION: Shingles is the cutaneous expression of the reactivation of latent varicella zoster virus infection in sensory ganglia. It presents as vesicles in the corresponding dermatome. The condition is called disseminated herpes zoster (DHZ) when more than 2 contiguous dermatomes are affected, more than 20 vesicles are observed outside the initial dermatome, or involvement is systemic. DHZ is rare and most frequently occurs in immunocompromised patients. OBJECTIVES: To describe the epidemiology, predisposing factors, clinical presentation, laboratory findings, and clinical course of patients with DHZ, and to compare the findings in immunocompromised and immunocompetent patients. METHODOLOGY: We analyzed a retrospective case series of adults hospitalized between February 2010 and October 2015. RESULTS: Forty-one patients with virologically confirmed manifestations of DHZ were included. Stress as a trigger factor was detected in 39% and immunodepression in 58.5%. Immunocompromised patients were younger than the immunocompetent patients (mean ages, 60.5 vs 82 years, P<.01). The 8 immunocompetent patients with no detectable trigger factors were older (mean age, 85 years). In 95% of cases, DHZ was initially limited to a single dermatome and then spread to other dermatomes or became disseminated. Thrombocytopenia was detected in 56% of cases. Complication rates were similar in immunocompromised and immunocompetent patients (54% vs 59%, P>.01). Six patients died; there was no difference in mortality between the 2 groups. CONCLUSION: This study provides evidence on the relationship between DHZ, the presence of underlying immunodepression, and complications. Immunosenescence may play an important role in the onset of this disease in older immunocompetent patients.

Nedd9/Hef1/Cas-L mediates the effects of environmental pollutants on cell migration and plasticity.

Aryl hydrocarbon receptor (AhR), or dioxin receptor, is a transcription factor that induces adaptive metabolic pathways in response to environmental pollutants. Recently, other pathways were found to be altered by AhR and its ligands. Indeed, developmental defects elicited by AhR ligands suggest that additional cellular functions may be targeted by this receptor, including cell migration and plasticity. Here, we show that dioxin-mediated activation of Ahr induces Nedd9/Hef1/Cas-L, a member of the Cas protein family recently identified as a metastasis marker. The Hef1 gene induction is mediated by two xenobiotic responsive elements present in this gene promoter. Moreover, using RNA interference, we show that Nedd9/Hef1/Cas-L mediates the dioxin-elicited changes related to cell plasticity, including alterations of cellular adhesion and shape, cytoskeleton reorganization, and increased cell migration. Furthermore, we show that both E-cadherin repression and Jun N-terminal kinases activation by dioxin and AhR also depend on the expression of Nedd9/Hef1/Cas-L. Our study unveils, for the first time, a link between pollutants exposure and the induced expression of a metastasis marker and shows that cellular migration and plasticity markers are regulated by AhR and its toxic ligands.

Eritema elevatum diutinum / Erythema elevatum diutinum

El eritema elevatum diutinum (EED) es una dermatosis crónica, poco frecuente, de causa desconocida, que al comienzo presenta histopatológicamente una vasculitis leucocitoclásticade la piel y luego evoluciona hacia una fibrosis. Presentamos una paciente con lesiones dermatológicas características y generalizadas de esta rara entidad, en la que se obtuvo una buena respuesta con la sulfonoterapia.

Erythema Elevatum Diutinum (EED) is a rare chronic cutaneous condition of unknow origin, that initially presents histological as leucocytoclastic vasculitis of the skin and later resolves with fibrosis. We present a patient of this uncommon condition with typical cutaneous lesions and widespread involvement. The disease was initially misinterpreted on clinical examination. Dapsone therapy was followed by a remarkable recovery.

Acroqueratoelastoidosis: caso clínico corto / Acrokeratoelastoidosis: short clinical case

We present a 45 years old woman, with papules traslucent keratotic in lateral margins of hands, non familial, whit diagnosis histologically of acrokeratoelastoidosis, this rare condition

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces insulin-like growth factor binding protein-1 gene expression and counteracts the negative effect of insulin.

