Early and transient gene expression changes in pressure overload-induced cardiac hypertrophy in mice.

Cardiac hypertrophy is an important risk factor for cardiac morbidity and mortality. To unravel the underlying pathogenic genetic pathways, we hybridized left ventricular RNA from Transverse Aortic Constriction mice at 48 h, 1 week, and 2, 3, and 8 weeks after surgery to microarrays containing a 15K fetal cDNA collection. Key processes involved an early restriction in the expression of metabolic genes, accompanied by increased expression of genes related to growth and reactivation of fetal genes. Most of these genes returned to basal expression levels during the later, compensated hypertrophic phase. Our findings suggest that compensated hypertrophy in these mice is established by rapid adaptation of the heart at the cost of gene expression associated with metabolic activity, with only temporary expression of possible maladaptive processes. Therefore, the transient early changes may reflect a beneficial response to pressure overload, as deterioration of cardiac hemodynamic function or heart failure does not occur.

LPP, an actin cytoskeleton protein related to zyxin, harbors a nuclear export signal and transcriptional activation capacity.

The LPP gene is the preferred translocation partner of the HMGIC gene in a subclass of human benign mesenchymal tumors known as lipomas. Here we have characterized the LPP gene product that shares 41% of sequence identity with the focal adhesion protein zyxin. LPP localizes in focal adhesions as well as in cell-to-cell contacts, and it binds VASP, a protein implicated in the control of actin organization. In addition, LPP accumulates in the nucleus of cells upon treatment with leptomycin B, an inhibitor of the export factor CRM1. The nuclear export of LPP depends on an N-terminally located leucine-rich sequence that shares sequence homology with well-defined nuclear export signals. Moreover, LPP displays transcriptional activation capacity, as measured by GAL4-based assays. Altogether, these results show that the LPP protein has multifunctional domains and may serve as a scaffold upon which distinct protein complexes are assembled in the cytoplasm and in the nucleus.

Regulation of HMGIC expression: an architectural transcription factor involved in growth control and development.

The HMGIC gene has been implicated in the control of cell proliferation and development. We show here that HMGIC has multiple mRNA isoforms that arise by transcription initiation from alternative tandem promoters. These transcripts are not only differentially expressed between cell lines, but they can also differ within an individual cell line, in response to particular stimuli. Whereas quiescent 3T3-L1 preadipocytes express low levels of HMGIC mRNA, stimulation by serum results in a dramatic upregulation with the characteristics of a delayed-early response gene. Characterization of involved signal transduction pathways showed that both FGF-1 and PDGF-BB are strong inducers of HMGIC expression mediated via both the PI-3 kinase and MAP kinase pathways. In order to characterize the regulatory elements, sequences upstream of the translation initiation site of HMGIC were assayed for promoter activity. The HMGIC 5' flanking sequences had constitutive promoter activity in all cell lines tested, suggesting that HMGIC is regulated by negative regulatory elements that were not present in the 5'-flanking regions analysed here.

Nucleotide sequence of the chicken HMGI-C cDNA and expression of the HMGI-C and IGF1 genes in autosomal dwarf chicken embryos.

Mutations in the genes for high mobility group protein I-C (HMGI-C) and insulin-like growth factor 1 (IGF1) are known to be responsible for dwarf phenotypes in the mouse. Because the locus for autosomal dwarfism (adw) in the chicken maps to a region which is syntenic to a region in the human and mouse in which the HMGI-C and IGF1 genes are located, HMGI-C and IGF1 are likely candidate genes for adw in the chicken. In this study their possible role in the establishment of this phenotype has been investigated. We have cloned and sequenced the complete coding region of the chicken HMGI-C cDNA. Comparison with its human counterpart revealed a nucleotide sequence conservation of 84%. Only nine amino acids are present principally in the N-terminal segment before the first DNA-binding domain. Northern blot analysis showed no difference in the expression of the HMGI-C gene between adw and wild-type chicken embryos. Also no mutations in either the HMGI-C or the IGF1 RNA nucleotide sequence were detected in adw chicken embryos.

Cell type-specific protein-DNA interactions at the cAMP response elements of the prohormone convertase 1 promoter. Evidence for additional transactivators distinct from CREB/ATF family members.

