Dejan Nikolic

Class A scavenger receptors mediate cell adhesion via activation of Gi/o and formation of focal adhesion complexes

Class A macrophage scavenger receptors (SR-A) are multifunctional receptors with roles in modified lipoprotein uptake, innate immunity, and macrophage adhesion. Our previous studies conducted in mouse peritoneal macrophages demonstrated that pertussis toxin (PTX) mediated inhibition of Gi/o attenuated SR-A-dependent uptake of modified lipoprotein. The finding that SR-A-mediated lipoprotein internalization was PTX-sensitive led us to hypothesize that SR-A-mediated cell adhesion might be similarly regulated by Gi/o-dependent signaling pathways. To test this hypothesis, SR-A was expressed in HEK cells under inducible control. Relative to HEK cells that lack SR-A, SR-A expressing cells displayed enhanced adhesion to tissue culture dishes. SR-A-mediated adhesion was significantly reduced following PTX treatment and was insensitive to chelating divalent cations with EDTA. SR-A-expressing cells exhibited a distinct cell morphology characterized by fine filopodia-like projections. Both polymerized actin and vinculin were codistributed with SR-A in the filopodia-like projections indicating the formation of focal adhesion complexes. Overall, our results indicate that the ability of SR-A to enhance cell adhesion involves Gi/o activation and formation of focal adhesion complexes.

Class A Scavenger Receptor-mediated Macrophage Adhesion Requires Coupling of Calcium-independent Phospholipase A2 and 12/15-Lipoxygenase to Rac and Cdc42 Activation

Class A scavenger receptors (SR-A) participate in multiple macrophage functions including adhesion to modified extracellular matrix proteins present in various inflammatory disorders such as atherosclerosis and diabetes. By mediating macrophage adhesion to modified proteins and increasing macrophage retention, SR-A may contribute to the inflammatory process. Eicosanoids produced after phospholipase A2 (PLA2)-catalyzed release of arachidonic acid (AA) are important regulators of macrophage function and inflammatory responses. The potential roles of AA release and metabolism in SR-A-mediated macrophage adhesion were determined using macrophages adherent to modified protein. SR-A-dependent macrophage adhesion was abolished by selectively inhibiting calcium-independent PLA2 (iPLA2) activity and absent in macrophages isolated from iPLA2 β–/– mice. Our results further demonstrate that 12/15-lipoxygenase (12/15-LOX)-derived, but not cyclooxygenase- or cytochrome P450-dependent epoxygenase-derived AA metabolites, are specifically required for SR-A-dependent adhesion. Because of their role in regulating actin polymerization and cell adhesion, Rac and Cdc42 activation were also examined and shown to be increased via an iPLA2- and LOX-dependent pathway. Together, our results identify a novel role for iPLA2-catalyzed AA release and its metabolism by 12/15-LOX in coupling SR-A-mediated macrophage adhesion to Rac and Cdc42 activation.

Overexpression of Constitutively Active PKG‐I Protects Female, But Not Male Mice From Diet‐Induced Obesity

Cyclic guanosine monophosphate (cGMP)–dependent protein kinase I (PKG‐I) is a multifunctional protein. The direct effects of PKG‐I activation on energy homeostasis and obesity development are not well understood. Herein, we generated transgenic mice with expression of the constitutively active PKG‐I in adipose tissue as well as in other tissues. Male and female PKG‐I overexpressing mice were fed a low‐fat (LF) or high‐fat (HF) diet for 16 weeks. HF‐fed female PKG‐I transgenic mice had decreased body weight gain, lower percentage of body fat, and improved glucose tolerance compared to HF‐fed wild‐type (WT) controls. In contrast, male transgenic PKG‐I mice were not resistant to the development of HF‐diet‐induced obesity, and exhibited similar levels of adiposity and glucose intolerance as HF‐fed WT controls. Furthermore, we found that HF‐fed female transgenic PKG‐I mice had increased energy expenditure and cold‐induced adaptive thermogenesis compared to HF‐fed WT controls, which was associated with increased expression of uncoupling protein‐1 (UCP1) in brown adipose tissue (BAT). In addition, the rates of lipolysis in white adipose tissue (WAT) were also increased in female transgenic PKG‐I mice compared to WT controls due to increased phosphorylation of hormone‐sensitive lipase (HSL). However, in male mice, adaptive thermogenesis or WAT lipolysis was similar between transgenic PKG‐I mice and WT controls. Together, these data demonstrate sex differences in effects of PKG‐I activation on the regulation of adipose tissue function and its contribution to diet induced obesity.

