Alexander V. Bocharov

Lipopolysaccharide Down Regulates Both Scavenger Receptor B1 and ATP Binding Cassette Transporter A1 in RAW Cells

ABSTRACT Lipopolysaccharide (LPS) has recently been shown to facilitate macrophage foam cell formation and has been suggested to be a proatherogenic factor. The mechanism of LPS induced cholesterol accumulation, however, is unclear. In this report, using the macrophage-like RAW 264.7 cell line, we provide experimental evidence that LPSs proatherogenic effects may at least in part reflect altered cholesterol metabolism. Data presented demonstrate that in a dose-dependent manner, LPS is able to down regulate the mRNA expression of the two primary high-density lipoprotein (HDL) receptors, scavenger receptor B1 (SR-B1) and ATP binding cassette A1 (ABCA1), with a 50% inhibitory concentration of less than 0.2 ng/ml, as well as to decrease SR-B1 protein expression by 80%. We also found that LPS treatment resulted in a significant decrease (to 20% of the control level) of the specific 125I-HDL binding as well as in 50% inhibition of the HDL-mediated cholesterol efflux compared to untreated cells. In addition, we compared the potencies of various modified LPS preparations and demonstrated that the phosphorylated lipid A portion of LPS, which is highly conserved among gram-negative microorganisms, including Chlamydia, is primarily responsible for the effects of LPS on SR-B1 and ABCA1 expression. Inhibitors of NF-κB activation were observed to efficiently block the suppressive effect of LPS on SR-B1 and ABCA1, suggesting a mechanism involving NF-κB. These data indicate that the LPS effects on cholesterol metabolism may contribute to the proatherogenic properties of LPS.

Role of Human CD36 in Bacterial Recognition, Phagocytosis, and Pathogen-Induced JNK-Mediated Signaling

Scavenger receptor CD36 mediates Staphylococcus aureus phagocytosis and initiates TLR2/6 signaling. We analyzed the role of CD36 in the uptake and TLR-independent signaling of various bacterium, including Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, S. aureus, and Enterococcus faecalis. Expression of human CD36 in HeLa cells increased the uptake of both Gram-positive and Gram-negative bacteria compared with the control mock-transfected cells. Bacterial adhesion was associated with pathogen phagocytosis. Upon CD36 transfection, HEK293 cells, which demonstrate no TLR2/4 expression, acquired LPS responsiveness as assessed by IL-8 production. The cells demonstrated a marked 5- to 15-fold increase in cytokine release upon exposure to Gram-negative bacteria, while the increase was much smaller (1.5- to 3-fold) with Gram-positive bacteria and lipoteichoic acid. CD36 down-regulation utilizing CD36 small interfering RNA reduced cytokine release by 40–50% in human fibroblasts induced by both Gram-negative and Gram-positive bacteria as well as LPS. Of all MAPK signaling cascade inhibitors tested, only the inhibitor of JNK, a stress-activated protein kinase, potently blocked E. coli/LPS-stimulated cytokine production. NF-κB inhibitors were ineffective, indicating direct TLR-independent signaling. JNK activation was confirmed by Western blot analyses of phosphorylated JKN1/2 products. Synthetic amphipathic peptides with an α-helical motif were shown to be efficient inhibitors of E. coli- and LPS-induced IL-8 secretion as well as JNK1/2 activation/phosphorylation in CD36-overexpressing cells. These results indicate that CD36 functions as a phagocytic receptor for a variety of bacteria and mediates signaling induced by Gram-negative bacteria and LPS via a JNK-mediated signaling pathway in a TLR2/4-independent manner.

Parkin is a lipid-responsive regulator of fat uptake in mice and mutant human cells

It has long been hypothesized that abnormalities in lipid biology contribute to degenerative brain diseases. Consistent with this, emerging epidemiologic evidence links lipid alterations with Parkinson disease (PD), and disruption of lipid metabolism has been found to predispose to α-synuclein toxicity. We therefore investigated whether Parkin, an E3 ubiquitin ligase found to be defective in patients with early onset PD, regulates systemic lipid metabolism. We perturbed lipid levels by exposing Parkin+/+ and Parkin-/- mice to a high-fat and -cholesterol diet (HFD). Parkin-/- mice resisted weight gain, steatohepatitis, and insulin resistance. In wild-type mice, the HFD markedly increased hepatic Parkin levels in parallel with lipid transport proteins, including CD36, Sr-B1, and FABP. These lipid transport proteins were not induced in Parkin-/- mice. The role of Parkin in fat uptake was confirmed by increased oleate accumulation in hepatocytes overexpressing Parkin and decreased uptake in Parkin-/- mouse embryonic fibroblasts and patient cells harboring complex heterozygous mutations in the Parkin-encoding gene PARK2. Parkin conferred this effect, in part, via ubiquitin-mediated stabilization of the lipid transporter CD36. Reconstitution of Parkin restored hepatic fat uptake and CD36 levels in Parkin-/- mice, and Parkin augmented fat accumulation during adipocyte differentiation. These results demonstrate that Parkin is regulated in a lipid-dependent manner and modulates systemic fat uptake via ubiquitin ligase-dependent effects. Whether this metabolic regulation contributes to premature Parkinsonism warrants investigation.