Recent epidemiological studies have revealed a possible correlation between exposure to high levels of dioxins or dioxin-like compounds and diabetes. Yet the interaction between insulin and dioxin actions remains elusive. We studied the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), a protein involved in glucose homeostasis and whose expression is down-regulated by insulin. We showed that 2,3,7,8-tetrachorodibenzo-p-dioxin (TCDD) specifically induced IGFBP-1 mRNA in human hepatocytes and HepG2 human hepatoma cells (2.5- and 8-fold, respectively). Cellular and secreted IGFBP-1 protein levels were also up-regulated. Transfection and reporter assays showed that the IGFBP-1 promoter was activated by TCDD and that this activation was dependent on the integrity of a proximal xenobiotic-responsive element (XRE). This XRE, located near the insulin-glucocorticoid regulatory region, binds the aryl-hydrocarbon receptor. In agreement with previous studies, the IGFBP-1 promoter was down-regulated by insulin (50%); we show here that although TCDD activated the IGFBP-1 promoter 5- to 6-fold, the combination of TCDD and insulin led to an expression level of IGFBP-1 that was higher than basal level (2- to 3-fold activation). Similar regulations were observed for the endogenous IGFBP-1 mRNA. These data suggest that the xenobiotic-hormonal regulatory region of the IGFBP-1 promoter mediates an up-regulation of IGFBP-1 expression by TCDD even in the presence of insulin. Because IGFBP-1 modulates blood glucose levels, the up-regulation of IGFBP-1 by dioxins might account for the disruptive effects of these pollutants on glucose metabolism.

Regulation of NAD(P)H:quinone oxidoreductase 1 gene expression by CYP1A1 activity.

The dioxin 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) induces phase I and II xenobiotic metabolizing enzymes (XME) which act sequentially to eliminate different classes of xenobiotics. The transcriptional effects of TCDD are generally mediated by the arylhydrocarbon receptor (AhR). We hypothesized that TCDD could also act indirectly, by increasing the activity of cytochrome P450 1A1 (CYP1A1), a phase I gene, which could then mediate the induction of other XME genes, such as the NAD(P)H:quinone oxidoreductase 1 (NQO1). To test this hypothesis, NQO1 gene expression was monitored after either overexpression of CYP1A1 or siRNA-mediated knock-down of CYP1A1 activity in the hepatoma cell line HepG2. Overexpression of CYP1A1 in the absence of TCDD was carried out using either adenoviral infection or the "Tet-off" system. Recombinant adenoviruses were produced encoding no protein, CYP1A1 (Ad1A1), or a mutated inactive CYP1A1 (Ad1A1mut). In the HepG2 Tet-off cell line, CYP1A1 expression was induced by the removal of doxycycline (dox) from the cell medium. Ad1A1 infection or dox removal induced CYP1A1 activity and H(2)O(2) production similarly to TCDD treatment. Moreover, in both systems, the amount of NQO1 mRNA increased to the same level as after TCDD treatment (approximately 2-fold). The UDP-glucuronosyl transferase 1A6 (UGT1A6) gene is also similarly regulated. NQO1 gene expression was not induced when mutant, inactive CYP1A1 was overexpressed or when the antioxidant N-acetyl cysteine (NAC) was added to Ad1A1. Finally, either NAC or siRNA directed against CYP1A1 mRNA decreased the induction of NQO1 gene expression by TCDD. We conclude that, after exposure to TCDD, the NQO1 gene expression can be controlled by CYP1A1 activity through an oxidative stress mediated pathway.

A natural sequence consisting of overlapping glucocorticoid-responsive elements mediates glucocorticoid, but not androgen, regulation of gene expression.

