The tissue-specific regulation of the carboxyl ester lipase gene in exocrine pancreas differs significantly between mouse and human.

The carboxyl ester lipase (CEL) gene is highly expressed in exocrine pancreas and expression of the human CEL gene is mediated by a strong tissue-specific enhancer, which is absolutely necessary for high-level expression. The mouse promoter, on the other hand, does not contain a corresponding enhancer element, but instead is totally dependent on another pancreas-specific element. This element is identified as a pancreatic transcription factor 1 (PTF 1)-binding site. The human CEL promoter also contains a putative PTF 1 element located at a position corresponding to the essential PTF 1 site in the mouse promoter. However, nucleotide changes in the human promoter 5' flanking this PTF 1 site have created an overlapping CCAAT/enhancer-binding protein (C/EBP)-like binding motif, interfering with the binding of PTF 1. Hence, our findings provide an example of genetic divergence between species not accompanied by difference in function.

Characterization of the gorilla carboxyl ester lipase locus, and the appearance of the carboxyl ester lipase pseudogene during primate evolution.

In this study we report on the isolation and characterization of the gorilla carboxyl ester lipase gene, CEL, and the corresponding CEL pseudogene. We also report on the age of the CEL pseudogene. The gorilla CEL gene is 10.5kb long and comprises 11exons intervened by introns similar to the situation in man, mouse and rat. The encoded protein is 998amino acids long and includes a 23amino acid-long leader peptide. Comparison of the coding sequence, excluding exon 11, of CEL from gorilla and man reveals a 97% similarity. Exon 11, which encodes the characteristic proline rich repeats, contains 39 repeated units in gorilla compared to 16 in man. A truncated CEL pseudogene, with the same organization as that found in man, is also shown to be present in the gorilla genome. The gorilla CEL pseudogene is 4.9kb in length and consists of 5exons interrupted by introns. Southern analysis of the gorilla CEL locus shows that the locus is arranged in a similar way as in man with the functional CEL gene being the most 5' one. To bring further insight to the events involved in the rearrangement of the CEL locus, genomic Southern analyses were performed across several primates; Homo sapiens, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus and Macaca arctoides. Results presented show that the CEL gene duplication occurred prior to the separation of Hominidae (man, chimpanzee, gorilla and orangutan) from Old World monkeys (macaque). The deletion of the original CEL gene giving rise to the truncated version of the CEL gene seems, however, to be restricted to man and the great apes only.

Studies of the regulation of the mouse carboxyl ester lipase gene in mammary gland.

The lactating mammary gland and pancreas of mouse constitute the main tissues for synthesis and secretion of a bile-salt-stimulated lipase called carboxyl ester lipase (CEL). In this paper we have analysed the endogenous CEL gene expression in mammary gland. It is shown that the gene is expressed at day 14 of pregnancy, which is synchronous with that of the whey acidic protein (WAP) gene. Even though the CEL and WAP genes are induced at the same time during mammary gland differentiation, their regulation is different with respect to dependence on lactogenic hormones. The high induction of the WAP gene expression due to the activation of signal transducer and activator of transcription (STAT)5 by prolactin has not been observed for the CEL gene, even though it has been demonstrated that both STAT5 isoforms interact with one of the gamma-interferon activation sequence sites in the promoter of the CEL gene. Hence we have demonstrated that the prolactin/STAT5 signal is not involved in a general and significant activation of 'milk genes'. Instead of a direct effect of the lactogenic hormones, the up-regulation of the CEL gene is correlated with an increase in the number of differentiated epithelial cells. Furthermore, promoter studies using the mammary-gland-derived cell line, HC11, show that a major positive element in the CEL gene promoter interacts with a member(s) of the CCAAT-binding transcription factor/nuclear factor 1 family, binding to a palindromic site. Binding of this factor(s) is important for the tissue-specific activation of the CEL gene in the mammary gland, because no activation by this factor(s) was seen in cells of pancreatic origin.

Transcriptional regulation of the human carboxyl ester lipase gene in exocrine pancreas. Evidence for a unique tissue-specific enhancer.

