Dual mechanisms of ABCA1 regulation by geranylgeranyl pyrophosphate.

ATP-binding cassette transporter A1 (ABCA1) mediates an active efflux of cholesterol and phospholipids and is mutated in patients with Tangier disease. Expression of ABCA1 may be increased by certain oxysterols such as 22(R)-hydroxycholesterol via activation of the nuclear hormone receptor liver X receptor (LXR). In searching for potential modulators of ABCA1 expression, we have studied the effects of various mevalonate metabolites on the expression of ABCA1 in two human cell lines, THP-1 and Caco-2 cells. Most of the tested metabolites, including mevalonate, geranyl pyrophosphate, farnesyl pyrophosphate, and ubiquinone, failed to significantly change the expression levels of ABCA1. However, treatment with geranylgeranyl pyrophosphate resulted in a dose- and time-dependent reduction of ABCA1 expression. Geranylgeranyl pyrophosphate appears to reduce ABCA1 expression via two different mechanisms. One of these mechanisms is by acting directly as an antagonist of LXR since it reduces the interaction between LXR alpha or -beta with nuclear coactivator SRC-1. Another mechanism appears to involve activation of the Rho GTP-binding proteins since treatment of Caco-2 cells with inhibitors of geranylgeranyl transferase or the Rho proteins significantly increased the expression and promoter activity of ABCA1. Further studies showed that mutations in the DR4 element of the ABCA1 promoter completely eliminate the inducible activities of these inhibitors. These data indicate that activation of the Rho proteins may change the activation status of LXR.

27-hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells.

The nuclear receptors liver X receptor alpha (LXRalpha) (NR1H3) and LXRbeta (NR1H2) are important regulators of genes involved in lipid metabolism, including ABCA1, ABCG1, and sterol regulatory element-binding protein-1c (SREBP-1c). Although it has been demonstrated that oxysterols are LXR ligands, little is known about the identity of the physiological activators of these receptors. Here we confirm earlier studies demonstrating a dose-dependent induction of ABCA1 and ABCG1 in human monocyte-derived macrophages by cholesterol loading. In addition, we show that formation of 27-hydroxycholesterol and cholestenoic acid, products of CYP27 action on cholesterol, is dependent on the dose of cholesterol used to load the cells. Other proposed LXR ligands, including 20(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 24(S),25-epoxycholesterol, could not be detected under these conditions. A role for CYP27 in regulation of cholesterol-induced genes was demonstrated by the following findings. 1) Introduction of CYP27 into HEK-293 cells conferred an induction of ABCG1 and SREBP-1c; 2) upon cholesterol loading, CYP27-expressing cells induce these genes to a greater extent than in control cells; 3) in CYP27-deficient human skin fibroblasts, the induction of ABCA1 in response to cholesterol loading was ablated; and 4) in a coactivator association assay, 27-hydroxycholesterol functionally activated LXR. We conclude that 27-hydroxylation of cholesterol is an important pathway for LXR activation in response to cholesterol overload.

Inhibition of adipocyte differentiation by HIV protease inhibitors.

Patients with AIDS who are receiving therapy with HIV protease inhibitors have been widely reported to be afflicted with a syndrome characterized by lipodystrophy (fat redistribution favoring the accumulation of abdominal and cervical adipose tissue), hyperlipidemia, and insulin resistance. HIV protease inhibitors have been suggested to have a direct role in modulating adipocyte differentiation. To address this hypothesis, several HIV protease inhibitors were studied for their ability to either augment or inhibit the differentiation of murine 3T3-L1 preadipocytes. Dose-responsive inhibition of adipogenesis by several protease inhibitors was noted as measured by reduced triglyceride accumulation and attenuated induction of three differentiation marker genes -- aP2, lipoprotein lipase, and Adipo Q. Potential mechanisms for altered adipocyte function, including direct binding to PPARgamma or inhibition of PPARgamma-mediated gene transcription were effectively excluded.

Targeted disruption of the tumor necrosis factor-alpha gene: metabolic consequences in obese and nonobese mice.

