Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL.

Apolipoprotein A-I (apoA-I), the major constituent of HDL, plays an essential role in regulating cholesterol metabolism, acting as the physiological activator of lecithin: cholesterol acyltransferase, which converts cholesterol to cholesterol ester. Thiol-reactive fluorescent probes attached to cysteine-containing apoA-I mutants are currently being used to investigate the "LCAT active" conformation of lipid-bound apoA-I. Herein, we report new methodologies allowing rapid expression, fluorescent labeling, and recombinant HDL (rHDL) preparation for use in apoA-I in fluorescence resonance energy transfer (FRET) studies. Cysteine-containing mutant forms of human apoA-I were cloned into the pTYB12 vector containing a T7 promoter, a modified self-splicing protein element (intein), and a small affinity tag [chitin binding domain (CBD)]. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates, and bound to a chitin-affinity column. Release of mature human apoA-I was initiated by the addition of DTT, which induced self-cleavage at the COOH terminus of the intein - CBD fusion protein. ApoA-I was further purified by Q-sepharose and then used for fluorescent probe labeling. Discoidal rHDL were then prepared with donor and/or acceptor labeled apoA-I and characterized with respect to their size, composition and ability to activate LCAT.

Isoprostane levels in lipids extracted from atherosclerotic arteries of nonhuman primates.

Nonhuman primates used in these studies had been fed for 5 years diets enriched with cholesterol and one of three classes of fatty acids: saturated, monounsaturated, or polyunsaturated fatty acids. Atherosclerotic iliac artery lipid extracts were quantitatively analyzed for cholesterol, cholesteryl esters, fatty acid composition, and a marker of lipid oxidation, the F(2)-isoprostanes. There was no significant difference in the mean accumulation of F(2)-isoprostanes among the different diet groups. To account for the small, individual variation in the arachidonate concentration the F(2)-isoprostane mass from each sample was normalized by dividing by arachidonate mass: F(2)-isoprostane mass/(mass arachidonate). At lower levels of cholesterol accumulation, the F(2)-isoprostane mass/(mass arachidonate) ratio was greater in lipids from POLY arteries compared to SAT arteries, but the reverse was true at high levels of cholesterol. F(2)-isoprostane/(mass arachidonate) increased with mole fraction linoleate for the SAT group, but decreased for the POLY group. In summary, these studies demonstrated that there is no simple explanation of how F(2)-isoprostane accumulation did not depend on the concentration of oxidizable lipids that promote free-radical lipid oxidation.

Is the oxidation of high-density lipoprotein lipids different than the oxidation of low-density lipoprotein lipids?

This article gives detailed insight into the kinetics of high-density lipoprotein (HDL) oxidation catalyzed by azobis(2-amidinopropane).dihydrochloride (ABAP) or by copper. ABAP initialized oxidation of human HDL 3-4 times faster than non-human primate HDL with a similar composition. The oxidizability of non-human primate HDL was 1000 times lower than the oxidizability calculated from rate constants derived from liposome oxidation, suggesting that there is a slow step in HDL oxidation not present in liposomes. Saturable binding of copper to HDL was a significant feature of copper-catalyzed oxidation. Binding constants (K(m)) for non-human primate HDL were 2-3-fold lower than those for human HDL. Copper-catalyzed oxidation of non-human primate HDL was slower than that of human HDL, but human HDL(2) and HDL(3) oxidized at about the same rate. Overall, the kinetics describing the oxidation of HDL were mechanistically similar to those reported for LDL, suggesting that HDL lipids were as easily oxidized as LDL lipids and that HDL will be easily oxidized in vivo when exposed to agents that oxidize LDL.

Structural determination of lipid-bound ApoA-I using fluorescence resonance energy transfer.