The human carboxyl ester lipase (CEL) is an important enzyme for the intestinal absorption of dietary lipids. The gene is highly expressed in exocrine pancreas and in the mammary gland during pregnancy and lactation. In this paper, we have focused on its transcriptional regulation in exocrine pancreas. Reporter gene analysis in cell cultures reveals that a high level of tissue-specific expression is established by the proximal 839 base pairs of the 5'-flanking region. This is due to a strong enhancer, located at -672 to -637. Transfections in mammary gland-derived cells reveal that the enhancer is pancreas-specific and does not contribute to the mammary gland expression. This indicates that the expression of the CEL gene in the mammary gland and pancreas, respectively, is due to two different regulatory systems. Further characterizations of the enhancer reveal that it is composed of two closely located cis-elements. The proximal element mediates a positive effect, whereas the distal element exerts a silencing effect on the positive proximal element. The functional enhancer complex is composed of ubiquitously expressed factors, since similar interactions are achieved with nuclear extracts from cells derived from other tissues. However, no enhancer activity is achieved in such cells. Hence, the net enhancer activity is the result of a tissue-specific balance between factors interacting with the two elements. Since none of the described cis-elements show any clear homology to known cis-elements, we propose that the interacting complex is composed of yet unidentified transcription factors.

Molecular cloning and characterization of the mouse carboxyl ester lipase gene and evidence for expression in the lactating mammary gland.

DNA hybridization was used to isolate a 2.04-kb cDNA encoding carboxyl ester lipase (CEL) from a mouse lactating mammary gland, lambda gt10 cDNA library. The cDNA sequence translated into a protein of 599 amino acids, including 20 amino acids of a putative signal peptide. Comparison of the deduced amino acid sequence of the mouse CEL with CEL from five other species revealed that there is a high degree of homology between the different species. The mouse CEL gene was also isolated and found to span approximately 7.2 kb and to include 11 exons. This organization is similar to those of the recently reported human and rat CEL genes. We have also analyzed expression of the CEL gene in the mammary glands from other species by performing a Northern blot analysis with RNA from goat and cow. The results show that the gene is expressed in both species.

The human carboxyl ester lipase-like (CELL) gene is ubiquitously expressed and contains a hypervariable region.

In this paper we characterize the human carboxyl ester lipase-like (CELL) transcript. An analysis of the tissue distribution of the expression of the gene shows that it is expressed in low amounts in all tissues analyzed. This is in contrast to its closely related and functional gene, the carboxyl ester lipase (CEL) gene, which is expressed only in human lactating mammary gland and pancreas. The primary structure of the cDNA encoding the carboxyl ester lipase-like transcript has been determined. The average length of the cDNA is 1214 bases. This sequence includes several termination codons in all three reading frames. The longest open reading frame with the same start of translation as that of the CEL transcript could encode a 59-amino-acid-long peptide, presumably without any function. The CELL gene may have arisen as a result of a gene duplication of the CEL gene followed by deletions and point mutations. However, the mutations are unevenly distributed. In the first three exons no mutations are found compared to the corresponding exons of the CEL gene. On the other hand, in the next exon several point mutations and a 2-base insertion are found and are present in all individuals analyzed. A hypervariable region present in the last exon of the CELL gene is also characterized. Several allelic variants can be resolved by polymerase chain reaction amplification of this region followed by sequencing using an automated laser fluorescent sequencer.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the human lipoprotein lipase (LPL) promoter: evidence of two cis-regulatory regions, LP-alpha and LP-beta, of importance for the differentiation-linked induction of the LPL gene during adipogenesis.

When preadipocytes differentiate into adipocytes, several differentiation-linked genes are activated. Lipoprotein lipase (LPL) is one of the first genes induced during this process. To investigate early events in adipocyte development, we have focused on the transcriptional activation of the LPL gene. For this purpose, we have cloned and fused different parts of intragenic and flanking sequences with a chloramphenicol acetyltransferase reporter gene. Transient transfection experiments and DNase I hypersensitivity assays indicate that several positive as well as negative elements contribute to transcriptional regulation of the LPL gene. When reporter gene constructs were stably introduced into preadipocytes, we were able to monitor and compare the activation patterns of different promoter deletion mutants at selected time points representing the process of adipocyte development. We could delimit two cis-regulatory elements important for gradual activation of the LPL gene during adipocyte development in vitro. These elements, LP-alpha (-702 to -666) and LP-beta (-468 to -430), contain a striking similarity to a consensus sequence known to bind the transcription factors HNF-3 and fork head. Results of gel mobility shift assays and DNase I and exonuclease III in vitro protection assays indicate that factors with DNA-binding properties similar to those of the HNF-3/fork head family of transcription factors are present in adipocytes and interact with LP-alpha and LP-beta. We also demonstrate that LP-alpha and LP-beta were both capable of conferring a differentiation-linked expression pattern to a heterolog promoter, thus mimicking the expression of the endogenous LPL gene during adipocyte differentiation. These findings indicate that interactions with LP-alpha and LP-beta could be a part of a differentiation switch governing induction of the LPL gene during adipocyte differentiation.

Genomic organization, sequence analysis, and chromosomal localization of the human carboxyl ester lipase (CEL) gene and a CEL-like (CELL) gene.

The gene encoding human carboxyl ester lipase (CEL), including 1628 bp of the 5'-flanking region, has been isolated and characterized from two overlapping lambda phage clones. The gene spans 9832 bp and contains 11 exons interrupted by 10 introns. The exons range in size from 88 to 204 bp, except for the last exon, which is 841 bp. A major and a minor transcription initiation site were determined 13 and 7 bp, respectively, upstream of the initiator methionine. The nucleotide sequence is identical with that of the previously reported cDNA, except for the third nucleotide in the 5'-untranslated sequence, a C, which in the cDNA is a T. A TAAATA sequence is present 26 nt upstream from the major CAP site, and within the 5'-flanking region there are several putative transcription factor binding sites. Seven Alu repetitive sequence elements are present in the region analyzed. The organization of the human CEL gene is similar to that of the recently reported rat pancreatic cholesterol esterase gene. The CEL gene was assigned to chromosome 9q34-qter, which confirms the recently reported results of Tayler et al. (1991, Genomics 10: 425-431). A previously unknown gene with a striking homology to the human CEL gene, here called the CEL-like gene (CELL), has also been isolated and characterized, including 1724 bp of the 5'-flanking region. The CELL gene, which most likely is a psuedogene, spans 4846 bp, and due to the absence of a 4.8-kb segment, the CEL gene exons 2-7 are not present in the CELL gene. Despite these differences, the CELL gene is transcribed. We have also assigned the CELL gene to a separate locus at chromosome 9q34-qter.

The CCAAT/enhancer binding protein and its role in adipocyte differentiation: evidence for direct involvement in terminal adipocyte development.

During the course of differentiation of preadipocytes into adipocytes, several differentiation-linked genes are activated synchronously with morphological changes. To follow this process we have used 3T3-F442A cells, known to undergo adipocyte conversion with high frequency. Accumulation of lipid droplets in the cytoplasm constitutes an easily visualized sign of the terminally differentiated phenotype. In this report we demonstrate that expression of the CCAAT/enhancer binding protein (C/EBP) is an important factor in determining the ability to accumulate lipid droplets in terminally differentiated adipocytes. In one experiment we can suppress C/EBP expression through administration of hydrocortisone to differentiating 3T3-F442A cells, which is accompanied by an inability of the cells to accumulate lipid. In another experiment a C/EBP antisense expression vector has been stably introduced into 3T3-F442A cells and as compared with control cells, a 62% decrease of C/EBP mRNA (p less than 0.01) is demonstrated. This decrease of C/EBP mRNA is accompanied by a change in cellular morphology characterized by a reduced ability to form lipid droplets. We can also demonstrate a correlation between the degree of reduction of C/EBP mRNA and the amount of lipid present in the cells. These findings strongly support the view that C/EBP is a necessary component of terminal adipocyte differentiation.

Two nuclear proteins bind to the major positive element of the apolipoprotein B gene promoter.

The promoter of the apolipoprotein B-encoding gene (apoB) contains a number of regulatory elements, which together produce a high level of expression that is restricted to two tissues: liver and intestine. In this paper we have used the gel retardation and methylation interference assays to identify two nuclear proteins, LIT1 and LIT2, which bind to the major positive element (MPE) of the apoB promoter. LIT1 is large protein, estimated to be approx. 200 kDa by gel filtration, which binds to the apoB promoter between positions of -79 and -65 bp in relation to the transcription start point. Its binding site is identical to the region responsible for cell-specific transcriptional activation. However, whereas the MPE has no influence on expression from a heterologous promoter in the non-apoB-expressing HeLa cells, these cells still contain a DNA-binding activity indistinguishable from LIT1. LIT2 binds to the apoB promoter immediately downstream from the LIT1 site. It is present in nuclear extracts from the apoB-expressing cell lines of hepatic (HepG2) and intestinal (CaCo-2) origin, but absent from HeLa cells. CCAAT/enhancer binding protein (C/EBP), expressed in bacteria, binds to the LIT2 site and produces a methylation interference pattern indistinguishable from that of LIT2. That C/EBP binds to and activates the apoB promoter in vivo, is shown by the increased chloramphenicol acetyltransferase activity observed when HepG2 cells, transfected with apoB-promoter-cat constructs, are cotransfected with a plasmid expressing c/ebp; an effect that depends on the presence in the apoB promoter of the LIT2 site.(ABSTRACT TRUNCATED AT 250 WORDS)

cDNA cloning of human-milk bile-salt-stimulated lipase and evidence for its identity to pancreatic carboxylic ester hydrolase.

We have isolated and sequenced cDNA clones covering the entire coding sequence of human-milk bile-salt-stimulated lipase, as well as 996 nucleotides of the 3' end of the pancreatic enzyme carboxylic ester hydrolase. The deduced amino acid sequence of the lipase starts with a 23-residue leader peptide. The open reading frame continues with 722 amino acid residues. The sequence contains in the C-terminal part a proline-rich repeat, 16 repeats of 11 amino acid residues each. The mRNA was estimated to be approximately 2500 nucleotides from Northern blot and of similar size in mammary and pancreatic tissues. Data obtained indicate that the lipase and the carboxylesterase are identical and coded for by the same gene. The cDNA is 2428 bases long, which indicates that a near full-length copy of the transcript has been isolated. Comparisons with other enzymes show that the lipase is a new member of the supergene family of serine hydrolases. It is not only closely related (and in its N-terminal half virtually identical) to lysophospholipase from rat pancreas and cholesterol esterase from bovine pancreas, but also shows a high degree of similarity to several esterases, e.g. acetylcholine esterase. In contrast, no such similarity could be found to typical lipases.

Hyperlipoproteinemia type I in a patient with active lipoprotein lipase in adipose tissue and indications of defective transport of the enzyme.

This paper presents a case of typical hyperlipoproteinemia type I in a young woman. Her serum triglycerides varied between 2 and 90 mmol/l and she had substantial amounts of apolipoprotein B-48 in fasting plasma. She had no detectable lipoprotein lipase (LPL) activity in post-heparin plasma (less than 0.2 percent of normal). Southern blot analysis suggested no major defect in her LPL gene and Northern blot analysis of adipose tissue RNA showed normal-sized LPL-mRNA. A 2-h [35S]methionine incorporation experiment with adipose tissue pieces in vitro showed that she produced normal-sized LPL and had LPL catalytic activity in the tissue. The amounts were, however, only 5-10% of control. No detectable LPL radioactivity or catalytic activity was released from patient tissue even in the presence of heparin in the incubations. Immunofluorescent staining of adipose tissue biopsies from the patient showed LPL immunoreactivity only in adipocytes and little or none within the capillaries. Treatment of immunoprecipitated labeled LPL with endoglycosidase H showed that the oligosaccharide chains on her enzyme were of the high-mannose type and not processed as in controls. Taken together the data suggest that the patient synthesizes a relatively normal LPL protein which is core-glycosylated and folded into active enzyme as in normal subjects, but is not effectively transported via the Golgi to the cell surface.

Expression and regulation of pOb24 and lipoprotein lipase genes during adipose conversion.

Lipoprotein lipase (LPL) and pOb24 mRNAs are known to be early markers of adipose cell differentiation. Comparative studies of the expression of pOb24 and LPL genes during adipose conversion of Ob1771 preadipocyte cells and in mouse adipose tissue have shown the following: 1) the expression of both genes takes place at confluence; this event can also be triggered by growth arrest of exponentially growing cells at the G1/S stage of the cell cycle; 2) In contrast to glycerol-3-phosphate dehydrogenase mRNA, the emergence of pOb24 and lipoprotein lipase mRNAs requires neither growth hormone or tri-iodothyronine as obligatory hormones nor insulin as a modulating hormone; 3) in mouse adipose tissue, pOb24 mRNA is present at a high level in stromal-vascular cells and at a low level in mature adipocytes, and in contrast LPL mRNAs are preferentially expressed in mature adipocytes. Thus, these two genes do not appear to be regulated in a similar manner, as also shown by the differential inhibition of their expression by tumor necrosis factor (TNF) and transforming growth factor-beta (TGF-beta).

X-linked myotubular myopathy: a linkage study.