CD36 Is a Novel Serum Amyloid A (SAA) Receptor Mediating SAA Binding and SAA-induced Signaling in Human and Rodent Cells

Serum amyloid A (SAA) is a major acute phase protein involved in multiple physiological and pathological processes. This study provides experimental evidence that CD36, a phagocyte class B scavenger receptor, functions as a novel SAA receptor mediating SAA proinflammatory activity. The uptake of Alexa Fluor® 488 SAA as well as of other well established CD36 ligands was increased 5–10-fold in HeLa cells stably transfected with CD36 when compared with mock-transfected cells. Unlike other apolipoproteins that bind to CD36, only SAA induced a 10–50-fold increase of interleukin-8 secretion in CD36-overexpressing HEK293 cells when compared with control cells. SAA-mediated effects were thermolabile, inhibitable by anti-SAA antibody, and also neutralized by association with high density lipoprotein but not by association with bovine serum albumin. SAA-induced cell activation was inhibited by a CD36 peptide based on the CD36 hexarelin-binding site but not by a peptide based on the thrombospondin-1-binding site. A pronounced reduction (up to 60–75%) of SAA-induced pro-inflammatory cytokine secretion was observed in cd36−/− rat macrophages and Kupffer cells when compared with wild type rat cells. The results of the MAPK phosphorylation assay as well as of the studies with NF-κB and MAPK inhibitors revealed that two MAPKs, JNK and to a lesser extent ERK1/2, primarily contribute to elevated cytokine production in CD36-overexpressing HEK293 cells. In macrophages, four signaling pathways involving NF-κB and three MAPKs all appeared to contribute to SAA-induced cytokine release. These observations indicate that CD36 is a receptor mediating SAA binding and SAA-induced pro-inflammatory cytokine secretion predominantly through JNK- and ERK1/2-mediated signaling.

CLA-1 and its splicing variant CLA-2 mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells

CD36 and LIMPII analog 1, CLA-1, and its splicing variant, CLA-2 (SR-BI and SR-BII in rodents), are human high density lipoprotein receptors with an identical extracellular domain which binds a spectrum of ligands including bacterial cell wall components. In this study, CLA-1- and CLA-2-stably transfected HeLa and HEK293 cells demonstrated several-fold increases in the uptake of various bacteria over mock-transfected cells. All bacteria tested, including both Gram-negatives (Escherichia coli K12, K1 and Salmonella typhimurium) and Gram-positives (Staphylococcus aureus and Listeria monocytogenes), demonstrated various degrees of lower uptake in control cells. This result is consistent with the presence of high-density lipoprotein-receptor-independent bacterial uptake that is enhanced by CLA-1/CLA-2 overexpression. Bacterial lipopolysaccharides, lipoteichoic acid, and synthetic amphipathic helical peptides (L-37pA and D-37pA) competed with E. coli K12 for CLA-1 and CLA-2 binding. Transmission electron microscopy and confocal microscopy revealed cytosolic accumulation of bacteria in CLA-1/CLA-2-overexpressing HeLa cells. The antibiotic protection assay confirmed that E. coli K12 was able to survive and replicate intracellularly in CLA-1- and CLA-2-overexpressing HeLa, but both L-37pA and D-37pA prevented E. coli K12 invasion. Peritoneal macrophages isolated from SR-BI/BII-knockout mice demonstrated a 30% decrease in bacterial uptake when compared with macrophages from normal mice. Knockout macrophages were also characterized by decreased bacterial cytosolic invasion, ubiquitination, and proteasome mobilization while retaining bacterial lysosomal accumulation. These results indicate that, by facilitating bacterial adhesion and cytosolic invasion, CLA-1 and CLA-2 may play an important role in infection and sepsis.

TLR4 mutant mice are protected from renal fibrosis and chronic kidney disease progression

Chronic kidney disease (CKD) is associated with persistent low‐grade inflammation and immunosuppression. In this study we tested the role of Toll‐like receptor 4, the main receptor for endotoxin (LPS), in a mouse model of renal fibrosis and in a model of progressive CKD that better resembles the human disease. C3HeJ (TLR4 mutant) mice have a missense point mutation in the TLR4 gene, rendering the receptor nonfunctional. In a model of renal fibrosis after folic acid injection, TLR4 mutant mice developed less interstititial fibrosis in comparison to wild‐type (WT) mice. Furthermore, 4 weeks after 5/6 nephrectomy with continuous low‐dose angiotensin II infusion, C3HeOuJ (TLR4 WT) mice developed progressive CKD with albuminuria, increased serum levels of BUN and creatinine, glomerulosclerosis, and interstitial fibrosis, whereas TLR4 mutant mice were significantly protected from CKD progression. TLR4 WT mice also developed low‐grade systemic inflammation, splenocyte apoptosis and increased expression of the immune inhibitory receptor PD‐1 in the spleen, which were not observed in TLR4 mutant mice. In vitro, endotoxin (LPS) directly upregulated NLRP3 inflammasome expression in renal epithelial cells via TLR4. In summary, TLR4 contributes to renal fibrosis and CKD progression, at least in part, via inflammasome activation in renal epithelial cells, and may also participate in the dysregulated immune response that is associated with CKD.