To address the hypothesis that tumor necrosis factor (TNF)-alpha has a role in obesity-associated insulin resistance or the regulation of in vivo lipid metabolism, mice with targeted disruption of the TNF-alpha gene were generated and studied. The absence of TNF-alpha protein in TNF-null (-/-) mice was confirmed. Lean or obese (gold-thioglucose [GTG]-injected) homozygous (-/-) mice were compared with lean or obese age- and sex-matched wild-type (+/+) mice derived from the same line at 13, 19, and 28 weeks of age. The following parameters were significantly affected in lean -/- versus +/+ mice: Body weight was not affected until week 28 (decreased by 14%); epididymal fat pad weight also decreased (25%) at this time, as did percentage body fat (16%), while percentage body protein was increased 13%. Fed plasma insulin levels decreased 47% (28 weeks), triglyceride levels decreased (all three ages; maximum 35% at 19 weeks), and fed plasma leptin decreased 33% (28 weeks). Fasting glucose was slightly (10%) reduced, but the glucose response to an oral glucose tolerance test (OGTT) was not affected. There was a trend (NS) toward increased total adipose tissue lipoprotein lipase in -/- versus +/+ mice. GTG-treatment resulted in obese -/- and +/+ mice with equal mean body weights (42 and 58% increased weight versus lean mice). The following parameters were significantly different in obese -/- mice: fasting plasma glucose decreased 13% (28 weeks), fed plasma insulin decreased 67% (28 weeks), and insulin response to OGTT was decreased by 50%. For both groups of obese mice, glucose levels during the OGTT were substantially increased compared with those in lean mice; however, mean stimulated glucose levels were 20% lower in obese -/- versus +/+ mice. We conclude 1) that TNF-alpha functions to regulate plasma triglycerides and body adiposity and 2) that although TNF-alpha contributes to reduced insulin sensitivity in older or obese mice, the absence of TNF-alpha is not sufficient to substantially protect against insulin resistance in the GTG hyperphagic model of rodent obesity.

Expression and immunoaffinity purification of human inducible nitric-oxide synthase. Inhibition studies with 2-amino-5,6-dihydro-4H-1,3-thiazine.

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37 degrees C and contained 1.15 +/- 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret lambdamax at 396 nm with a shoulder at 460 nm and contained 0. 28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 +/- 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.

Active-site structure analysis of recombinant human inducible nitric oxide synthase using imidazole.

Nitric oxide synthase catalyzes the pyridine nucleotide-dependent oxidation of L-arginine to nitric oxide and L-citrulline. It is a specialized cytochrome P450 monooxygenase that is sensitive to inhibition by imidazole. Steady-state kinetic studies on recombinant human inducible nitric oxide synthase (rH-iNOS) demonstrate that imidazole and 1-phenylimidazole are competitive and reversible inhibitors versus L-arginine. Structure-activity relationship and pH dependence studies on the inhibition suggest that the neutral form of imidazole may be the preferred species and that the only modifications allowed without the loss of inhibition are at the N-1 position of imidazole. Optical spectrophotometric studies of rH-iNOS with imidazole and 1-phenylimidazole yielded type II difference spectra exhibiting Kd values of 63 +/- 2 and 28 +/- 3 microM, respectively. These values were in good agreement with the steady-state Ki of 95 +/- 10 and 38 +/- 4 microM, respectively, and confirms the site of binding is at the sixth axial ligand of the heme. Imidazole (2.2 mM) also perturbed the Kd of L-arginine from 3.03 +/- 0.45 to 209 +/- 10 microM. The observed increase in the Kd for L-arginine is consistent with imidazole being a competitive inhibitor versus L-arginine. The IC50 values of imidazole and 1-phenylimidazole were lower in the absence of exogenous BH4, and both inhibitors also competitively inhibited the BH4-dependent activation of the enzyme. These data taken together suggest that the L-arginine, dioxygen, and the BH4 binding sites are in close proximity in rH-iNOS. Furthermore, these studies demonstrate the usefulness of imidazole compounds as active site probes for recombinant human iNOS.

Use of an antibody against the matrix metalloproteinase-generated aggrecan neoepitope FVDIPEN-COOH to assess the effects of stromelysin in a rabbit model of cartilage degradation.

