Biomedical subjects

S Enerbäck

Publications and source records attributed to S Enerbäck.

At least 37 records · Page 2Linked to original sources

PubMed1998

The winged helix transcription factor Fkh10 is required for normal development of the inner ear.

Fkhl0 is a member of the forkhead family of winged helix transcriptional regulators. Genes encoding forkhead proteins are instrumental during embryogenesis in mammals, in particular during development of the nervous system. Here we report that mice with a targeted disruption of the Fkh10 locus exhibit circling behaviour, poor swimming ability and abnormal reaching response-all common findings in mice with vestibular dysfunction. These animals also fail to elicit a Preyer reflex in response to a suprathreshold auditory stimulation, as seen in mice with profound hearing impairment. Histological examination of the inner ear reveals a gross structural malformation of the vestibulum as well as the cochlea. These structures have been replaced by a single irregular cavity in which neither proper semicircular ducts nor cochlea can be identified. We also show that at 9.5 days post coitum (dpc), Fkh10 is exclusively expressed in the otic vesicle. These findings implicate Fkh10 as an early regulator necessary for development of both cochlea and vestibulum and identify its human homologue FKHL10 as a previously unknown candidate deafness gene at 5q34.

Acoustic Stimulation↗

PubMed1998

Mutations of the forkhead/winged-helix gene, FKHL7, in patients with Axenfeld-Rieger anomaly.

Genetic linkage, genome mismatch scanning, and analysis of patients with alterations of chromosome 6 have indicated that a major locus for development of the anterior segment of the eye, IRID1, is located at 6p25. Abnormalities of this locus lead to glaucoma. FKHL7 (also called "FREAC3"), a member of the forkhead/winged-helix transcription-factor family, has also been mapped to 6p25. DNA sequencing of FKHL7 in five IRID1 families and 16 sporadic patients with anterior-segment defects revealed three mutations: a 10-bp deletion predicted to cause a frameshift and premature protein truncation prior to the FKHL7 forkhead DNA-binding domain, as well as two missense mutations of conserved amino acids within the FKHL7 forkhead domain. Mf1, the murine homologue of FKHL7, is expressed in the developing brain, skeletal system, and eye, consistent with FKHL7 having a role in ocular development. However, mutational screening and genetic-linkage analyses excluded FKHL7 from underlying the anterior-segment disorders in two IRID1 families with linkage to 6p25. Our findings demonstrate that, although mutations of FKHL7 result in anterior-segment defects and glaucoma in some patients, it is probable that at least one more locus involved in the regulation of eye development is also located at 6p25.

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of freac-9 (FKHL17), a novel kidney-expressed human forkhead gene that maps to chromosome 1p32-p34.

We describe the cloning of a near full-length cDNA of 4258 nucleotides encoding freac-9 (HGMW-approved symbol FKHL17), a novel human forkhead gene. The 5' untranslated region is unusual since it is very long, 2127 nucleotides, and contains 15 upstream AUG codons. Hybridization to a panel consisting of RNA derived from 50 different tissues showed that freac-9 is transcribed exclusively in the kidney. The kidney-derived cell lines COS-7 and 293 are shown to express freac-9. A combination of fluorescence in situ hybridization and somatic cell hybrids localizes freac-9 to the chromosomal region of 1p32-p34. The conceptual translation product predicts a protein of 372 amino acids with an N-terminal domain rich in acidic amino acids and with a high likelihood of forming an amphipatic helix, a DNA binding forkhead domain, and a C-terminal region that has a high probability of forming an amphipatic beta-sheet. The amino acid sequence of the DNA binding forkhead motif of FREAC-9 is identical to that of another forkhead protein, FREAC-4, whereas 12 substitutions are present at the nucleotide level. There are no similarities in regions outside of the DNA binding domains of FREAC-9 and FREAC-4 and since freac-4 maps to a different chromosome (5q12-q13) it is likely that an evolutionary selection has acted to maintain identical DNA binding domains between these two kidney expressed transcription factors.