Based on the x-ray crystal structure of lipid-free Delta43 apoA-I, two monomers of apoA-I were suggested to bind to a phospholipid bilayer in an antiparallel paired dimer, or "belt orientation." This hypothesis challenges the currently held model in which each of the two apoA-I monomers fold as antiparallel alpha-helices or "picket fence orientation." When apoA-I is bound to a phospholipid disc, the first model predicts that the glutamine at position 132 on one apoA-I molecule lies within 16 A of glutamine 132 in the second monomer, whereas, the second model predicts glutamines at position 132 to be 104 A apart. To distinguish between these models, glutamine at position 132 was mutated to cysteine in wild-type apoA-I to produce Q132C apoA-I, which were labeled with thiol-reactive fluorescent probes. Q132C apoA-I was labeled with either fluorescein (donor probe) or tetramethylrhodamine (acceptor probe) and then used to make recombinant phospholipid discs (recombinant high density lipoprotein (rHDL)). The rHDL containing donor- and acceptor-labeled Q132C apoA-I were of similar size, composition, and lecithin:cholesterol acyltransferase reactivity when compared to rHDL-containing human plasma apoA-I. Analysis of donor probe fluorescence showed highly efficient quenching in rHDL containing one donor- and one acceptor-labeled Q132C apoA-I. rHDL containing only acceptor probe-labeled Q132C apoA-I showed rhodamine self-quenching. Both of these observations demonstrate that position 132 in two lipid-bound apoA-I monomers were in close proximity, supporting the "belt conformation" hypothesis for apoA-I on rHDL.

Single repeat deletion in ApoA-I blocks cholesterol esterification and results in rapid catabolism of delta6 and wild-type ApoA-I in transgenic mice.

The deletion mutation Delta6 apolipoprotein A-I lacks residues 143-164 or repeat 6 in the mature apoA-I protein. In vitro studies show this mutation dramatically reduces the rate of lecithin:cholesterol acyltransferase (LCAT) catalyzed cholesterol esterification. The present study was initiated to investigate the effect of this mutation on in vivo high density lipoprotein (HDL) cholesterol esterification and metabolism. Transgenic mice expressing human Delta6 apoA-I (TgDelta6 +/+) were created and then crossed with apoA-I knockout mice (-/-) to generate mice expressing only human Delta6 apoA-I (TgDelta6 -/-). Human Delta6 apoA-I was associated with homogeneous sized alpha-HDL, when wild-type mouse apoA-I was present (in TgDelta6 +/+ and +/- mice). However, in the absence of endogenous mouse apoA-I, Delta6 apoA-I was found exclusively in cholesterol ester-poor HDL, and lipid-free HDL fractions. This observation coincides with the 6-fold lower cholesterol ester mass in TgDelta6 -/- mouse plasma compared with control. Structural studies show that despite the structural perturbation of a domain extending from repeat 5 to repeat 8 (137-178), Delta6 apoA-I binds to spherical unilamellar vesicles with only 2-fold less binding affinity. In summary, these data show a domain corresponding to apoA-I repeat 6 is responsible for providing an essential conformation for LCAT catalyzed generation of cholesterol esters. Deletion of apoA-I repeat 6 not only blocks normal levels of cholesterol esterification but also exerts a dominant inhibition on the ability of wild-type apoA-I to activate LCAT in vivo.

The hydrophobic face orientation of apolipoprotein A-I amphipathic helix domain 143-164 regulates lecithin:cholesterol acyltransferase activation.

Apolipoprotein A-I (apoA-I) activates the plasma enzyme lecithin:cholesterol acyltransferase (LCAT), catalyzing the rapid conversion of lipoprotein cholesterol to cholesterol ester. Structural mutants of apoA-I have been used to study the details of apoA-I-LCAT-catalyzed cholesterol ester formation. Several studies have shown that the alpha-helical segments corresponding to amino acids 143-164 and 165-186 (repeats 6 and 7) are essential for LCAT activation. In the present studies, we examined how the orientation of the hydrophobic face, independent of an increase in overall hydrophobicity, affects LCAT activation. We designed, expressed, and characterized a mutant, reverse of 6 apoA-I (RO6 apoA-I), in which the primary amino acid sequence of repeat 6 (amino acids 143-164) was reversed from its normal orientation. This mutation rotates the hydrophobic face of repeat 6 approximately 80 degrees. Lipid-free RO6 apoA-I showed a marked stabilization when denatured by guanidine hydrochloride, but showed significant destabilization to guanidine hydrochloride denaturation in the lipid-bound state compared with wild-type apoA-I. Recombinant high density lipoprotein discs (rHDL) formed from RO6 apoA-I, sn-1-palmitoyl-sn-2-oleoyl phosphati-dylcholine, and cholesterol were approximately 12 A smaller than wild-type apoA-I rHDL. The reduced size suggests that one of the repeats did not effectively participate in phospholipid binding and organization. The sn-1-palmitoyl-sn-2-oleoyl phosphatidylcholine RO6 rHDL were a less effective substrate for LCAT. Mapping the entire lipid-free and lipid-bound RO6 apoA-I with a series of monoclonal antibodies revealed that both the lipid-free and lipid-bound RO6 apoA-I displayed altered or absent epitopes in domains within and adjacent to repeat 6. Together, these results suggest that the proper alignment and orientation of the hydrophobic face of repeat 6 is an important determinant for maintaining and stabilizing helix-bilayer and helix-helix interactions.