OBJECTIVE: To define the stromelysin cleavage site in the interglobular domain of rabbit aggrecan, and to determine whether the stromelysin-generated neoepitope can be used as a marker of matrix metalloproteinase (MMP) activity in vivo. METHODS: The carboxy-terminus sequence of the stromelysin-generated hyaluronic acid-binding region (HABR) of rabbit aggrecan was determined by reverse transcription-polymerase chain reaction complementary DNA cloning and DNA sequence analysis, followed by purification and mass spectral protein sequence analysis of the HABR fragment. Active stromelysin was injected into the stifle joints of rabbits, and a stromelysin-generated aggrecan neoepitope was analyzed by Western blotting and localized in situ by indirect immunofluorescence. Proteoglycan fragments in joint fluids were quantified by a dimethylmethylene blue dye-binding assay. RESULTS: Stromelysin cleavage of rabbit aggrecan generated a 55-kd HABR fragment that terminated in the sequence FMDIPEN: An anti-FVDIPEN antibody recognized the FMDIPEN neoepitope in situ in cartilage from stromelysin-injected joints. The appearance of the FMDIPEN neoepitope corresponded to the release of cartilage proteoglycan fragments into the joint fluid, and could be inhibited by pretreatment of the rabbits with a synthetic stromelysin inhibitor. CONCLUSION: These results indicate that the anti-FVDIPEN antibody can be used to assess the role of MMPs in cartilage degradation in vivo.

Induction of increased levels of proteoglycan fragments in synovial fluid (SF) and increased levels of stromelysin in cartilage, synovium and SF by intraarticular injection of canine monocyte conditioned medium into dogs.

OBJECTIVE: To study the effects of the intraarticular injection of canine monocyte conditioned medium (cMCM) into dogs on proteoglycan fragment and stromelysin levels in the joint. METHODS: cMCM was injected intraarticularly into dogs, and the levels of proteoglycan fragments in synovial fluid (SF) as well as stromelysin levels in cartilage, synovium, and SF were assessed after 12 h. RESULTS: There was a 4-fold increase of proteoglycan fragment levels and a 6-fold increase in stromelysin levels in SF, and a 4.4-fold increase in stromelysin levels in cartilage extracts. Elevated mRNA levels were detected in both synovium and cartilage. By immunofluorescence staining, stromelysin was localized in chondrocytes throughout the cartilage and in synovial cells. CONCLUSION: Intraarticular injection of cMCM stimulated the expression of stromelysin mRNA and protein in cartilage and synovium and caused marked increases in stromelysin protein and proteoglycan fragment levels in SF.

Differential in vivo expression of collagenase messenger RNA in synovium and cartilage. Quantitative comparison with stromelysin messenger RNA levels in human rheumatoid arthritis and osteoarthritis patients and in two animal models of acute inflammatory arthritis.

OBJECTIVE: To compare quantitatively the in vivo expression of collagenase messenger RNA (mRNA) and stromelysin mRNA in the joint tissues of human osteoarthritis (OA) and rheumatoid arthritis (RA) patients and in two animal models of acute inflammatory arthritis. METHODS: In vivo levels of metalloproteinase mRNA and protein were determined by quantitative Northern hybridization and by enzyme-linked immunosorbent assay, respectively. RESULTS: In synovium, mean levels of collagenase mRNA were similar to those of stromelysin mRNA; however, in cartilage, mean levels of collagenase mRNA were significantly lower. The ratios of collagenase mRNA to stromelysin mRNA levels in RA and OA cartilage reflected similar ratios of collagenase protein to stromelysin protein levels in synovial fluid. CONCLUSION: The regulation of collagenase mRNA expression in cartilage is distinct from that of stromelysin, suggesting distinct roles for these two metallo-proteinases in normal and abnormal physiologic functioning of cartilage.

Differential expression of iNOS and cNOS mRNA in human vascular smooth muscle cells and endothelial cells under normal and inflammatory conditions.

To examine the potential contribution of endothelial cell cNOS (ec-cNOS) and inducible NOS (iNOS) in controlling vascular tone under normal versus inflammatory conditions, we performed Northern hybridizations to examine the differential expression of each NOS mRNA in human aortic endothelial cells (AOEC) and human aortic smooth muscle cells (AOSMC) cultured for 8 h in the presence or absence of cytokines (IL-1 beta, TNF-alpha, and IFN-gamma) and LPS. Cytokine/LPS treatment induced a 4.4 kb iNOS mRNA in the human AOSMC; in contrast, cytokine/LPS treatment down regulated the expression of ec-cNOS mRNA in the AOEC. No iNOS mRNA was detected in the AOEC under the conditions examined. These results suggest that under specific inflammatory conditions the generation of NO in vascular tissue switches from ec-cNOS in the endothelium to iNOS in the smooth muscle.