Amino Acid Sequence↗

PubMed1997

Chromosome localization, sequence analysis, and expression pattern identify FKHL 18 as a novel human forkhead gene.

The forkhead gene family of transcription factors belongs to the "winged helix" class of DNA-binding proteins. Today over 40 members of this gene family have been identified. Forkhead genes have been shown to be involved in embryonic development, tumorigenesis, and direction of tissue specificity of gene expression. Here we describe a new human forkhead gene called freac-10 (HGMW-approved symbol FKHL 18). A combination of fluorescence in situ hybridization and somatic cell hybrids localizes freac-10 to the chromosomal region of 20q11.1-q11.2. Hybridization to a panel consisting of RNA derived from 50 different tissues shows that freac-10 is transcribed predominantly in the aorta, thus having a unique expression pattern compared with other forkhead genes. Sequence comparison reveals a striking similarity, over the conserved DNA binding region, to a murine forkhead gene-fkh-3. We propose, based on sequence differences in the N- and C-terminal regions of the forkhead domain and a clear difference in expression pattern between freac-10 and fkh-3, that freac-10 represents a novel member of this gene family.

Amino Acid Sequence↗

PubMed1997

The transcription factor C/EBP-beta and its role in ovarian function; evidence for direct involvement in the ovulatory process.

Gonadotropins are responsible for maturation of the ovarian follicle and the oocyte. Ovulation is the ultimate step in this process and involves disintegration of the follicular wall and subsequent release of an oocyte into the oviduct. These events are triggered by a surge of luteinizing hormone (LH). Genes expressed in the ovary, that respond to LH, are likely to be involved in the biochemical pathways that regulate ovulation. The transcription factor C/EBP-beta is induced promptly in the ovary, as a response to an ovulatory dose of gonadotropins. We used an ex vivo perfusion system to demonstrate that a specific reduction in ovarian C/EBP-beta expression inhibits ovulation. In such ovaries the oocytes appeared to be entrapped within the follicle. We have found a correlation between the expression level of the activating isoform of C/EBP-beta and the number of oocytes ovulated in response to gonadotropins. Since a reduction in C/EBP-beta expression does not affect the level of the ovulatory mediator prostaglandin endoperoxide synthase-2 (PGS-2), these findings support the view of C/EBP-beta as an important factor in the ovulatory process and highlight a C/EBP-beta-dependent and PGS-2-independent pathway that takes part in regulation of ovulation.

Animals↗

PubMed1997

Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese.

The mitochondrial uncoupling protein (UCP) in the mitochondrial inner membrane of mammalian brown adipose tissue generates heat by uncoupling oxidative phosphorylation. This process protects against cold and regulates energy balance. Manipulation of thermogenesis could be an effective strategy against obesity. Here we determine the role of UCP in the regulation of body mass by targeted inactivation of the gene encoding it. We find that UCP-deficient mice consume less oxygen after treatment with a beta3-adrenergic-receptor agonist and that they are sensitive to cold, indicating that their thermoregulation is defective. However, this deficiency caused neither hyperphagia nor obesity in mice fed on either a standard or a high-fat diet. We propose that the loss of UCP may be compensated by UCP2, a newly discovered homologue of UCP; this gene is ubiquitously expressed and is induced in the brown fat of UCP-deficient mice.

Adipose Tissue, Brown↗

PubMed1997

Changes in expression of CCAAT/enhancer binding protein alpha (C/EBP alpha) and C/EBP beta in rat liver after partial hepatectomy but not after treatment with cyproterone acetate.