Cytosolic aspartate aminotransferase (cAspAT) is regulated by glucocorticoids in rat liver and kidney. Part of this regulation is mediated by an unusual glucocorticoid-responsive element (GRE)-like sequence called GRE A. GRE A is composed of two overlapping imperfect GREs, each comprising a conserved half-site (half-sites 1 and 4 respectively) and a poorly conserved half-site (half-sites 2 and 3 respectively). The sequence binds co-operatively two dimers of the glucocorticoid receptor (GR) and mediates efficient glucocorticoid regulation of gene expression. Analysis of deletions of the cAspAT gene promoter and subcloning of GRE A upstream of the thymidine kinase promoter indicate that this sequence is responsive to glucocorticoids, but not to androgens. Electrophoretic mobility shift assays indicate that the GRE A unit does not bind the androgen receptor (AR). The modification of three nucleotides in the poorly conserved half-sites 2 and 3, converting GRE A into two overlapping high-affinity GREs (ov-cGRE), resulted in co-operative binding of the AR. Furthermore, ov-cGRE efficiently mediated androgen regulation of the thymidine kinase promoter. A single base modification in half-site 2 or 3 in GRE A allowed the binding of the AR as one or two dimers respectively, and restored transcriptional activation by androgens only in the latter case. Thus the poor affinity of the AR for half-sites 2 and 3 prevented its binding to GRE A, indicating that the overlapping GRE A sequence of the cAspAT gene promoter discriminates a glucocorticoid-mediated from an androgen-mediated response.

Properties of overlapping EREs: synergistic activation of transcription and cooperative binding of ER.

We have designed a novel estrogen-responsive unit, overERE, which consists of two overlapping ERE separated by 5 bp (center-to-center). In gel retardation assays, this sequence forms a low-mobility complex that migrates like an estrogen receptor tetramer. The receptor-overERE complex was specific and was supershifted by anti-ER H222 antibodies. Dose response studies showed that the formation of the receptor tetramer-overERE complex was cooperative. Truncated receptors were used to assess the contribution of the receptor domains. Deletion of the E domain of the ER prevented the formation of an ER-tetramer complex, which reflects a novel function of this receptor domain. In transfection experiments, 17-beta-estradiol activated transcription from an overERE-containing promoter 4-6 times better than from an ERE-containing promoter. This synergistic effect was observed using either the natural hormone (17-beta-estradiol) or xenoestrogens (phenol red, chlordane). We conclude that two overlapping estrogen-responsive elements can elicit synergistic induction of transcription.

Contribution of a nuclear factor 1 binding site to the glucocorticoid regulation of the cytosolic aspartate aminotransferase gene promoter.

Two regions of the cAspAT gene promoter mediate the glucocorticoid regulation of this gene in the Fao hepatoma cell line. The proximal region was localized by deletion studies and stable transfections in the Fao cells to the sequence -553/-398. This region includes the glucocorticoid-responsive element (GRE) A sequence, which consists of two overlapping GREs and which can mediate the glucocorticoid regulation of a heterologous promoter. DNase I footprinting studies have shown that a site 80 base pairs upstream of the GRE A was protected by liver and brain nuclear extracts (site P8). The binding was displaced by an excess of an oligonucleotide containing a typical NF1 binding site and by NF1-specific antibodies. In electrophoretic mobility shift assay using the P8 oligonucleotide as a probe, several complexes were formed. Most complexes were common to liver and brain but were less abundant when testis extracts were used. At least one complex was specific to the liver. All complexes, with the exception of two, were competed for by the NF1 oligonucleotide. Furthermore, the sequence of the P8 site showed a 7/9-base pair homology with a typical NF1 site. A mutation of the P8 site, which prevents the binding of NF1-like proteins to it, considerably decreases the regulation of the cAspAT promoter fragment by glucocorticoids. Surprisingly, the basal activity of the mutant promoter was increased 2-fold. Thus, the regulation of the cAspAT gene promoter is mediated by a regulatory unit comprising the GRE A and a NF1 binding site.

Insulin down-regulates cytochrome P450 2B and 2E expression at the post-transcriptional level in the rat hepatoma cell line.