Sex steroids increase cholesterol 7alpha-hydroxylase mRNA in nonhuman primates.

One mechanism that may account for our prior observation that oral contraceptives decrease the hepatic cholesterol concentration independently of the low-density lipoprotein (LDL) receptor in sexually intact nonhuman primates is that sex hormones increase biliary cholesterol secretion by increasing hepatic mRNA abundance for cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in the conversion of cholesterol into bile acids. To examine the independent effect of estrogen, progestin, and combined estrogen and progestin on the hepatic cholesterol concentration and cholesterol 7alpha-hydroxylase mRNA abundance, 34 ovariectomized adult female cynomolgus monkeys were fed a moderately atherogenic diet for 12 weeks with either oral conjugated equine estrogen ([CEE] n = 8), medroxyprogesterone acetate ([MPA] n = 9), or combined CEE + MPA (n = 9) and compared with a control group (n = 8) that did not receive exogenous sex hormones. After 12 weeks, hepatic cholesterol was significantly lower in CEE-treated (6.2 +/- 1.2 mg/g liver) and CEE + MPA-treated (6.4 +/- 0.9 mg/g liver) animals compared with the control (12.6 +/- 1.9 mg/g liver) and MPA-treated (14.6 +/- 1.6 mg/g liver) groups. Hepatic cholesterol 7alpha-hydroxylase mRNA abundance was significantly increased in CEE-treated (0.553 +/- 0.08 pg/microg RNA), MPA-treated (0.734 +/- 0.12 pg/microg RNA), and CEE + MPA-treated (0.487 +/- 0.07 pg/microg RNA) animals compared with the controls (0.318 +/- 0.03 pg/microg RNA). There was no significant difference in the plasma LDL cholesterol concentration and hepatic LDL receptor mRNA abundance between the groups. These data support but do not prove the hypothesis that low-dose oral estrogen induces an increase in cholesterol 7alpha-hydroxylase mRNA abundance, which is correlated with biliary cholesterol secretion and may result in depletion of hepatic cholesterol.

Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase reactivity.

Apolipoprotein A-I contains eight 22-amino acid and two 11-amino acid tandem repeats that comprise 80% of the mature protein. These repeating units are believed to be the basic motif responsible for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. Computer analysis indicates that despite a fairly high degree of compositional similarity among the tandem repeats, significant differences in hydrophobic and amphipathic character exist. Our previous studies demonstrated that deletion of repeat 6 (143-164) or repeat 7 (165-186) resulted in a 98-99% reduction of LCAT activation as compared with wild-type apoA-I. To determine the effects of substituting one of these repeats with a more hydrophobic repeat we constructed a mutant apoA-I protein in which residues 143-164 (repeat 6) were replaced with repeat 10 (residues 220-241). The cloned mutant protein, 10F6 apoA-I, was expressed and purified from an Sf-9 cell baculoviral system and then analyzed using a number of biophysical and biochemical techniques. Recombinant complexes prepared at a 100:5:1 molar ratio of L-alpha-dimyristoylphosphatidylcholine:cholesterol:wild-type or 10F6 apoA-I showed a doublet corresponding to Stokes diameters of 114 and 108 A on nondenaturing 4-30% polyacrylamide gel electrophoresis. L-alpha-Dimyristoylphosphatidylcholine 10F6 apoA-I complexes had a 5-6-fold lower apparent Vmax/apparent Km as compared with wild-type apoA-I containing particles. As expected, monoclonal antibody epitope mapping of the lipid-free and lipid-bound 10F6 apoA-I confirmed that a domain expressed between residues 143 and 165 normally found in wild-type apoA-I was absent. The region between residues 119 and 144 in 10F6 apoA-I showed a marked reduction in monoclonal antibody binding capacity. Therefore, we speculate that the 5-6-fold lower LCAT reactivity in 10F6 compared with wild-type apoA-I recombinant particles results from increased stabilization within the 121-165 amino acid domain due to more stable apoprotein helix phospholipid interactions as well as from conformational alterations among adjacent amphipathic helix repeats.