BACKGROUND/AIMS: The proliferation rate of adult rat liver is normally very low. It is markedly enhanced during compensatory regeneration, e.g. after partial hepatectomy, or after administration of certain growth promoters, e.g. cyproterone acetate. These two types of liver cell proliferation appear to differ, since the expression of immediate early genes is induced during compensatory regeneration but not after cyproterone acetate treatment. The transcription factor C/EBP alpha, which has been associated with hepatocyte differentiation and growth arrest, is suppressed during compensatory regeneration. In contrast, C/EBP beta, associated with acute phase reaction, is increased during regeneration. We have investigated the effects of the liver growth promoter cyproterone acetate on the hepatic expression of C/EBP alpha and C/EBP beta. METHODS: Adult male rats received either cyproterone acetate treatment or were subjected to partial hepatectomy. Livers were obtained at different time intervals for measurement of C/EBP alpha and C/EBP beta mRNA with solution hybridization/RNAse protection assay, and C/EBP alpha and C/EBP beta content with immunoblotting. RESULTS: The levels of both C/EBP alpha and C/EBP beta mRNA and the corresponding immunoreactivities were unchanged 2-48 h after injection of cyproterone acetate. The levels of C/EBP alpha mRNA and immunoreactivity were significantly suppressed 10-18 h and 18-26 h after partial hepatectomy, respectively. The levels of C/EBP beta mRNA and immunoreactivity were enhanced during compensatory regeneration 2 h after partial hepatectomy. CONCLUSIONS: Liver cell proliferation during regeneration, but not in response to cyproterone acetate treatment, is associated with changes in C/EBP alpha and C/EBP beta expression. This further supports the notion that changes in expression of transcription factors during liver growth in vivo are dependent on the growth inducer.

Animals↗

PubMed1997

The CCAAT/enhancer-binding protein-alpha is expressed in the germinal layer of the growth plate: colocalisation with the growth hormone receptor.

The transcription factor C/EBP alpha, a member of the CCAAT/enhancer-binding protein family, is highly expressed in the liver and in adipose tissue. The aim of this study was to determine if C/EBP alpha is expressed in rat growth cartilage. The expression pattern of C/EBP alpha in monolayer-cultured growth plate chondrocytes was similar to that of C/EBP alpha during hepatocyte and preadipocyte differentiation. Immunohistochemistry with a polyclonal antibody for C/EBP alpha revealed that the C/EBP alpha protein is present in the perichondrial ring, in the germinal layer of the growth plate and on the surface of the articular cartilage. The growth hormone (GH) receptor has a similar distribution in the rat tibial growth plate, and hypophysectomised rats were used to investigate a possible connection between C/EBP alpha and GH. C/EBP alpha mRNA levels were decreased in rib cartilage after hypophysectomy. However, GH treatment did not counteract this effect, indicating that other pituitary hormones regulate the C/EBP alpha mRNA levels in growth plate cartilage. We thus demonstrate, for the first time, that C/EBP alpha is expressed in cartilage. The finding that C/EBP alpha, like the GH receptor, is predominantly expressed in stem cell areas of the rat growth plate indicates a possible functional role for C/EBP alpha during early chondrogenic differentiation.

Animals↗

PubMed1996

Expression of CCAAT enhancer binding protein-alpha (C/EBP alpha) in the rat ovary: implications for follicular development and ovulation.

The development of follicles from the antral to the preovulatory stage in the mammalian ovary involves both proliferation and differentiation of cells. These processes are coordinated by endocrine, paracrine, and autocrine factors in a time- and cell-specific pattern. Each stage of development is characterized by expression of specific genes the products of which are involved in different cellular processes, e.g., signal transduction (cAMP-dependent protein kinases), steroidogenesis (cytochrome P450 enzymes). At the nuclear level, the signaling pathway from external stimuli converges to modify the mechanisms of transcription factors. One such factor, CCAAT enhancer binding protein-alpha (C/EBPalpha), participates in differentiation processes of several organs, e.g., liver, adipose tissue, and gut. We have previously demonstrated that C/EBPalpha is expressed in rat ovarian follicles in a cell-, time-, and hormone-specific manner. This increase in granulosa cells was concomitant with the more differentiated phenotype. The aim of the present study was to explore the function of C/EBPalpha in the rat ovary. To achieve this, the expression of C/EBPalpha in follicular cells was attenuated in vivo by the local administration of antisense oligonucleotides (AS) into the ovarian bursa, i.e., the sac-like structure surrounding the ovary of immature rats. This administration resulted in an impaired response to subsequent injections of exogenous gonadotropins (PMSG, hCG) with an attenuated expression of C/EBPalpha protein and finally a decreased ovulation rate. Furthermore, the morphology of the AS-treated ovary was altered with large, oocyte-containing follicles at a time when ovaries exposed to sense (S) oligonucleotides demonstrated newly formed corpora lutea. AS also affected the expression of the proto-oncogene c-myc, which was elevated by this treatment. The administration of S was without effects. Thus, C/EBPalpha seems to be a necessary factor for follicular development in the rat ovary.