Cytochromes P450 (P450s) are inducible drug-metabolizing enzymes involved in the metabolism of numerous endogenous and exogenous substrates. The regulation of some of these enzymes during experimental diabetes has been reported, but the direct involvement of insulin and the mechanism of its action remain unclear. The aim of our work was to study the effects of insulin on P450 2B and 2E expression in differentiated Fao hepatoma cells. Exposure of the cells to 0.1 microM insulin caused 60% and 80% decreases in the steady state levels of P450 2B and 2E proteins, respectively, within 24 hr. Before this, a rapid decrease in the corresponding messages was observed. Indeed, 5-6 hr of insulin treatment produced 80 and 50% decreases in P450 2B and 2E mRNA levels, respectively. Nuclear run-on transcription and mRNA turnover studies were performed to determine the mechanism (transcriptional and/or post-transcriptional) by which insulin modulated these mRNA levels. From our results, it can be concluded that insulin down-regulates the expression of P450 2B by shortening the half-life of its mRNA (half-lives of 6.9 hr without insulin and 3.6 hr with insulin), whereas it down-regulates the expression of P450 2E both by weak repression of the transcription rate (-30%) and, in particular, by acceleration of its mRNA turnover (half-lives of 8.5 hr without insulin and 3.3 hr with insulin).

A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer.

An unusual glucocorticoid-responsive element (called GRE A) was found to mediate the induction of the cytosolic aspartate aminotransferase gene by glucocorticoids and was bound by the glucocorticoid receptor in a DNase I footprinting assay. GRE A consists of two overlapping GREs, each comprising a conserved half-site and an imperfect half-site. The complete unit was able to confer glucocorticoid inducibility to a heterologous promoter (delta MTV-CAT). Mutation of any of the half-sites, including the imperfect ones, abolished inducibility by the hormone, demonstrating that each of the isolated GREs was inactive. In electrophoretic mobility shift assays, purified rat liver glucocorticoid receptor (GR) formed a low-mobility complex with GRE A, presumably containing a GR tetramer. When purified bacterially expressed DBD was used, low-mobility complexes as well as dimer and monomer complexes were formed. In inactive mutated oligonucleotides, no GR tetramer formation was detected. Modification of the imperfect half-sites in order to increase their affinity for GR gave a DNA sequence that bound a GR tetramer in a highly cooperative manner. This activated unit consisting of two overlapping consensus GREs mediated glucocorticoid induction with a higher efficiency than consensus GRE.

Functional characterization of the rat gamma-glutamyl transpeptidase promoter that is expressed and regulated in the liver and hepatoma cells.

gamma-Glutamyl transpeptidase (GGT) is an enzyme encoded by multiple mRNAs (mRNAI to mRNAIV) that, in the rat, are transcribed from a single copy gene in a tissue-specific manner. In the liver, GGT expression is up-regulated in transformed cells, and this induction is the most widely used marker of liver cell transformation. We characterized the GGT mRNA species expressed in the liver (mRNAIII), and we report that this mRNA differs from the other GGT mRNA species by a 275-base alternate 5'-end sequence. Its transcription occurs on a specific promoter (promoter III) that maps on the GGT gene upstream of the two promoters coding for the GGT mRNAI and mRNAII. In hepatoma cells, mRNAIII expression is related to the differentiation state of the cells. We have shown that, in Reuber H-35-derived cell lines, the GGT mRNAIII is transcribed in cells that express a differentiated phenotype (Fao), but not in the dedifferentiated C2 and H5 variants. Moreover, we observed a reexpression of the GGT mRNAIII species in the C2 Rev7 variant, which has reverted from C2 toward a differentiated hepatocyte phenotype. In the proximal promoter III region, we identified a sequence that strongly enhances transcriptional activity in Fao and C2 Rev7 cells, but not in the dedifferentiated C2 variant. This motif interacts with nuclear proteins belonging to the NF-1 and NF-Y families that govern GGT promoter III expression in differentiated hepatoma cells.

Testis-specific transcription start site in the aspartate aminotransferase housekeeping gene promoter.

We have studied the expression and regulation of the rat testis cytosolic aspartate aminotransferase gene. The cytosolic aspartate aminotransferase activity was 5-fold lower in the testis than in the liver and kidney. A 1.9-kilobase mRNA form was detected in the rat testis in contrast to the 2.1- and 1.8-kilobase forms present in other organs. Using Northern blot and S1 mapping analyses, we found that the proximal polyadenylation site was almost exclusively used in the testis as opposed to other organs where the distal site was preferentially used. RNase protection and primer extension analysis showed that transcription was initiated at multiple sites in all organs, but the pattern of those start sites was different in the testis; in particular, a novel transcription start site was specifically detected in this organ (at position -115 from the translation start site). This site was first observed in 29-day-old rats and was maximally utilized in the adult testis. DNase I footprinting using testis nuclear extracts revealed the presence of three sites of DNA-protein interaction in the 250-base pair proximal promoter, a pattern similar to the one found using liver nuclear extracts. However, the proteins bound had different properties as shown by gel retardation experiments. We conclude that the pattern of transcription initiation and the polyadenylation site selection of a housekeeping gene can be tissue-specific.

Acinar zonation of the hormonal regulation of cytosolic aspartate aminotransferase in the liver.

The zonation of the expression and regulation of the cytosolic aspartate aminotransferase (cAspAT) mRNAs in the liver acinus was investigated in diabetic and/or adrenalectomized rats. Dexamethasone increased cAspAT activity two- to threefold alone and up to sixfold in combination with streptozotocin-induced diabetes. Northern blot analysis showed that the cAspAT mRNAs were increased by those treatments; the effect of streptozotocin was reversed by the administration of insulin. In situ hybridization experiments showed that basal cAspAT mRNAs were uniformly distributed within the liver acinus. However, cAspAT mRNAs were induced by glucocorticoids specifically in the periportal zone and by streptozotocin in a larger area including the periportal and intermediary zone. The alpha 2u-globulin mRNAs which are specifically expressed in the perivenous hepatocytes are also induced by glucocorticoids in this zone, suggesting that the specific regulation of the cAspAT gene by glucocorticoids in the periportal zone is not due to the absence of functional glucocorticoid receptors in the other zones. We conclude that the regulation of the cAspAT housekeeping gene is zone specific in the liver. Furthermore, this zonation depends on the gene and on the type of hormonal or pharmacological treatment.

Phorbol esters inhibit the glucocorticoid-mediated stimulation of cytosolic aspartate aminotransferase gene transcription.

The regulation of cytosolic aspartate aminotransferase (cAspAT) gene expression by phorbol esters was investigated in the highly differentiated hepatoma cell line Fao. Phorbol 12,13-dibutyrate (PdBu) had no effect on basal activity but partially inhibited the induction of cAspAT by dexamethasone. The extent of inhibition (40%) was similar to that obtained with insulin or vanadate. The inhibitory effects of PdBu and vanadate were additive. In the case of PdBu, the inhibitory effects could be eliminated by first incubating the cells with PdBu, which down-regulates protein kinase C. In contrast, inhibition by insulin was not modified by this treatment. The molecular mechanism of PdBu action was investigated. Northern blot analysis showed that the steady-state mRNA levels of cAspAT were decreased by PdBu in the presence of dexamethasone. In addition, the transcription rate, as measured by run-on experiments, was also decreased under the same conditions. Finally, a 2.4 kb promoter fragment driving the chloramphenicol acetyltransferase gene was stably transfected into the Fao cells. The regulation of the activity of this promoter fragment by dexamethasone and PdBu was similar to the regulation of the endogenous cAspAT activity. We conclude that PdBu acts by regulating the promoter activity of the cAsPAT gene.

Cell-specific regulation of cytosolic aspartate aminotransferase by glucocorticoids in the rat kidney.