Glycosylation structure and enzyme activity of lecithin:cholesterol acyltransferase from human plasma, HepG2 cells, and baculoviral and Chinese hamster ovary cell expression systems.

The glycosylation state of lecithin:cholesterol acyltransferase (LCAT) may be important in determining its enzymatic activity. We compared glycosylation structure, enzyme kinetics, and phosphatidylcholine (PC) acyl specificity of human LCAT from four sources: human plasma (pLCAT), media from HepG2 cells (HepG2 LCAT), media from SF21 cells infected with a recombinant baculovirus (bLCAT) and media from stably transfected Chinese hamster ovary (CHO) cells (CHO LCAT). bLCAT was underglycosylated (molecular weight approximately 50 kDa) and resistant to digestion by N-glycanase F, endoglycosidase F, and neuraminidase. CHO and HepG2 LCAT were overglycosylated (approximately 68 kDa and approximately 70-75 kDa) compared to pLCAT (approximately 65 kDa). CHO LCAT, like pLCAT, was sensitive to N-glycanase F and neuraminidase but not to endoglycosidase F. HepG2 LCAT demonstrated resistance to N-glycanase F and endoglycosidase F. Apparent Km values for all four enzymes were similar (1.4-9.2 microM cholesterol) for recombinant high density lipoproteins (rHDL) containing sn-1 16:0, sn-2 18:1 PC (POPC). Apparent Vmax values (nmol cholesteryl ester formed/h per micrograms) were 52.6 for pLCAT, 48.6 for CHO LCAT, 15.3 for bLCAT, and 8.3 for HepG2 LCAT. Changes in PC acyl specificity in the presence and absence of cholesterol were characterized by comparing the ratio of LCAT activity on rHDL containing sn-1 16:0, sn-2 20:4 PC (PAPC) or POPC (PAPC/POPC activity ratio). The ratios for pLCAT, bLCAT, CHO LCAT, and HepG2 LCAT activity were 0.63, 0.49, 0.56, and 0.51 with cholesterol and 0.34, 0.29, 0.36, and 0.99 without cholesterol, respectively. We conclude that LCAT source influences glycosylation structure, which affects the apparent Vmax for cholesteryl ester formation with only minor changes in apparent Km or acyl substrate specificity.

High level secretion of wild-type and mutant forms of human proapoA-I using baculovirus-mediated Sf-9 cell expression.

To facilitate the investigation of apoA-I structure:function relationships as they relate to LCAT activation and lipid binding, we have developed an apoA-I baculoviral expression and purification system that yields milligram quantities of wild-type or mutant proapoA-I. Baculovirus-infected Sf-9 cells, grown in suspension, were found to secrete high levels of human wild-type (40-50 mg/l) or mutant apoA-I protein (1-38 mg/l), which was determined to be > 95% pure following a two-step purification procedure. In the case of wild-type apoA-I, ELISA showed that approximately 13-18% of the total protein secreted into the culture medium was apoA-I. To isolate pure protein from culture medium, 72 h post-infection medium was subjected to preparative reverse phase high performance liquid chromatography (HPLC), followed by DEAE ion-exchange chromatography. Purity and molecular size determination of wild-type proapoA-I protein was verified by SDS polyacrylamide gel electrophoresis, electrospray mass spectrometry, and N-terminal sequencing. In addition, recombinant discoidal apoA-I:phospholipid complexes prepared from wild-type or plasma apoA-I showed similar particle size and LCAT activation properties. To fully characterize the utility of this expression system, the expression levels of various mutant apoA-I proteins were compared to wild-type. Despite a lower production level seen with selected apoA-I mutants, milligram quantities of these purified mutant proteins were also obtained. In summary, we show that baculovirus-derived wild-type proapoA-I shows properties similar to plasma apoA-I relative to recombinant HDL formation, LCAT reactivity, and alpha-helical content. In addition, we show that a variety of mutant forms of human proapoA-I can be expressed and purified in abundant quantity from baculoviral-infected Sf-9 cells.