Animals↗

PubMed1996

Characterization of the human forkhead gene FREAC-4. Evidence for regulation by Wilms' tumor suppressor gene (WT-1) and p53.

We describe the cloning and sequence analysis of a nearly full-length cDNA as well as a corresponding 5.2-kilobase pair genomic fragment encoding FREAC-4, a member of the forkhead family of transcription factors. The cDNA is collinear with respect to the coding region of the intronless genomic clone. The conceptual translation product predicts a protein of 465 amino acids with a hyperacidic amino-terminal end, a DNA binding forkhead domain and a carboxyl-terminal part that is rich in homopolymeric runs of prolines and alanines. The transcription start is identified using an RNase protection assay. A 2.7-kilobase pair genomic DNA fragment, located immediately upstream of the translation start, was fused to a luciferase reporter gene. Significant levels of luciferase activity were detected when this construct was transfected into two kidney-derived cell lines, 293 and COS-7 cells, whereas only background reporter gene expression was observed in a cell line of nonkidney origin. Cotransfections with plasmids expressing WT-1, WTAR (a mutated form of WT-1), p53, and a mutated form of p53 revealed a complex pattern of regulation with a 3-fold induction with WT-1, a 7-fold induction with mutated p53, and a 4-fold repression with wild-type p53. A 5'-promoter deletion series delimits a DNA fragment necessary for WT-1 inducibility in cotransfection experiments. This fragment is shown to contain at least one cis-element that is capable of interacting with recombinant WT-1.

Amino Acid Sequence↗

PubMed1996

Differential activation of lung-specific genes by two forkhead proteins, FREAC-1 and FREAC-2.

We describe the cDNA sequences for two human transcription factors, Forkhead RElated ACtivator (FREAC)-1 and -2, that belong to the forkhead family of eukaryotic DNA binding proteins. FREAC-1 and -2 are encoded by distinct genes, are almost identical within their DNA binding domains and in the COOH termini, but are otherwise divergent. Cotransfections with a reporter carrying FREAC binding sites showed that both proteins are transcriptional activators, and deletions located the activation domains to the COOH-terminal side of the forkhead domains. Expression of FREAC-1 and FREAC-2 is restricted to lung and placenta. We show that the promoters of genes for lung-specific proteins such as pulmonary surfactant proteins A, B, and C (SPA, SPB, and SPC) and the Clara cell 10-kDa protein (CC10) contain potential binding sites for FREAC-1 and FREAC-2. DNaseI footprinting verified that FREAC proteins bind to the predicted sites in the CC10 and SPB promoters. While an SPB promoter construct could be transactivated by both FREAC-1 and FREAC-2, CC10 was only activated by FREAC-1. Efficient activation of CC10 by FREAC-1 is shown to be specific for a lung cell line with Clara cell characteristics (H441) and to involve a region of the FREAC-1 protein unable to activate in other cell types.

Amino Acid Sequence↗

PubMed1995

Chromosomal localization of six human forkhead genes, freac-1 (FKHL5), -3 (FKHL7), -4 (FKHL8), -5 (FKHL9), -6 (FKHL10), and -8 (FKHL12).

The forkhead family of transcription factors has been shown to be involved in the regulation of embryonic development in organisms from Drosophila to humans. Genes encoding forkhead proteins are also emerging as a new class of oncogenes with relevance for human cancer. We have used a combination of fluorescence in situ hybridization and somatic cell hybrids to map the chromosomal localizations of five previously cloned human forkhead genes, freac-1, -3, -4, -5, and -6. (HGMW-approved symbols FKHL5, FKHL7, FKHL8, FKHL9 and FKHL10). We also report the sequence and the chromosomal position of a novel human forkhead gene, freac-8 (FKHL12). The freac genes have been localized to the following positions: freac-1, 16q24; freac-3, 6p25; freac-4, 5q12-q13; freac-5, 9 pericen; freac-6, 5q34, and freac-8, 1p32.

Amino Acid Sequence↗

PubMed1995

Selection of high-affinity binding sites for sequence-specific, DNA binding proteins from random sequence oligonucleotides.

We describe a rapid and sensitive method to isolate sets of high-affinity binding sites for sequence-specific DNA binding proteins. The DNA binding domain of the protein is expressed in Escherichia coli as a fusion with glutathione S-transferase (GST). Binding reactions are set up with total soluble extract from induced bacteria and a double-stranded oligonucleotide for which the central 32 bp have been randomized. To ensure stringent conditions, binding is done in the presence of high levels of poly(dI:C). The GST fusion protein is recovered by the addition of glutathione-Sepharose. Following extensive washing of the Sepharose beads, the bound oligonucleotides are rescued by polymerase chain reaction amplification. The amplified material is used in the next cycle of selection and amplification. Approximately five cycles are needed to obtain a pure population of high-affinity sites, which are then cloned and sequenced. This procedure should be applicable to any sequence-specific DNA binding protein for which the cDNA is available and which can be expressed in bacteria in a functional form.

Base Sequence↗

PubMed1995

Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity.

The brown fat-specific mitochondrial uncoupling protein (UCP) provides a mechanism for generating heat by uncoupling respiration and oxidative phosphorylation. It has been suggested that this system of thermogenesis can provide a defense against obesity. To test this idea, we created a transgenic mouse in which the fat-specific aP2 gene promoter directed Ucp expression in white fat and provided for the constitutive expression of Ucp in brown fat. Transgenic mice showed both Ucp mRNA and immunoreactive UCP in white fat at 2-10% the level normally measured in brown fat. A reduction in subcutaneous fat of aP2-Ucp C57BL/6J mice was observed at 3 mo of age. When the transgene was expressed in Avy genetically obese mice reductions in total body weight and subcutaneous fat stores were observed. Female transgenic Avy mice at 13 mo of age weighed 35 grams, a weight indistinguishable from nontransgenic C57BL/6J mice. Gonadal fat showed an increase in a novel adipocyte derivative that did not accumulate lipids and that constituted approximately 80% of the mass of the tissue in Avy transgenic. A major effect of aP2-Ucp in brown fat was to reduce endogenous gene expression by as much as 95%. The results suggest that UCP synthesized from the aP2 gene promoter is thermogenically active and capable of reducing fat stores.

Adipose Tissue↗

PubMed1995

Growth hormone inhibits lipoprotein lipase activity in human adipose tissue.

The in vitro effects of GH on human adipose tissue lipoprotein lipase (LPL) activity and messenger ribonucleic acid (mRNA) levels were studied using a tissue incubation technique. After preincubation for 3 days, abdominal sc adipose tissue pieces were exposed to cortisol (1000 nmol/L) for 3 days to induce LPL activity. Addition of GH (50 micrograms/L) to the cortisol-containing medium during the last 24 h (day 6) caused a decrease by 84 +/- 4% (P < 0.01) in heparin-releasable LPL activity and by 65 +/- 4% (P < 0.01) in total LPL activity. Moreover, the heparin-releasable fraction was reduced from 42% of the total LPL activity with cortisol alone to 17% when both GH and cortisol were present in the incubation medium during the last 24 h (P < 0.01). The reduction in LPL activity in response to GH was not accompanied by a decrease in the level of LPL mRNA measured by a solution hybridization ribonuclease protection assay. In adipose tissue incubated in the control medium for 6 days, the addition of GH alone during the last 24 h caused an insignificant decrease in heparin-releasable LPL activity. Low control activities limited the scope for further decrease. It is concluded that GH counteracts the potent stimulatory effect of glucocorticoids on LPL activity without affecting LPL mRNA levels. Therefore, the inhibition of LPL activity by GH probably occurs during translation and/or posttranslational processing of the enzyme, and the mechanism may involve a decreased channeling of the lipase to the cell surface.