The basal expression and hormonal regulation of cytosolic aspartate aminotransferase (cAspAT) were investigated in the rat kidney. In adrenalectomized animals, the basal activity was highest in the renal cortex and in the inner stripe of the outer medulla (0.1-0.15 U/mg protein). The glucocorticoid analogue dexamethasone increased cAspAT activity about twofold in the cortex and in the inner stripe of the outer medulla but not in the papilla. A half-maximal increase in the activity was achieved at doses of approximately 5 micrograms/100 g body wt. The mineralocorticoid aldosterone did not modify the cAspAT activity. The cell specificity of the hormonal regulation was analyzed by in situ hybridization. In untreated adrenalectomized rats, a cAspAT cRNA probe labeled mainly the inner stripe of the outer medulla. After dexamethasone or hydrocortisone treatment, labeling was uniformly increased in this part of the medulla and was heterogeneously increased in the renal cortex. The specific increase in labeling within the cortex was shown to be confined to the distal convoluted tubule and the thick ascending limb. We conclude that, in addition to widespread basal expression, cAspAT is regulated by glucocorticoids in a highly cell-specific manner in the renal cortex. The enzyme may thus participate in the increased energy metabolism elicited by these hormones in these cells.

Regulation of the cytosolic aspartate aminotransferase housekeeping gene promoter by glucocorticoids, cAMP, and insulin.

The cytosolic aspartate aminotransferase (cAspAT) is a ubiquitous enzyme that displays liver-specific hormonal regulation. In the hepatoma cell line Fao, both the activity and the mRNA level of cAspAT are increased by glucocorticoids. This effect is potentiated by cAMP and inhibited by insulin. Using in vivo run-on experiments, we showed that these effectors act at the transcriptional level. A cAspAT gene fragment containing 2405 bp of the promoter was sequenced. Deletion fragments of this promoter were inserted upstream of the CAT gene, and the regulation of their activity was assayed following transfection in Fao cells. Stable transfection experiments established that the construct including the entire 2.405-kb fragment undergoes positive regulation by glucocorticoids and cAMP and negative regulation by insulin similar to the regulation of the endogenous gene. A physical separation of the positive and negative control elements is suggested by the fact that cAMP acted on the -682/-26-bp fragment (a 2-fold increase of the stimulation by dexamethasone), whereas the negative regulation by insulin (50% of the stimulation by dexamethasone) required the -1983/-1718-bp fragment. Both regions were required for maximal glucocorticoid activity (6-9-fold increase of CAT activity). We conclude that at least two regulatory regions, a proximal and a distal one, are required for full hormonal regulation of the cAspAT gene.

CCAAT/enhancer-binding protein-related proteins bind to the unusual promoter of the aspartate aminotransferase housekeeping gene.

The cytosolic aspartate aminotransferase (cAspAT) gene is ubiquitously expressed but it is regulated by hormones in a tissue-specific manner. In vitro DNase I footprinting studies of a 260-base pair fragment carrying the basal promoter activity revealed that three CCAAT sequences bind liver nuclear proteins (protected regions P2, P3, P4). Competition studies, the heat resistance of these proteins, and identical footprints obtained using a recombinant CCAAT/enhancer-binding protein (C/EBP) alpha fragment indicate that they belong to the C/EBP family of transcription factors. A fourth protected region P1, overlapping the P2 region, was observed in the liver in the presence of competing oligonucleotides containing the C/EBP site. In cotransfection experiments, the C/EBP beta protein trans-activated 10-15-fold the cAspAT gene promoter in HepG2 cells. Deletion studies revealed that regions P2 and P4 are critical for promoter activity. In gel retardation experiments, the P4 region bound different C/EBP-related proteins in different tissues: the brain protein is heat sensitive in contrast to the liver protein. The synthetic oligonucleotide OL 1-2, which covers the P1 and P2 regions, binds C/EBP-like proteins as well as NF1 and CP1 (or NFY) transcription factors, but the preferential binding of one of these protein is tissue-specific. In summary, using the cAspAT gene promoter, we have shown that ubiquitously active promoters may be recognized by different proteins in different tissues.