SR-A ligand and M-CSF dynamically regulate SR-A expression and function in primary macrophages via p38 MAPK activation

BackgroundInflammation is characterized by dynamic changes in the expression of cytokines, such as M-CSF, and modifications of lipids and proteins that result in the formation of ligands for Class A Scavenger Receptors (SR-A). These changes are associated with altered SR-A expression in macrophages; however, the intracellular signal pathways involved and the extent to which SR-A ligands regulate SR-A expression are not well defined. To address these questions, SR-A expression and function were examined in resident mouse peritoneal macrophages incubated with M-CSF or the selective SR-A ligand acetylated-LDL (AcLDL).ResultsM-CSF increased SR-A expression and function, and required the specific activation of p38 MAPK, but not ERK1/2 or JNK. Increased SR-A expression and function returned to basal levels 72 hours after removing M-CSF. We next determined whether prolonged incubation of macrophages with SR-A ligand alters SR-A expression. In contrast to most receptors, which are down-regulated by chronic exposure to ligand, SR-A expression was reversibly increased by incubating macrophages with AcLDL. AcLDL activated p38 in wild-type macrophages but not in SR-A-/- macrophages, and p38 activation was specifically required for AcLDL-induced SR-A expression.ConclusionsThese results demonstrate that in resident macrophages SR-A expression and function can be dynamically regulated by changes in the macrophage microenvironment that are typical of inflammatory processes. In particular, our results indicate a previously unrecognized role for ligand binding to SR-A in up-regulating SR-A expression and activating p38 MAPK. In this way, SR-A may modulate inflammatory responses by enhancing macrophage uptake of modified protein/lipid, bacteria, and cell debris; and by regulating the production of inflammatory cytokines, growth factors, and proteolytic enzymes.

High glucose levels upregulate upstream stimulatory factor 2 gene transcription in mesangial cells

Previously, we demonstrated that upstream stimulatory factor 2 (USF2) mediates high glucose‐induced thrombospondin1 (TSP1) gene expression and TGF‐β activity in glomerular mesangial cells and plays a role in diabetic renal complications. In the present studies, we further determined the molecular mechanisms by which high glucose levels regulate USF2 gene expression. In primary rat mesangial cells, we found that glucose treatment time and dose‐dependently up‐regulated USF2 expression (mRNA and protein). By using cycloheximide to block the de novo protein synthesis, similar rate of USF2 degradation was found under either normal glucose or high glucose conditions. USF2 mRNA stability was not altered by high glucose treatment. Furthermore, high glucose treatment stimulated USF2 gene promoter activity. By using the luciferase‐promoter deletion assay, site‐directed mutagenesis, and transactivation assay, we identified a glucose‐responsive element in the USF2 gene promoter (−1,740 to −1,620, relative to the transcription start site) and demonstrated that glucose‐induced USF2 expression is mediated through a cAMP‐response element‐binding protein (CREB)‐dependent transactivation of the USF2 promoter. Furthermore, siRNA‐mediated CREB knock down abolished glucose‐induced USF2 expression. Taken together, these data indicate that high glucose levels up‐regulate USF2 gene transcription in mesangial cells through CREB‐dependent transactivation of the USF2 promoter. J. Cell. Biochem. 103: 1952–1961, 2007.

Regulation of Class A scavenger receptor-mediated cell adhesion and surface localization by PI3K: identification of a regulatory cytoplasmic motif