Cortisol up-regulates corticotropin releasing factor gene expression in the fetal ovine brainstem at 0.70 gestation.

Glucocorticoids are important for the development of the central nervous system. In the ovine fetus, increased levels of plasma cortisol at term provide a stimulus to initiate parturition. CRF is central to this event in that it is one of the main modulators of the hypothalamic-pituitary-adrenal (HPA) axis. The purpose of the present study was to determine the effect of physiological increases in fetal plasma cortisol levels on corticotropin-releasing factor (CRF) gene expression in the developing ovine brain. Fetal plasma cortisol levels were chronically elevated at 0.70 gestation (100 days) to physiological levels found at 0.90 gestation (130 days; term 145 +/- 2 days) when glucocorticoid-induced maturational changes are known to occur in the HPA axis. The 3' end of the ovine CRF gene encodes 4 putative polyadenylation (poly(A)) signals that may post-transcriptionally regulate gene expression through stability, translation and localization of the mRNA in a temporal and spatial manner. To determine whether CRF mRNA levels or poly(A) site usage are differentially regulated by cortisol in a region-specific manner, we used an RNase protection assay with an antisense CRF RNA probe from the 3' coding and untranslated regions of the gene to quantify changes in mRNA levels in the hypothalamus (Hypo), hippocampal-amygdala complex (H and A), frontal cerebral cortex (FCC) and brainstem. Our novel finding was a 3.5-fold increase in CRF mRNA levels in the medulla oblongata of fetuses from the cortisol group compared to those from the saline group (P = 0.001). CRF mRNA levels in the Hypo, H and A and FCC did not change significantly in fetuses from the cortisol group.(ABSTRACT TRUNCATED AT 250 WORDS)

Species-specific polymorphism in the promoter of the apolipoprotein A-I gene: restoration of human transcriptional efficiency by substitution at positions -189, -144 and -48 bp.

Previous studies indicate that species-specific differences in apolipoprotein A-I (apo A-I) expression could be largely explained by cis-acting factors located within or near the 5' flanking region (-231 to +223 bp, where +1 is the start site of transcription). In the present studies, we have localized 7 sites within the (-231 to -15 bp) region of the African green monkey apo A-I gene that differ from the human apo A-I gene 5' flanking region. To identify which of the 7 polymorphic sites were essential for the species-specific differences in apo A-I gene expression, mutated promoter constructs were transfected into HepG2 cells and reporter gene expression was measured. Each of the 7 sites within a defined 5' flanking region of the human gene was individually mutated to the African green nucleotide sequence found at that position. Three of the sites (-189, -144 and -48) were found to raise the human apo A-I promoter activity to approx. 60-65% of the African green promoter. While double mutations (-144/-48 bp and -189/-144 bp), restored the human apo A-I promoter activity to 100% of that found with the African green monkey promoter. Additional studies revealed similar DNA: protein interactions with DNA probes from either human or African green monkey and HepG2 cell nuclear extract. In conclusion, these studies demonstrate that double and triple nucleotide substitutions within the human apo A-I promoter are sufficient to restore gene expression in HepG2 cells to levels seen with the African green monkey promoter. These data suggest that sites -189, -144 and -48 bp are involved in significantly altering the binding affinity of a nuclear factor determining the species-specific level of apo A-I gene transcription.

Estrogen modulates the inducible expression of platelet-derived growth factor mRNA by monocyte/macrophages.

We examined the effects of estrogen, 12-O-tetradecanoylphorbol 13 acetate (TPA), and lipopolysaccharide (LPS) on the gene expression of platelet-derived growth factor (PDGF) by the monocyte/macrophage cell line, THP-1. THP-1 cells were exposed to TPA for 48 or 96 hours to induce differentiation. Some were treated with LPS in the last 3 hours and/or ethinyl estradiol (estrogen) (10(-9) M) in the last 20 hours. Total cellular RNA was isolated and cDNA was synthesized and then coamplified (with an internal control, beta-actin, product size 1126 bp) using polymerase chain reaction (PCR) and a set of primers for PDGF-A (product size 225 bp), PDGF-B (217 bp), or PDGF beta-receptor (PDGF-R) (228 bp). The products were separated on an agarose gel and the ratios of radioactivity incorporated into PDGF PCR products to beta-actin products were used to assess the relative changes in the levels of PDGF mRNA abundance in response to various inducers. TPA induced the expression of PDGF-A mRNA, whereas LPS had no effect. Treatment of TPA-stimulated cells with estrogen caused a 61% and 190% increase in PDGF-A mRNA (p < 0.05) at 48 and 96 hours, respectively. Addition of estrogen to cells treated with both TPA and LPS did not cause any significant change in the amounts of the transcripts. In contrast to PDGF-A mRNA, attempts to visualize and estimate PDGF-B and PDGF-R mRNA were unsuccessful. This was probably due to low levels of these transcripts in THP-1 cells. The results indicate that estrogen modulates PDGF-A gene expression by monocyte/macrophages and suggest that estrogen may influence atherogenesis at the vascular level.

Estrogen modulates the expression of tumor necrosis factor alpha mRNA in phorbol ester-stimulated human monocytic THP-1 cells.

Monokines, including tumor necrosis factor alpha (TNF-alpha), have been implicated in the pathogenesis of several pathologic processes, including atherosclerosis. Because estrogen has been found to offer a certain degree of protection against atherosclerotic progression, we examined the effect of estrogen on the expression of TNF-alpha mRNA in a monocyte-macrophage cell line, THP-1. Cells were exposed to 12-O-tetradecanoylphorbol 13-acetate (TPA, 50 ng/ml) for 48 or 96 h to induce differentiation. Some of the cells were treated with lipopolysaccharide (LPS, 10 micrograms/ml) in the last 3 h and/or ethinyl estradiol (estrogen, 10(-9) M) in the last 20 h. Total cellular RNA was isolated and cDNA synthesized and than coamplified using the polymerase chain reaction (PCR) in the presence of two sets (pairs) of 32P-labeled primers, one for TNF-alpha (product size 325 bp) and the second for the internal control, glyceraldehyde 3-phosphate dehydrogenase (G3PDH; 983 bp). The resultant PCR products were separated by agarose gel electrophoresis, and the ratios of radioactivity incorporated into TNF-alpha PCR products to G3PDH products were used to assess the relative changes in the levels of TNF-alpha mRNA abundance in response to various substances. Treatment with TPA for 48 h induced the expression of TNF-alpha mRNA. Treatment of these TPA-stimulated cells with estrogen caused a 62% decrease in TNF-alpha message abundance (p < 0.01). Similar results were obtained with cells stimulated with TPA for 96 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticotropin releasing factor mRNA and peptide levels are differentially regulated in the developing ovine brain.

The regulation of CRF mRNA and protein in the developing ovine brain has been studied to assess the hypothesis that CRF is differentially regulated in the hypothalamus (Hypo), hippocampal-amygdala complex (H & A), frontal cerebral cortex (FCC) and brainstem (BS). We used a quantitative RNase protection assay and radioimmunoassay to determine mRNA and peptide concentrations, respectively, from the last third of gestation until term (i.e., from 95 to 142 days gestation (dg); term approximately 145 days). The major findings from this study are: (1) Hypothalamic CRF mRNA was increased by 2-fold in 140-142 dg fetuses compared to 128-138 and 95-123 dg fetuses; P = 0.016. (2) In the hypothalamus of 140-142 dg fetuses, there was a 2.5-fold increase in CRF mRNA derived from polyadenylation at poly(A) sites 2, 3 or 4; P = 0.005. (3) In 128-138 dg fetuses, CRF mRNA in the frontal cortex was 2-fold higher than in the other brain regions during this time period; P = 0.008. (4) CRF peptide concentrations in the Hypo were 2.5-fold higher in 140-142 dg fetuses compared to 95-106 and 128-138 dg fetuses; P = 0.007. (5) CRF peptide concentrations in the frontal cortex were 5.5-fold higher in 140-142 dg fetuses compared to fetuses at 95-106 dg; P = 0.004. (6) CRF peptide concentrations in the H & A were 5-fold higher in 140-142 dg fetuses compared to 95-106 dg fetuses; P = 0.029. The results from the present study demonstrate for the first time that CRF mRNA and peptide are differentially regulated in a region-specific manner during development.