Two families with the congenital X-linked infantile form of myotubular myopathy have been investigated by linkage analysis using markers from the X-chromosome. Linkage was found at the locus Xq28 (with DXS52). The analysis gave a peak lod score of 2.41 at the recombination fraction zero. Free recombinations (theta = 0.50) were seen using the markers DXS84, DXS14 and DXS146 from the p arm of the X-chromosome. Since the disorder is very rare, it is important to add cumulative linkage data from the few families that do exist.

Genomic organization of the region encoding guinea pig lipoprotein lipase; evidence for exon fusion and unconventional splicing.

The coding sequence of guinea pig lipoprotein lipase (LPL) is organized into nine exons and spans a region of approximately 14 kb of the guinea pig genome. A non-conforming 5'-splice site is located on the first intron, which exhibits a 12-nucleotide perfect match with the 5'-end of the second exon. A previously described tryptic cleavage site is located on exon V, close to the 3' end of this exon. A similarity to vitellogenin resides on exons IV and V, and a putative active site is found on exon IV. A novel similarity to a fatty-acid-binding protein is noted on exon VI, adjacent to the postulated heparin-binding region. We suggest that free fatty acids (FFA) and heparin to some extent share the same site of interaction on the LPL molecule; and that a high local concentration of FFA can displace LPL from its site of action--the vascular endothelium--by competing for binding to heparan sulfate.

XbaI restriction fragment length polymorphism of apolipoprotein B in Swedish myocardial infarction patients.

Apolipoprotein B (apoB) is a major importance to the metabolism of lipoproteins, and there is also evidence which suggests that apoB plays a central role in atherogenesis. In order to study whether there is a link between one of the mutations of the apoB gene and premature coronary heart disease, the frequency of the XbaI RFLP for the apoB gene was analysed in 52 male myocardial infarction patients. These were compared with a control group matched for age and sex (n = 52), and a random population sample of middle-aged men (n = 106). Two alleles were identified by the presence (X2) or the absence (X1) of an XbaI cleavage site. A somewhat higher frequency of the X2 allele was seen among the patients, however there was no significant difference between patients and controls regarding the genotypes or allele frequencies. This observation does not confirm one earlier report where a higher frequency of the X1 allele was seen in myocardial infarction patients. Differences between the studied populations or epidemiological designs of the studies might explain the diverging results. Further studies are evidently needed to fully resolve the relation between the XbaI RFLP and risk of atherosclerotic disease or lipoprotein metabolism.

Negative and positive promoter elements contribute to tissue specificity of apolipoprotein B expression.

Apolipoprotein B (ApoB) is a major constituent of the plasma lipoproteins. In adult mammals it is synthesized in two different tissues, liver and intestine. We have examined the promoter elements involved in determining the cell specificity of apoB expression, using a chloramphenicol acetyltransferase assay and the cell lines HepG2, CaCo-2 and HeLa. The human apoB promoter contains: (i) a strong, cell-specific, positive element which can act on a heterologous promoter. This element is located between pos -111 and -33 and is built up by three subdomains, two positive and one negative; (ii) a large, negative element between pos -639 and -129, which reduces promoter activity in apoB expressing cells (HepG2 and CaCo-2) and block activation of the promoter by the SV40 enhancer in non-expressing cells (HeLa); (iii) a positive element in the noncoding part of exon 1 which retains its activity if placed upstream from the other regulatory elements and stimulates transcription from the simian virus 40 promoter in all three cell lines. The same sequence elements appear to be important for expression in cells of hepatic (HepG2) and intestinal (CaCo-2) origin.

Lack of correlation between the apolipoprotein B XbaI polymorphism and blood lipid levels in a Swedish population.

The possible connections between the apolipoprotein B (apo B) XbaI polymorphism and the serum levels of total cholesterol, triglycerides, LDL, HDL and apo B have been investigated among 187 randomly selected subjects from Gothenburg, Sweden. The interferences of age and sex on the serum lipoproteins and apo B concentrations were considered. Using multiple regression analysis, we compared the different lipid levels and the levels of apo B with the genotypes X1X1, X1X2 and X2X2 (X1 = without the XbaI restriction site, X2 = with the site), with age and with sex and with those factors combined with each other. A significantly higher concentration of serum cholesterol and LDL among men than among women was found and total serum cholesterol, LDL and apo B were positively correlated with age. The allele frequency of the XbaI polymorphism in the sample was 0.45 for the allele without the XbaI restriction site. No correlation was found between the apo B genotypes and the levels of serum lipoproteins or apo B.