The importance of cytoplasmic motifs in differentially regulating SR‐A function was demonstrated by deleting the first 49 cytoplasmic aa (SR‐AΔ1–49), which abolished SR‐A‐mediated ligand internalization without reducing cell adhesion. To identify additional cytoplasmic motifs within the first 49 aa that regulate SR‐A function, the acidic residues in a conserved motif (EDAD) were changed to their amide derivatives (SR‐AQNAN). The function and regulation of SR‐AQNAN were compared with that of SR‐AΔ1–49 and SR‐A in transfected HEK‐293 cells. Blocking PI3K activation inhibited SR‐A, but not SR‐AΔ1–49‐ or SR‐AQNAN‐mediated cell adhesion. Although deleting (SR‐AΔ1–49) or mutating (SR‐AQNAN) the EDAD motif abolished the PI3K sensitivity of SR‐A‐mediated cell adhesion, these mutations did not affect ligand internalization or PI3K activation during cell adhesion. To define the mechanism by which PI3K regulates SR‐A‐mediated cell adhesion, the cellular localization of wild‐type and mutant SR‐A was examined. PI3K inhibition reduced surface localization of SR‐A but not of SR‐AΔ1–49 or SR‐AQNAN. The regulation of SR‐A surface localization by PI3K was confirmed in peritoneal macrophages, which endogenously express SR‐A. Together, these results suggest a pathway in which SR‐A binding to an immobilized ligand activates PI3K to recruit more receptor to the plasma membrane and enhances cell adhesion.

Activation of renal renin-angiotensin system in upstream stimulatory factor 2 transgenic mice

Previously we demonstrated that upstream stimulatory factor 2 (USF2) transgenic (Tg) mice developed nephropathy including albuminuria and glomerular hypertrophy, accompanied by increased transforming growth factor (TGF)-beta and fibronectin accumulation in the glomeruli. However, the mechanisms by which overexpression of USF2 induces kidney injury are unknown. USF has been shown to regulate renin expression. Moreover, the renin-angiotensin system (RAS) plays important roles in renal diseases. Therefore, in the present studies the effects of USF2 on the regulation of RAS in the kidney as well as in mesangial cells from USF2 (Tg) mice were examined. The role of USF2-mediated regulation of RAS in TGF-beta production in mesangial cells was also determined. Our data demonstrate that USF2 (Tg) mice exhibit increased renin and angiotensin (ANG) II levels in the kidney. In contrast, renal expression of other components of RAS such as renin receptor, angiotensinogen, angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1a (AT(1a)) receptor, and AT(2) receptor was not altered in USF2 (Tg) mice. Similarly, mesangial cells isolated from USF2 (Tg) mice had increased renin and ANG II levels. Mesangial cells overexpressing USF2 also had increased TGF-beta production, which was blocked by small interfering RNA-mediated renin gene knockdown or RAS blockade (enalapril or losartan). Collectively, these results suggest that USF2 promotes renal renin expression and stimulates ANG II generation, leading to activation of the intrarenal RAS. In addition, renin-dependent ANG II generation mediates the effect of USF2 on TGF-beta production in mesangial cells, which may contribute to the development of nephropathy in USF2 (Tg) mice.

Prostaglandins produced during class A scavenger receptor-mediated macrophage adhesion differentially regulate cytokine production

Inflammation is associated with modification of the extracellular environment, changes in cytokine expression, and the accumulation of immune cells. Such modifications create ligands that support SR‐A‐mediated macrophage adhesion and retention. This may be particularly important in settings, such as atherosclerosis and diabetes, as modified lipoproteins and gluc‐collagen are ligands for SR‐A. SR‐A‐mediated adhesion requires the PLA2‐dependent generation of AA and its metabolism by 12/15 LOX. In contrast, the inhibition of the COX‐dependent conversion of AA to PG had no effect on SR‐A‐mediated adhesion. In this study, macrophages were isolated from SR‐A+/+ and SR‐A−/− mice and plated on gluc‐collagen to test the hypothesis that COX‐derived PGs are produced during SR‐A‐mediated adhesion and regulate macrophage function. SR‐A‐mediated binding to gluc‐collagen induced a rapid but transient increase in PG production, which required the activation of PLA2 and Src kinase but not PI3K. SR‐A+/+ macrophages cultured on gluc‐collagen for 24 h secreted a similar amount of TNF‐α and 2.5‐fold more IL‐10 than SR‐A−/− macrophages. The inhibition of COX substantially increased TNF‐α production but reduced IL‐10 levels in SR‐A+/+ macrophages. These effects of COX inhibition were reversed by exogenous PGE2 and mimicked by specific antagonism of the EP4 receptor. Thus, in addition to the enhancement of macrophage adhesion, SR‐A binding to gluc‐collagen stimulates PG production, which in turn, differentially regulates the expression of inflammatory cytokines.

Development of an unbiased, semi-automated approach for classifying plasma cell immunophenotype following multicolor flow cytometry of bone marrow aspirates: OPTIMIZED APPROACH TO CLASSIFY PLASMA CELL PHENOTYPES

Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes.