Neutrophil-derived azurocidin cleaves insulin-like growth factor-binding protein-1, -2 and -4.

OBJECTIVE: Azurocidin is an important inflammatory mediator and considered to be an inactive serine protease homologue. It has previously been reported that azurocidin is a possible IGFBP-1 specific protease; however, the protease-activity of azurocidin was not isolated in its active form. The aim of this study was to determine the effect of neutrophil-derived azurocidin on the six different IGFBPs, focusing especially on IGFBP-1. METHODS: IGFBPs were incubated with azurocidin in phosphate-buffered saline for 2 h and proteolysis was studied by SDS-PAGE. Analysis of azurocidin was performed by MALDI-TOF peptide mass fingerprint and MALDI-TOF/TOF peptide sequencing. RESULTS: The neutrophil-derived preparation of azurocidin cleaved IGFBP-1, IGFBP-2 and IGFBP-4. IGFBP-1 bound to IGF-I was also degraded whereas IGF-II was shown to have an inhibitory effect on proteolysis of IGFBP-1. The proteolytically active preparation of neutrophil-derived azurocidin was found to be glycosylated and determined to be 31 kDa by SDS-PAGE. CONCLUSIONS: Our results indicate that the neutrophil-derived preparation of azurocidin contains a protease activity which cleaves IGFBP-1, IGFBP-2 and IGFBP-4. These findings are of interest since both IGFBP-1 and azurocidin increase during inflammation. The effect of azurocidin on IGFBP- and IGF-activity needs to be further investigated.

Allele-specific regulation of MTTP expression influences the risk of ischemic heart disease.

Promoter polymorphisms in microsomal triglyceride transfer protein (MTTP) have been associated with decreased plasma lipids but an increased risk for ischemic heart disease (IHD), indicating that MTTP influences the susceptibility for IHD independent of plasma lipids. The objective of this study was to characterize the functional promoter polymorphism in MTTP predisposing to IHD and its underlying mechanism. Use of pyrosequencing technology revealed that presence of the minor alleles of the promoter polymorphisms -493G>T and -164T>C result in lower transcription of MTTP in vivo in the heart, liver, and macrophages. In vitro experiments indicated that the minor -164C allele mediates the lower gene expression and that C/EBP binds to the polymorphic region in an allele-specific manner. Furthermore, homozygous carriers of the -164C were found to have increased risk for IHD as shown in a case-control study including a total of 544 IHD patients and 544 healthy control subjects. We concluded that carriers of the minor -164C allele have lower expression of MTTP in the heart, mediated at least partly by the transcription factor CCAAT/enhancer binding protein, and that reduced concentration of MTTP in the myocardium may contribute to IHD upon ischemic damage.

IGFBP-1 protease activity and IGFBP-1 fragments in a patient with multiple myeloma.

OBJECTIVE: Cleavage of IGFBPs by proteases results in IGFBP fragments that have altered IGF-binding affinity, and IGF-independent roles. We have previously purified a specific IGFBP-1 protease activity from the urine of an individual with multiple myeloma and dermatitis. The aim of this study was to determine whether IGFBP-1 protease activity and/or IGFBP-1 fragments were present in the circulation of this patient. METHODS: The size of immunoreactive IGFBP-1 in serum samples was determined after Superose 12 chromatography. Intact IGFBP-1 and IGFBP-1 fragments were characterized in four RIAs and after SDS-PAGE. RESULTS: Specific proteolysis of IGFBP-1 generated an N-terminal fragment (IGFBP-1(1-130)) with a predicted molecular mass of 13kDa but an apparent mass of 21kDa on SDS-PAGE. A C-terminal fragment (IGFBP-1(131-234)) produced in vitro migrated at 11.4kDa, close to its predicted size. However a C-terminal fragment of cleaved IGFBP-1 (IGFBP-1(142-234)) migrated at 14kDa on SDS-PAGE. Serum from the patient inhibited IGFBP-1 protease activity. Immunoreactive IGFBP-1 in patient serum was present at molecular masses consistent with IGFBP-1 fragments, in addition to intact IGFBP-1. CONCLUSIONS: Specific cleavage of IGFBP-1 occurs at the tissue level and not in the circulation in a patient with multiple myeloma and dermatitis. The fragments that are generated may have endocrine roles.