The proximal promoter region of the neuroendocrine-specific human prohormone convertase 1 (PC1) gene contains two distinct cAMP response elements (CRE-1 and CRE-2). Both elements are essential in directing the cAMP-mediated hormonal regulation of PC1 gene transcription. In this study, we have demonstrated that CRE-1 binds several trans-acting factors. In electrophoretic mobility shift assay experiments with nuclear extracts prepared from neuroendocrine AtT-20 and beta-TC3 cells and non-neuroendocrine COS-1 cells, three specific protein-DNA complexes (I-III) were detected. Complexes II and III were shown to contain CREB-1 and ATF-1, respectively. The most slowly migrating complex I was only detected with the neuroendocrine cell lines and appeared to comprise a c-Jun-containing heterodimer. In addition, CRE-2 was shown to bind a protein that was only detected in nuclear extracts derived from the neuroendocrine cell lines. Antibody supershift experiments indicated that both the c-Jun-interacting protein in CRE-1 complex I and the CRE-2-interacting protein are distinct from known members of the basic domain, leucine zipper family of transcription factors. UV cross-linking experiments demonstrated that these potential novel proteins are approximately 100 and 60 kDa in size, respectively. Site-specific mutagenesis experiments demonstrated that the formation of both CRE-1 and CRE-2 complexes is correlated with the transcriptional activity of the proximal PC1 promoter as has been shown in transient transfections with wild-type and mutant promoter constructs. In addition, it was shown that both CREB-1 and ATF-1 transactivate the human PC1 promoter in transient transfection experiments.

Regulation of human prohormone convertase 2 promoter activity by the transcription factor EGR-1.

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5'-terminal region of the human prohormone convertase 2 (PC2) gene and established characteristics of the PC2 promoter region. The proximal promoter region is very G+C-rich and does not contain a canonical TATA box or a CAAT box. Transient expression assays with a set of human PC2 gene fragments containing progressive 5' deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, we show that the transcription factor EGR-1 interacts with two distinct elements within the proximal human PC2 promoter region. Transfection experiments also demonstrate that EGR-1 is able to enhance PC2 promoter activity.

BCL-6 expression in reactive lymphoid tissue and in B-cell non-Hodgkin's lymphomas.

Chromosomal abnormalities involving 3q27 have recently been associated with diffuse large B-cell lymphomas and, less frequently, with follicular lymphomas. Molecular studies have led to the identification of the BCL-6/LAZ-3 gene, located at 3q27 and coding for a putative zinc-finger protein that might act as a transcriptional regulator during cell differentiation and development. Rearrangement of BCL-6 results in truncation of the gene in its 5' portion, leaving the protein intact; a resultant deregulation of its expression has been hypothesized. In order to test this hypothesis, the expression of BCL-6 protein was investigated in human reactive lymphoid tissue and compared with a group of non-Hodgkin's lymphomas (NHLs) with or without 3q27 anomalies and/or BCL-6 gene rearrangement. BCL-6 protein is consistently expressed in reactive lymphoid tissues, where it is restricted to the follicle centre. The protein is also widely expressed in NHL: all follicular lymphomas tested showed a pattern of expression similar to the reactive B follicle, independently of the presence of BCL-6 gene rearrangement and/or 3q27 anomalies. In the diffuse large B-cell lymphomas, there was more variation in BCL-6 expression, but a correlation with 3q27 anomalies and/or BCL-6 rearrangement was not found. Deregulation of the BCL-6 gene did not result in an aberrant tissue expression as detected by immunohistochemistry.

Genomic organization of the human NSP gene, prototype of a novel gene family encoding reticulons.