In vivo expression of stromelysin in synovium and cartilage of rabbits injected intraarticularly with interleukin-1 beta.

OBJECTIVE: To examine the in vivo expression of the matrix metalloproteinase stromelysin in the synovium and articular cartilage of rabbits injected intraarticularly with recombinant human interleukin-1 beta (IL-1). METHODS: The direct isolation of messenger RNA (mRNA) from articular cartilage without the prior isolation of chondrocytes is described. The in vivo expression of stromelysin was examined at the mRNA level by Northern blot analysis, and at the protein level by in situ immunolocalization and by enzyme-linked immunosorbent assay. RESULTS: In the synovium of IL-1-injected joints, stromelysin mRNA levels were highest at 4 hours and declined to background levels within 24 hours. In the cartilage of IL-1-injected joints, stromelysin mRNA was elevated at 4 hours and continued to increase until 8 hours, before declining. Stromelysin mRNA expression preceded a similar increase in stromelysin protein levels in both synovium and cartilage. CONCLUSION: Intraarticular injection of IL-1 induced the endogenous expression of stromelysin mRNA and protein in both synovium and cartilage. The kinetics of stromelysin expression correlated well with the accumulation of stromelysin and proteoglycan in synovial fluids. Therefore, the de novo synthesis of stromelysin in cartilage may have contributed to the loss of proteoglycan from that tissue.

Production, purification and characterization of canine prostromelysin.

One of the best studied animal models of osteoarthritis is a dog model in which the anterior cruciate ligament of the hind limb stifle joint is transected (Pond-Nuki model). To determine whether stromelysin might play a role in this model, it was necessary to purify the enzyme for production of suitable probes. In the present study, dog synovial fibroblasts were stimulated to express a metalloproteinase that was demonstrated to be canine prostromelysin by Northern blot, protein purification and amino-terminal sequence analyses. Unlike rabbit synoviocytes, passaged dog synoviocytes did not express stromelysin mRNA in response to recombinant human IL-1, but expressed stromelysin mRNA only upon treatment with dog monocyte-conditioned medium (dMCM). The aminophenylmercuric acetate (APMA)-activatable metalloproteinase present in the culture supernatants of stimulated dog synoviocytes was purified using a combination of ion-exchange and dye matrix affinity chromatography. The purified canine metalloproteinase co-migrated on reducing SDS-PAGE with recombinant human prostromelysin-1 as a doublet with apparent molecular masses of 54 and 56 kDa. Similar to APMA-activated human prostromelysin-1, the APMA-activated canine metalloproteinase was inhibited by 1,10-phenanthroline or recombinant human tissue inhibitor of metalloproteinase (TIMP). The amino-terminal sequences of the canine pro- and APMA-activated enzymes were compared with those of human, rabbit and rat stromelysin. The striking homologies among the sequences demonstrated that the purified canine metalloproteinase was indeed canine prostromelysin. A rabbit anti-canine prostromelysin polyclonal antiserum was generated and used to localize the enzyme within cultured dog synoviocytes and articular cartilage stimulated with dMCM. The reagents developed in this study should be useful for examining the expression and distribution of prostromelysin in canine models of osteoarthritis.

Analysis of IL-1 and TNF-alpha gene expression in human rheumatoid synoviocytes and normal monocytes by in situ hybridization.