Species-specific and age-dependent bile acid composition: aspects on CYP8B and CYP4A subfamilies in bile acid biosynthesis.

The present review aims to give an overview of the cytochrome P450 8B (CYP8B) and cytochrome P450 4A (CYP4A) subfamilies in relation to biosynthesis of bile acids, in particular trihydroxy bile acids. Trihydroxy bile acids are basically required in most species and have an impact on cholesterol and lipid metabolism. The primary trihydroxy bile acid in most mammals is cholic acid. Some species produce other important trihydroxy bile acids, for example the adult pig which produce hyocholic acid instead of cholic acid. The position of the third hydroxyl group in cholic acid and hyocholic acid, 12alpha or 6alpha position, respectively, has a profound effect on the hydrophilic-hydrophobic property of the trihydroxy bile acids. The CYP8B subfamily is required for introduction of the 12alpha-hydroxyl group in cholic acid biosynthesis. The enzyme responsible for 6alpha-hydroxylation in hyocholic acid biosynthesis, however, varies among species. This review will discuss, in particular, porcine members of the CYP8B and CYP4A subfamilies because interesting findings regarding members of these subfamilies have recently been recognized in this species. CYP8B1 was for a long time believed to be absent in the pig but was recently found to be expressed in fetal pig liver. The enzyme catalyzing the 6alpha-hydroxylation in hyocholic acid biosynthesis in pig was found to be an atypical member of the CYP4A subfamily, denoted CYP4A21. The review presents bile acid biosynthesis in view of these findings and discusses physiochemical properties and developmental-dependent aspects related cholic acid and hyocholic acid biosynthesis.

PPARdelta increases expression of the human apolipoprotein A-II gene in human liver cells.

The peroxisome proliferator-activated receptor delta (PPARdelta) is a transcription factor that regulates genes of importance in lipid and glucose metabolism. ApoA-II is one of the major proteins of the HDL-particle. The aim of this study was to investigate the regulation of apoA-II gene expression by PPARdelta. Treatment of HepG2 cells with the PPARdelta specific agonist GW501516 increased apoA-II mRNA expression. Likewise, reporter gene assays using a construct containing 2.7 kb of the proximal apoA-II promoter showed increased activity after treatment with GW501516, both in HepG2 and in HuH-7 cells. Mutation of two putative PPAR response elements (PPREs) in this region showed that the PPRE at position -737/-717 is the functional site. Binding of PPARdelta to this site was confirmed by chromatin immunoprecipitation and gel retardation analyses. In conclusion, PPARdelta increases the expression of the human apoA-II gene in liver cells via a PPRE in the proximal promoter.

Alternative splicing of human peroxisome proliferator-activated receptor delta (PPAR delta): effects on translation efficiency and trans-activation ability.

BACKGROUND: Peroxisome proliferator-activated receptor delta (PPAR delta) is a member of the nuclear receptor superfamily. Numerous studies have aimed at unravelling the physiological role of PPAR delta as a transcriptional regulator whereas the regulation of PPAR delta gene expression has been less studied. RESULTS: The principal transcription start site in the human PPAR delta gene identified here is positioned upstream of exon 1, although four alternative 5'-ends related to downstream exons were identified. The demonstration of multiple 5'-UTR splice variants of PPAR delta mRNA, with an impact on translation efficiency, suggests a translational regulation of human PPAR delta expression. Five untranslated exons identified in this study contribute to the variability among the 5'-UTRs of human PPAR delta mRNAs. Moreover, in vitro studies of a 3'-splice transcript encoding a truncated variant of PPAR delta (designated PPAR delta 2) show that this isoform constitutes a potential dominant negative form of the receptor. CONCLUSION: We propose that alternative splicing of human PPAR delta constitutes an intrinsic role for the regulation of PPAR delta expression and thus activity, and highlight the significance of alternative splicing of this nuclear receptor in physiology and disease.

The porcine taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21) gene: evolution by gene duplication and gene conversion.