Endothelial Expression of Scavenger Receptor Class B, Type I Protects against Development of Atherosclerosis in Mice.

The role of scavenger receptor class B, type I (SR-BI) in endothelial cells (EC) was examined in several novel transgenic mouse models expressing SR-BI in endothelium of mice with normal C57Bl6/N, apoE-KO, or Scarb1-KO backgrounds. Mice were also created expressing SR-BI exclusively in endothelium and liver. Endothelial expression of the Tie2-Scarb1 transgene had no significant effect on plasma lipoprotein levels in mice on a normal chow diet but on an atherogenic diet, significantly decreased plasma cholesterol levels, increased plasma HDL cholesterol (HDL-C) levels, and protected mice against atherosclerosis. In 8-month-old apoE-KO mice fed a normal chow diet, the Tie2-Scarb1 transgene decreased aortic lesions by 24%. Mice expressing SR-BI only in EC and liver had a 1.5 ± 0.1-fold increase in plasma cholesterol compared to mice synthesizing SR-BI only in liver. This elevation was due mostly to increased HDL-C. In EC culture studies, SR-BI was found to be present in both basolateral and apical membranes but greater cellular uptake of cholesterol from HDL was found in the basolateral compartment. In summary, enhanced expression of SR-BI in EC resulted in a less atherogenic lipoprotein profile and decreased atherosclerosis, suggesting a possible role for endothelial SR-BI in the flux of cholesterol across EC.

Hypermutation induced by APOBEC-1 overexpression can be eliminated

APOBEC-1 overexpression in liver has been shown to effectively reduce apoB-100 levels. However, nonspecific hypermutation and liver tumor formation potentially related to hypermutation in transgenic animals compromise its potential use for gene therapy. In studying apoB mRNA editing regulation, we found that the core editing auxiliary factor ACF dose-dependently increases APOBEC-1 nonspecific hypermutation and specific editing with variable site sensitivity. Overexpression of APOBEC-1 together with ACF in human hepatic HepG2 cells hypermutated apoB mRNAs 20%-65% at sites 6639, 6648, 6655, 6762, 6802, and 6845, in addition to the normal 90% editing at 6666. The hypermutation activity of APOBEC-1 was decreased to background levels by a single point APOBEC-1 mutation of P29F or E181Q, while 50% of wild-type control editing at the normal site was retained. The hypermutations on both apoB and novel APOBEC-1 target 1 (NAT1) mRNA were also decreased to background levels with P29F and E181Q mutants in rat liver primary culture cells. The loss of hypermutation with the mutants was associated with significantly decreased APOBEC-1/ACF interaction. These data suggest that nonspecific hypermutation induced by overexpressing APOBEC-1 can be virtually eliminated by site-specific mutation, while maintaining specific editing activity at the normal site, reopening the potential use of APOBEC-1 gene therapy for hyperlipidemia.

Correction of acute hepatic failure by hepatocytes cultured on microcarriers

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Human SR-BII mediates SAA uptake and contributes to SAA pro-inflammatory signaling in vitro and in vivo

Serum amyloid A (SAA) is an acute phase protein with cytokine-like and chemotactic properties, that is markedly up-regulated during various inflammatory conditions. Several receptors, including FPRL-1, TLR2, TLR4, RAGE, class B scavenger receptors, SR-BI and CD36, have been identified as SAA receptors. This study provides new evidence that SR-BII, splice variant of SR-BI, could function as an SAA receptor mediating its uptake and pro-inflammatory signaling. The uptake of Alexa Fluor488 SAA was markedly (~3 fold) increased in hSR-BII-expressing HeLa cells when compared with mock-transfected cells. The levels of SAA-induced interleukin-8 secretion by hSR-BII-expressing HEK293 cells were also significantly (~3–3.5 fold) higher than those detected in control cells. Moderately enhanced levels of phosphorylation of all three mitogen-activated protein kinases, ERK1/2, and p38 and JNK, were observed in hSR-BII-expressing cells following SAA stimulation when compared with control wild type cells. Transgenic mice with pLiv-11-directed liver/kidney overexpression of hSR-BI or hSR-BII were used to assess the in vivo role of each receptor in SAA-induced pro-inflammatory response in these organs. Six hours after intraperitoneal SAA injection both groups of transgenic mice demonstrated markedly higher (~2-5-fold) expression levels of inflammatory mediators in the liver and kidney compared to wild type mice. Histological examinations of hepatic and renal tissue from SAA-treated mice revealed moderate level of damage in the liver of both transgenic but not in the wild type mice. Activities of plasma transaminases, biomarkers of liver injury, were also moderately higher in hSR-B transgenic mice when compared to wild type mice. Our findings identify hSR-BII as a functional SAA receptor that mediates SAA uptake and contributes to its pro-inflammatory signaling via the MAPKs-mediated signaling pathways.