The inducible expression of THP-1 cell interleukin-1 mRNA: effects of estrogen on differential response to phorbol ester and lipopolysaccharide.

Monokines, such as interleukin-1, have been implicated in the pathogenesis of several pathologic processes, including the initiation and progression of atherosclerosis. Since estrogen has been identified as a modulator of atherosclerosis progression, we sought to examine the effect of estrogen on the inducible expression of interleukin-1 beta (IL-1 beta) and interleukin-1 alpha (IL-1 alpha) mRNA in the monocytic cell line, THP-1. Cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) (50 ng/ml) for 48 or 96 h to induce differentiation. Some cells were treated with lipopolysaccharide (LPS) (10 micrograms/ml) in the last 3 h and/or 10(-9) M ethinyl estradiol (estrogen) in the last 20 h. Total cellular RNA was isolated, and cDNA was synthesized and amplified using the polymerase chain reaction (PCR) using two sets (pairs) of 32P-labeled primers, one for IL-1 beta (product size 388 bp) and the second for the internal control, beta-actin (1126 bp), or to detect another cytokine mRNA, a set of primers for IL-1 alpha (product size 420 bp) and beta-actin. The PCR products were separated on a 3.0% agarose gel and the ratio of radioactivity incorporated into cytokine PCR products and beta-actin products was determined to assess the relative changes in the relative levels of cytokine to beta-actin mRNA abundance in response to various inducers. Treatment with TPA for 48 h induced expression of IL-1 beta mRNA, an effect that was enhanced two fold by LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokines decrease apolipoprotein accumulation in medium from Hep G2 cells.

Cytokines, important biochemical mediators of inflammation, cause a rapid fall in the plasma concentration of cholesterol in vivo. One mechanism by which cytokines may cause acquired hypocholesterolemia is by decreasing the hepatic synthesis and secretion of apolipoproteins. To test this hypothesis, we incubated Hep G2 cells with human recombinant tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. Each of the cytokines resulted in a dose-related reduction in the concentrations of apolipoprotein (apo) A-I, apoB, and lecithin:cholesterol acyltransferase (LCAT) activity in the medium after 24 hours of incubation. The effect of cytokines on apolipoprotein accumulation was not affected by preincubation of Hep G2 cells with fatty acids. Cytokines decreased the concentration of cellular apoA-I mRNA in a dose-related fashion but did not affect cellular concentrations of apoB mRNA. The concentrations of triglyceride and cholesterol were also reduced in the medium of cells incubated with cytokines. Total cell sterol synthesis rates were calculated by [14C]acetate incorporation. Cells incubated with interleukin-6 had a 31% increase in sterol synthesis rate but a 41% decrease in sterol secretion. These data suggest that these cytokines can decrease the hepatic synthesis and/or secretion of apolipoproteins and that this may explain, in part, the acquired hypocholesterolemia seen during acute and chronic inflammation.

Oral contraceptives decrease hepatic cholesterol independent of the LDL receptor in nonhuman primates.