Recently, cDNA cloning and expression of three mRNA variants of the human NSP gene were described. This neuroendocrine-specific gene encodes three NSP protein isoforms with unique amino-terminal parts, but common carboxy-terminal parts. The proteins, with yet unknown function, are associated with the endoplasmic reticulum and therefore are named NSP reticulons. Potentially, these proteins are neuroendocrine markers of a novel category in human lung cancer diagnosis. Here, the genomic organization of this gene was studied by analysis of genomic clones isolated from lambda phage and YAC libraries. The NSP exons were found to be dispersed over a genomic region of about 275 kb. The present elucidation of the genomic organization of the NSP gene explains the generation of NSP mRNA variants encoding NSP protein isoforms. Multiple promoters rather than alternative splicing of internal exons seem to be involved in this diversity. Furthermore, comparison of NSP genomic and cDNA sequences with databank nucleotide sequences resulted in the discovery of other human members of this novel family of reticulons encoding genes.

Regulation of gene expression by alternative promoters.

Promoters have been defined as modulatory DNA structures containing a complex array of cis-acting regulatory elements required for accurate and efficient initiation of transcription and for controlling expression of a gene. It is becoming increasingly evident that they also constitute prime target elements through which diversity and flexibility in the complex patterns of gene expression in multicellular organisms are created. The use of multiple promoters and transcription start sites is apparently a frequently used mechanism, whereas at the same time there is considerable variation and complexity in the patterns of alternative promoter usage. This review discusses the use of alternative promoters as a versatile mechanism to create diversity and flexibility in the regulation of gene expression. Alternative promoter usage can influence gene expression in very diverse ways. The level of transcription initiation can vary between alternative promoters, the turnover or translation efficiency of mRNA isoforms with different leader exons can differ, alternative promoters can have different tissue specificity and react differently to some signals, and finally, alternative promoter usage can lead to the generation of protein isoforms differing at the amino terminus.

Production of recombinant proteins in Chinese hamster ovary cells overexpressing the subtilisin-like proprotein converting enzyme furin.

The proprotein processing enzyme furin is the mammalian prototype of a novel family of subtilisin-like serine endoproteases which possess cleavage specificity for sites involving multiple basic amino acid residues and are involved in the processing of precursor proteins of a variety of regulatory peptides and proteins. One of the limiting steps in the engineering of mammalian cells designed for the overproduction of secreted proteins is the endoproteolytic cleavage of the precursor molecule to its mature biologically active form. The extremely low level of endogenous furin is likely the reason why cells are not able to fully mature overexpressed precursor proteins to their mature form. Here, we report a CHO-derived cell line genetically engineered for the production of high levels of recombinant proteins that need such endoproteolytic maturation. First, the human furin cDNA under the control of the cytomegalovirus early promoter and enhancer was introduced and overexpressed in a DHFR-deficient CHO cell line. A permanent cell line CHO-D3-FUR was established that expressed biologically active furin. Subsequently, to demonstrate the capacity of CHO-D3-FUR cells to produce recombinant proteins in a fully matured form, two derivative cell lines were established that overexpressed the von Willebrand factor (vWF) and transforming growth factor beta 1 (TGF beta 1); CHO-D3-vWF and CHO-D3-TGF beta 1, respectively. Both derivative cell lines were able to produce relatively high levels of recombinant protein in a fully matured and biologically active form. Our results illustrate the potential of the CHO-D3-FUR cell line in the production of recombinant secretory proteins that need endoproteolytic activation at the consensus furin cleavage sequence Arg-X-Lys/Arg-Arg.

Neuroendocrine-specific expression of the human prohormone convertase 1 gene. Hormonal regulation of transcription through distinct cAMP response elements.

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5'-terminal region of the human prohormone convertase 1 (PC1) gene and identified and characterized the PC1 promoter region. We found multiple transcription start sites located within a 15-base pair region, 205 base pairs upstream of the translation start codon. The promoter region is not G+C-rich and does not contain a canonical TATA box nor a CAAT box. Transient expression assays with a set of human PC1 gene fragments containing progressive 5' deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, the proximal promoter region confers both basal and hormone-regulated promoter activity. Site-specific mutagenesis experiments demonstrate that two closely spaced cAMP response elements within the proximal promoter region direct cAMP-mediated hormonal regulation of transcription of the PC1 gene.

The Dfur2 gene of Drosophila melanogaster: genetic organization, expression during embryogenesis, and pro-protein processing activity of its translational product Dfurin2.