Porcine taurochenodeoxycholic acid 6alpha-hydroxylase, cytochrome P450 4A21 (CYP4A21), differs from other members of the CYP4A subfamily in terms of structural features and catalytic activity. CYP4A21 participates in the formation of hyocholic acid, a species-specific primary bile acid in the pig. The CYP4A21 gene was investigated and found to be approx. 13 kb in size and split into 12 exons. The intron-exon organization of the CYP4A21 gene corresponds to that of CYP4A fatty acid hydroxylase genes in other species. Comparison with a genomic segment of a pig CYP4A fatty acid hydroxylase gene ( CYP4A24 ) revealed a sequence identity with CYP4A21 that extends beyond the exons, indicating a common origin by gene duplication. A pronounced sequence identity was found also within the proximal 5'-flanking regions, whereas the patterns of mRNA expression of CYP4A21 and CYP4A fatty acid hydroxylases in pig liver differ. Sequence comparison aiming to elucidate the origin of the unique features of CYP4A21 revealed a region of decreased sequence identity from exon 6 to exon 8, strongly suggesting that gene conversion could have contributed to the evolution of CYP4A21.

Gene structure of pig sterol 12alpha-hydroxylase (CYP8B1) and expression in fetal liver: comparison with expression of taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21).

Cholic acid is the major trihydroxy bile acid formed in most mammals. The domestic pig (Sus scrofa) is an exception. The bile of adult pig is devoid of cholic acid whereas hyocholic acid is found in amounts equal to that of cholic acid in humans. The pathway leading to formation of hyocholic acid is believed to be species-specific and to have evolved in the pig to compensate for a nonexistent or deficient cholic acid biosynthesis. However, a high level of cholic acid has recently been found in the bile of fetal pig. Here we describe that a gene encoding the key enzyme in cholic acid biosynthesis, the sterol 12alpha-hydroxylase (CYP8B1), is in fact present in the pig genome. The deduced amino acid sequence shows 81% identity to the human and rabbit orthologues. CYP8B1 mRNA is expressed at significant levels in fetal pig liver. Both CYP8B1 and the key enzyme in hyocholic acid formation, taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21), were found to be expressed in pig liver in a developmental-dependent but opposite fashion.

Biochemical properties of platelet microparticle membranes formed during exocytosis resemble organelles more than plasma membrane.

Studies of [3H]glycerol turnover in phosphatidylcholine (PC) in platelets revealed two metabolic pools, a 'low turnover PC' in collagen-induced microparticles with specific radioactivity only 10% of that found in the 'high turnover PC' of bulk platelet PC. Isolated organelle fractions of [3H]glycerol-labelled platelets contained [3H]PC with specific radioactivities about 20% of that in membrane fractions. These results together with studies on distribution of concanavalin A-FITC and GPlb, a plasma membrane receptor, indicate that microparticles formed during exocytosis are not simple vesiculations of plasma membrane, but they seem rather to originate from a relatively metabolically static membrane pool not accessible to extracellular reagents.

Cloning and expression of two novel pig liver and kidney fatty acid hydroxylases [cytochrome P450 (CYP)4A24 and CYP4A25].

A new member of the cytochrome P450 (CYP) 4A subfamily (CYP4A21) was recently cloned by PCR from pig liver [Lundell, Hansson, and Wikvall (2001) J. Biol. Chem. 276, 9606-9612]. This enzyme does not catalyse omega- or (omega-1)-hydroxylation of lauric acid, the model substrate for CYP4A enzymes. Instead, CYP4A21 participates in bile acid biosynthesis in the pig. Extensive studies, primarily conducted to verify the aberrant amino acids found in CYP4A21 within a normally conserved CYP4A motif, revealed that besides CYP4A21 two additional sequences were co-amplified by PCR. These two sequences (designated CYP4A24 and CYP4A25), generated from both pig liver and kidney, were characterized by restriction-enzyme analysis and were subsequently cloned. The deduced amino acid sequences of CYP4A24 and CYP4A25 share extensive sequence identity (97%). Both enzymes, expressed in yeast cells, exhibit omega-and (omega-1)-hydroxylase activities towards lauric acid and palmitic acid. The positions of the variable regions between CYP4A24 and CYP4A25, which are confined to beta-sheets 1 and 4, indicate a possible difference in substrate specificity or regioselectivity. The porcine CYP4A21, CYP4A24 and CYP4A25 enzymes, with an overall identity of 94%, have probably evolved from a common ancestral gene, perhaps in conjunction with species-specific habits.