Pharmacological doses of estrogens have been reported to increase hepatic catabolism of low-density lipoprotein (LDL) by the LDL receptor (LDL-R) pathway and to increase the concentration of mRNA for the LDL receptor. The induction of LDL-Rs by large doses of estrogen may not be relevant to the role of estrogens under physiological conditions. Furthermore, the mechanisms by which oral contraceptives, a combination of synthetic estrogen and progestin, may modulate LDL metabolism remain largely unexplored. Adult female cynomolgus monkeys were given combination ethinyl estradiol/norgestrel preparations (n = 16) for 16 weeks and were compared with a control group that did not receive exogenous sex hormones (n = 7). All animals consumed a diet containing 0.25 mg cholesterol/kcal with 40% of calories from saturated fats. After 16 weeks of treatment there was no significant difference in LDL cholesterol (LDL-C) and hepatic LDL-R mRNA concentration between oral contraceptive-treated animals (LDL-C, 242 +/- 113 mg/dL; LDL-R mRNA, 0.60 +/- 0.31 pg/microgram RNA) and control animals (LDL-C, 277 +/- 100 mg/dL; LDL-R mRNA, 0.51 +/- 0.21 pg/microgram RNA). In contrast, the hepatic cholesteryl ester concentration was significantly lower in the oral contraceptive-treated animals (7.28 +/- 3.59 mg/g liver) compared with the control animals (16.07 +/- 11.86 mg/g liver; P = .01) with no significant difference in hepatic free cholesterol concentration between the groups. Thus, oral contraceptives decrease hepatic cholesterol concentration independent of LDL-R expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships.

A series of mutant apolipoprotein (apoA-I) constructs were designed and then expressed in cell culture to identify structural domains within the mature native apoA-I protein that participate in the activation of the plasma enzyme, lecithin-cholesterol acyltransferase (LCAT). Evolutionary conservation analysis has shown previously that apoA-I contains eight repeats containing 22 amino acids and two repeats containing 11 amino acids that are highly conserved among species as well as within the apolipoprotein supergene family. These tandem repeats begin at residue 44 and are usually marked by a proline residue, with six of the 22-mer repeats showing high amphipathic alpha-helical character. To determine if specific 11- or 22-amino acid domains are essential for maximal LCAT activation within the entire native protein, each of the 10 repeats was sequentially deleted using a polymerase chain reaction based method of mutagenesis. The wild-type and mutant apoA-I gene constructs were expressed in Chinese hamster ovary (CHO) cells and stable lines established. Wild-type and mutant apoA-I protein were purified from 48-96-h conditioned serum-free medium and characterized by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Wild-type apoA-I showed a single migrating band of 28,000 daltons that corresponded to the mobility of human plasma apoA-I, whereas apoA-I deletion mutants (lacking 22- or 11-mer repeats) showed the corresponding shift to lower molecular size. To measure the relative LCAT activation of all deletion mutant apoA-I proteins relative to wild-type apoA-I, an assay system utilizing small unilamellar vesicles as the lipid substrate was used. The results of these studies suggest that several central amphipathic alpha-helical regions within the mature protein are critical in LCAT activation.

Dexamethasone increases apolipoprotein A-I concentrations in medium and apolipoprotein A-I mRNA abundance from Hep G2 cells.

Glucocorticoid hormones increase high-density lipoprotein (HDL) levels in vivo. However, there is little known about the mechanism by which glucocorticoids alter HDL metabolism. Hep G2 cells were incubated with dexamethasone to determine the effect of glucocorticoids on apolipoprotein (apo) A-I secretion. Dexamethasone increased apo A-I concentration in a dose-dependent fashion. After 24 hours, 5.5 x 10(-5) mol/L dexamethasone increased apo A-I accumulation in culture medium by 54%. Detectable increases in apo A-I concentration were noted in medium by 5 hours of incubation and persisted up to 48 hours. Cellular apo A-I mRNA concentration increased by 28% after incubation with dexamethasone for 24 hours. The increase in apo A-I mRNA concentration was detectable within 3 hours after incubation with dexamethasone. In contrast, incubation with dexamethasone decreased apo B concentration by 43% in culture medium, but it had no effect on cellular apo B mRNA concentrations. Dexamethasone had little effect on cholesterol and triglyceride accumulation in the medium. Incubation with albumin alone did not affect apo A-I concentration, but it decreased apo B concentration by 30% in the medium. Incubation with albumin and dexamethasone had no effect on apo A-I concentration in medium and had no additive effect on apo B concentration. These data suggest dexamethasone increases secretion of apo A-I by Hep G2 cells by increasing mRNA levels.