The gene structure and expression of the Dfur2 gene of Drosophila melanogaster, which encodes the subtilisin-like serine endoprotease Dfurin2, was studied. The Dfur2 gene is very compact in contrast to the related Dfur1 gene, which has an estimated size of over 100 kbp. The 6-kb Dfur2 mRNA is encoded by 16 exons dispersed over a genomic region of about 9 kbp. The exon/intron organization shows conservation of intron positions not only in comparison with Dfur1, but also with the related mammalian genes FUR, PC1/PC3, PC2, and PC4. This conservation supports the hypothesis that all genes belonging to the family of subtilisin-like pro-protein processing enzymes are evolutionary related by descent from a common ancestral gene. In primer extension experiments, Dfur2 transcription initiation sites were identified in the presumed Dfur2 promoter region. This region was found to contain general RNA polymerase II promoter elements like a potential TATA box, a potential CAP signal, and several potential CCAAT boxes. Also, several sequence motifs putatively corresponding to binding sites for Drosophila transcription factors like zeste, bicoid, and engrailed were found to be present. RNA in situ hybridization experiments on Drosophila embryos revealed presumably maternal Dfur2 expression until the syncytial blastoderm (stage 5 of embryogenesis), no expression during gastrulation (stage 9), transient expression in a subset of neurons in the central nervous system of stage 12-13 embryos, and, from stage 13 onwards, expression in the developing tracheal tree. In a vaccinia expression system, the endoprotease Dfurin2 not only cleaved wild-type precursor of von Willebrand factor (pro-vWF) with pro-region cleavage site R-S-K-R decreases, but also, although to a lesser extent, pro-vWF mutants in which the P2 (vWFK-2A) or P4 (vWFR-4A) basic residue with respect to the pro-region cleavage site had been mutated. This cleavage specificity resembles that of human furin. The cleavage of pro-vWF by Dfurin2 shows that the previously reported lack of cleavage of the precursor of the beta A-chain of activin-A by Dfurin2 in this vaccinia expression system is substrate determined.

Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum.

The trans-Golgi network (TGN) proprotein convertase furin is synthesized in a zymogenic form and is activated by intramolecular, autoproteolytic cleavage of the propeptide from its precursor. To obtain insight in possible functions of the furin propeptide, we have studied biosynthesis, propeptide cleavage, biological activity, and intracellular localization of human and bovine furin. Analysis of autocatalytic cleavage site mutants of furin revealed that efficient propeptide cleavage requires the presence of the complete furin cleavage consensus sequence Arg-X-Lys-Arg. In studies of a mutant in which the P1 + P4 + P5 residues of the autoproteolytic cleavage site were substituted, no substrate processing activity could be demonstrated, indicating a complete block of maturation. In immunofluorescence analysis, this mutant was found in the endoplasmic reticulum (ER), suggesting ER retention of profurin. This ER retention, however, appeared saturable. Furin proteins encoded by oxyanion hole mutant N188A and negative side chain mutant D248L, which possess autoprocessing activity but lack substrate processing activity, were found in the Golgi and the ER, respectively. Finally, analysis of a furin mutant, in which all three potential sites for N-linked glycosylation were altered, revealed autocatalytic cleavage, substrate processing, and transport to the Golgi. Our results indicate that cleavage of the propeptide occurs in the endoplasmic reticulum and is necessary but not sufficient for transport of furin out of this compartment.

Expression of the dibasic proprotein processing enzyme furin is directed by multiple promoters.

The prototype mammalian proprotein processing enzyme furin is shown to be encoded by three distinct FUR mRNA isoforms which differ only in their 5'-untranslated regions. By primer extension analysis, the transcription start sites of the three mRNA isoforms were defined. The genomic regions located immediately upstream of the three alternative transcriptional start sites were shown to possess promoter activity in transfection experiments using the luciferase encoding gene as reporter. In a liver cell line, the P1 promoter appeared to be the strongest; in a lung cell line, the P1A promoter. Human FUR promoter P1 but not P1A or P1B was transactivated by transcription factor C/EBP beta. Other members of this family of bZIP transcription factors, C/EBP alpha and C/EBP delta, were not able to transactivate the P1 promoter. Promoter P1A and P1B have characteristics of promoters of housekeeping genes. They lack TATA or CAAT boxes upstream of the transcription start site but are very GC-rich and contain several SP1 sites. Promoter P1, on the other hand, has a TATA box in the proximal promoter region. In electromobility shift assays and DNase I footprinting analysis, transcription factor SP1 was found to bind to the proximal region of the P1 promoter. Altogether, our results indicate that expression of the human FUR gene is directed by alternative promoters, housekeeping (GC-rich) as well as regulated (TATA-containing) promoters, suggesting that their differential use may be a mechanism to modulate levels of the furin enzyme.