Human IL-1 alpha, IL-1 beta, and TNF-alpha mRNA expression was examined in peripheral blood monocytes (PBM) from normal individuals and in primary synoviocytes isolated from patients with rheumatoid arthritis by Northern blot and in situ hybridization. Cells cultured in the presence or absence of LPS were analyzed using in vitro synthesized 35S-labeled sense or antisense RNA probes to determine the relative abundance and the cell type expressing each of the mRNA for these potent inflammatory mediators. The results indicated that 72% of the LPS-stimulated PBM expressed detectable levels of IL-1 alpha mRNA, 89% IL-1 beta mRNA, and 10% TNF-alpha transcripts. Thus, the majority of activated PBM produced both IL-1 alpha and IL-1 beta. Experiments combining immunofluorescence for IL-1 beta protein with in situ hybridization for TNF-alpha mRNA demonstrated that monocytes expressing TNF-alpha mRNA also produced IL-1 beta. Primary synoviocytes from four patients with RA were also examined for the mRNA expression of each cytokine. Northern blot analyses of total RNA isolated from 0 to 72 h after LPS- or mock-stimulation showed that IL-1 beta mRNA was the most abundantly expressed, followed by TNF-alpha. In situ hybridization revealed that IL-1 beta and TNF-alpha transcripts were detected exclusively in synovial tissue macrophages. IL-1 alpha mRNA was not detected in these cultures by either method.

Discoordinate expression of stromelysin, collagenase, and tissue inhibitor of metalloproteinases-1 in rheumatoid human synovial fibroblasts. Synergistic effects of interleukin-1 and tumor necrosis factor-alpha on stromelysin expression.

Primary and passaged human synovial fibroblasts isolated from rheumatoid pannus were treated with recombinant interleukin-1 (IL-1) alpha or beta, tumor necrosis factor-alpha (TNF), or phorbol myristate acetate (PMA) to determine the effects of these stimuli on the relative expression of stromelysin, collagenase, and tissue inhibitor of metalloproteinases (TIMP). The steady-state mRNA levels for these genes and glyceraldehyde-3-phosphate dehydrogenase were determined on Northern blots. Immunoblot analyses of the conditioned media using monoclonal antibodies generated against recombinant human stromelysin, collagenase, or TIMP showed that protein levels reflected the corresponding steady-state mRNA levels. The results revealed that 1) stromelysin and collagenase were not always coordinately expressed; 2) IL-1 was more potent than TNF or PMA in the induction of stromelysin expression; 3) neither IL-1 nor TNF significantly affected TIMP expression; 4) PMA induced both metalloproteinase and TIMP expression; and 5) the combination of IL-1 plus TNF had a synergistic effect on stromelysin expression. Dose response and time course experiments demonstrated that the synergistic effect of IL-1 plus TNF occurred at saturating concentrations of each cytokine and lasted for 7 days. In summary, the ability of IL-1 and TNF to preferentially induce stromelysin and collagenase expression, versus TIMP, may define a pivotal role for these cytokines in the pathogenesis of rheumatoid arthritis.

Identification of a high-affinity receptor for interleukin 1 alpha and interleukin 1 beta on cultured human rheumatoid synovial cells.

In this report the binding of recombinant human interleukins 1 alpha and 1 beta (rIL-1 alpha and rIL-1 beta) to primary cultures of human rheumatoid synovial cells is measured and compared to the concentrations of these mediators required for stimulation of PGE2 production by these same cells. The average concentration of IL-1 alpha required for half-maximal stimulation of PGE2 was 4.6 +/- 1.5 pM (+/- SEM) (n = 6), whereas for IL-1 beta half-maximal stimulation was observed at a concentration of 1.3 +/- 0.24 pM (n = 6). Both direct and competitive binding experiments were performed. In direct binding experiments, IL-1 alpha bound with a Kd of 66 pM (n = 1), while IL-1 beta bound with a Kd of 4 pM (n = 2). In competitive binding experiments, IL-1 alpha inhibited binding of 125I-IL-1 alpha with a Ki of 33-36 pM (n = 2) and binding of 125I-IL-1 beta with a Ki of 51-63 pM (n = 2). IL-1 beta inhibited binding of 125I-IL-1 alpha with a Ki of 2-3 pM (n = 2) and binding of 125I-IL-1 beta with a Ki of 7 pM (n = 2). The binding data were best fit by a model specifying a single class of receptors with homogeneous affinity for either IL-1 alpha or IL-1 beta and with an abundance of 3,000-14,000 sites per cell. Autoradiography showed that the vast majority of the synoviocytes within the cultures possessed IL-1 receptors. Comparison of biological response curves with the binding curves indicates that the observed receptors exhibit sufficiently high affinity to mediate the response of human synoviocytes to low picomolar concentrations of IL-1 alpha and IL-1 beta.