Adipose Tissue↗

PubMed1994

Cloning and characterization of seven human forkhead proteins: binding site specificity and DNA bending.

The forkhead domain is a monomeric DNA binding motif that defines a rapidly growing family of eukaryotic transcriptional regulators. Genetic and biochemical data suggest a central role in embryonic development for genes encoding forkhead proteins. We have used PCR and low stringency hybridization to isolate clones from human cDNA and genomic libraries that represent seven novel forkhead genes, freac-1 to freac-7. The spatial patterns of expression for the seven freac genes range from specific for a single tissue to nearly ubiquitous. The DNA binding specificities of four of the FREAC proteins were determined by selection of binding sites from random sequence oligonucleotides. The binding sites for all four FREAC proteins share a core sequence, RTAAAYA, but differ in the positions flanking the core. Domain swaps between two FREAC proteins identified two subregions within the forkhead domain as responsible for creating differences in DNA binding specificity. Applying a circular permutation assay, we show that binding of FREAC proteins to their cognate sites results in bending of the DNA at an angle of 80-90 degrees.

Amino Acid Sequence↗

PubMed1994

An upstream enhancer regulating brown-fat-specific expression of the mitochondrial uncoupling protein gene.

Previous studies on the regulation of a Ucp minigene in transgenic mice demonstrated that the sequences necessary for brown-fat-specific expression and inducibility by norepinephrine were located in the 5' flanking region between 1 and 2.8 kb from the transcriptional start site. We have investigated this region in more detail in cultured mouse brown adipocyte tumor cells. Deletion analysis of two types of chloramphenicol acetyltransferase reporter gene constructs under control of either the Ucp promoter or a heterologous herpes simplex virus-tk promoter defined an enhancer in a 220-bp HindIII-XbaI fragment which was essential for both brown fat specificity and norepinephrine inducibility. Site-directed mutagenesis of the reporter gene constructs established that independent mutations to a cyclic AMP-responsive element (CRE-2) or one of two TTCC motifs (BRE [brown fat regulatory element]), all within 17 bp, eliminated transient expression. Competitive DNA mobility shift assays with probes of the CRE and BRE motifs indicate that nuclear proteins interact with these motifs in a cooperative, synergistic manner. While these CRE-BRE probes do not show changes in binding which is dependent on norepinephrine treatment, a probe containing a third TTCC motif located 130 bp downstream of BRE-1 does show this dependency. The results indicate that a complex interaction of the CRE and BRE motifs, which cannot be functionally separated, control Ucp expression.

Adipose Tissue, Brown↗

PubMed1994

The effects of cortisol on the regulation of lipoprotein lipase activity in human adipose tissue.

The influence of cortisol, in the presence of insulin, on the regulation of lipoprotein lipase (LPL) activity was studied in human adipose tissue, using a tissue incubation technique. Tissue pieces were preincubated for 3 days in a control medium containing insulin (7175 pmol/L), then incubated for 2 additional days in the control medium with and without cortisol (1000 nmol/L). After the 5 days of incubation, the levels of LPL messenger ribonucleic acid (mRNA), relative LPL synthesis, and LPL activity (total and heparin releasable) were studied. Cortisol exposure for 2 days increased all of the variables related to LPL. The average increase was 2.5-fold for LPL mRNA, 3.0-fold for relative LPL synthesis, 5.2-fold for total LPL activity, and 9.4-fold for heparin-releasable LPL activity compared to that in controls without cortisol. The results confirm previous findings that cortisol, in the presence of insulin, has a marked stimulatory effect on LPL activity in human adipose tissue in vitro. New data have been presented on the mechanisms of cortisol regulation of LPL activity. They involve both an increased level of LPL mRNA, leading to increased relative LPL synthesis, and additional posttranslational regulation.

Adipose Tissue↗