Furin-mediated proprotein processing activity: involvement of negatively charged amino acid residues in the substrate binding region.

Furin, which is encoded by the recently discovered FUR gene, appears to be the first known mammalian member of the subtilisin family of serine proteases with cleavage selectivity for paired or multiple basic residues. A consensus cleavage sequence, Arg-X-Lys/Arg-Arg has been proposed. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Homology modelling of the catalytic domain of this protein suggested that negatively charged amino acid residues near or in the substrate binding region might contribute to the observed specificity for substrate segments with paired and multiple basic amino acid residues. To investigate this hypothesis, furin mutants were generated in which negatively charged residues, predicted to be located near or in the substrate binding pockets and involved in interactions with basic residues of the substrate, were replaced by neutral residues. Analysis of processing by these furin mutants of wild-type and cleavage mutants of pro-von Willebrand factor (pro-vWF) revealed that particular negatively charged residues are critical for specific cleavage activity.

Modulation of furin-mediated proprotein processing activity by site-directed mutagenesis.

The proprotein processing activity of mutants of the subtilisin-like enzyme furin was studied in transfected mammalian cells. Our studies indicate that the three residues of the catalytic triad of furin, Asp46, His87, and Ser261, are critical not only for substrate processing but also for maturation of furin. Furthermore, evidence is provided that maturation of furin occurs through an intramolecular autocatalytic process. Substitution of the asparagine residue (Asn188) of the oxyanion hole by an alanine residue appears to block substrate processing but not furin maturation. Analysis of carboxyl-terminal deletion mutants revealed that the segment encompassing residues Glu449 to Glu469 of the "middle" domain, which is more than 100 residues downstream of the predicted catalytic domain, contains residues that seem to be critical for processing activity but that the more carboxyl-terminal cysteine-rich region, the transmembrane region, and the cytosolic tail are dispensable. Finally, we made mutants in the substrate binding region of human furin and studied their ability to process von Willebrand factor (pro-vWF) substrates, including wild-type pro-vWF as well as pro-vWF mutants in which the P1 (vWFR-1G), P2 (vWFK-2A), or P4 (vWFR-4A) basic residue with respect to the pro region cleavage site had been mutated. It is demonstrated that particular negatively charged residues in or near the substrate binding region of furin are critical for cleavage activity and specificity of the enzyme for multiple basic residues in the substrate. Furthermore, substrate binding region mutants of furin were obtained, which cleaved either the pro-vWFK-2A or pro-vWFR-4A mutant of pro-vWF more efficiently than wild-type pro-vWF.

Proopiomelanocortin gene expression as a neural marker during the embryonic development of Xenopus laevis.

Proopomelanocortin (POMC) is the precursor protein for a number of peptide hormones and neuropeptides, and the POMC gene is transcriptionally very active in the pars intermedia of the pituitary of the amphibian Xenopus laevis (Xenopus). We analysed the expression of this gene during Xenopus embryogenesis, in order to examine whether it can function as a (novel) neural marker. We investigated the spatio-temporal distribution of POMC mRNA, using a single-stranded probe that corresponds to the 3' untranslated region of Xenopus POMC gene B mRNA. Gene transcripts were first detected at stage 25 of development via RNase protection assays. In situ hybridization analysis performed at stage 46 showed clearly that these transcripts are localised in a region representing the future pars intermedia of the pituitary. Experiments using Xenopus explants indicate that the POMC gene can be used successfully as an indirect marker in studies on neural induction: in the absence of interactions with mesoderm, ectoderm fails to express the POMC gene, whereas POMC transcripts are readily detectable in conjugates of ectoderm and mesoderm. Artificial application of two different signals, which are likely to be relevant for neural differentiation (namely retinoic acid and the activation of protein kinase C via phorbol ester), was not effective in evoking POMC gene expression in cultured ectoderm explants. However, retinoic acid treatment of conjugates of Xenopus ectoderm and mesoderm successfully prevented POMC expression. We conclude that POMC gene expression can be used as an indirect marker for anterior neural differentiation in Xenopus.

Transcriptional and posttranscriptional regulation of the proopiomelanocortin gene in the pars intermedia of the pituitary gland of Xenopus laevis.

In the melanotrope cells of the intermediate pituitary gland of the amphibian Xenopus laevis, expression of the POMC gene is under physiological control, i.e. it is 20- to 30-fold higher in animals adapted to a black background compared to animals adapted to a white background. To investigate whether changes in POMC messenger RNA stability contribute to this difference in expression, a steady state kinetic model for mRNA degradation was used to determine the half-life of POMC mRNA during the induction and deinduction of POMC gene expression in melanotrope cells. During induction of the POMC gene the half-life of POMC mRNA was 3- to 4-fold longer than during deinduction. This difference in mRNA stability is, however, not sufficient to account for the 20- to 30-fold difference in the steady state levels of POMC mRNA between the two physiological conditions. Results from experiments with the protein synthesis inhibitor cycloheximide and the mRNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzidimidazole suggest 1) that during induction of POMC gene expression no de novo protein synthesis is required and, 2) that deinduction of POMC gene expression requires transcriptional activation of an mRNA-degradation system. Pulse-labeling experiments with [3H]uridine showed that in neurointermediate lobes of white-adapted animals there is an 8-fold higher amount of newly synthesized POMC mRNA than in lobes of black animals. This suggests a fast in vitro induction of POMC gene transcription. The dopamine D2-receptor agonist apomorphine decreased POMC mRNA biosynthesis about 5-fold, which confirms that regulation of POMC gene expression includes a transcriptional component. In conclusion, our results demonstrate that during the physiological process of background adaptation the regulation of POMC gene expression in Xenopus melanotrope cells is exerted by alterations in POMC gene transcription as well as in POMC mRNA stability.

Application of recombinant DNA technology in epitope mapping and targeting. Development and characterization of a panel of monoclonal antibodies against the 7B2 neuroendocrine protein.

Three mouse monoclonal antibodies (Moabs) have been obtained with specificity for the 7B2 protein, a proposed member of the granin family of neuroendocrine proteins. Bacterially produced hybrid proteins of 7B2 were used as immunogens. The Moabs were designated MON-100, MON-101, and MON-102. Furthermore, we report the construction of 35 deletion mutants of the glutathione S-transferase-7B2 (GST-7B2) fusion-gene using recombinant DNA technology. The hybrid proteins encoded by eleven of these mutants were used in epitope mapping experiments and the results of these studies strongly suggested that recognition of 7B2 by all three Moabs involved the same 16 amino acid region of 7B2 (from amino acid residue 128-135). This was further substantiated by the observation that MON-101 and MON-102 specifically recognized a conjugate between bovine serum albumin and the synthetic peptide Phe-Glu-Pro-Glu-His-Asp-Tyr-Pro-Gly-Leu-Gly-Lys based upon the deduced amino acid sequence of the predicted epitope region in 7B2. In an approach to generate a series of 7B2-specific Moabs targeted against another epitope region in the 7B2 protein, the hybrid protein encoded by deletion mutant pPV32 was used as the immunogen. This protein lacked the epitope region recognized by the first series of Moabs. A second series of three Moabs, designated MON-142, MON-143, and MON-144, was obtained and, in all three cases, the region of 7B2 from amino acid residue 64-94 appeared to be involved in specific recognition by the Moabs. The whole panel of six anti-7B2 antibodies appeared to be useful in immunoprecipitation and Western blot analysis of the 7B2 protein and specifically stained neuroendocrine cells in immunohistochemical experiments. Using a double determinant sandwich enzyme immunoassay, 7B2 protein levels in rat pituitary were determined as 20 ng/mg tissue.