The α1A-Adrenoceptor couples to Ca2+ mobilization in mouse peritoneal macrophages

Activation of the α₁-adrenoceptor (α₁-AR) in mouse peritoneal macrophages induces a Ca²⁺-dependent K⁺ current via release of Ca²⁺ from internal stores [Hara et al. (1991) Pflügers Arch 419: 371–379]. In order to characterize the α₁-AR subtype in macrophages, we examined potencies of the α₁-AR subtype-selective antagonists against epinephrine-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i). The K_i values for the α₁-AR in macrophages of WB4101, 5-methylurapidil and phentolamine were 0.13 nM, 0.15 nM and 7.0 nM, respectively. (+)-Niguldipine (10 nM) completely blocked the response. Treatment with chloroethylclonidine (CEC) did not abolish the response. These pharmacological properties of the α₁-AR in macrophages agree quite well with those of the α_1A subtype.     

The effects of TGF-β1 on the expression of type IV collagenases in mouse peritoneal macrophages

Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that plays a critical role in modulating immune response and inflammation. We have investigated the effects of TGF-β1 on the expression of type IV collagenases, matrix metalloproteinase (MMP)-2 and MMP-9, in mouse peritoneal macrophages. TGF-β1 alone enhanced the secretion of MMP-9, while it blocked lipopolysaccharide (LPS)-stimulated MMP-9 production. We have shown that this biphasic effect of TGF-β1 is exerted at the mRNA level of the MMP-9 gene. Although TGF-β1 increased both basal and LPS-induced MMP-2 production at the protein and mRNA levels, the extent of the increase was smaller in LPS-activated macrophages than in control macrophages. The expression of type I and type II receptors for TGF-β was significantly decreased upon activation, suggesting that the lesser effect of TGF-β1 in activated macrophages may result from the decreased expression of TGF-β receptors. In addition, the expression of endogenous TGF-β1 mRNA was decreased significantly in activated macrophages. These findings suggest that activated macrophages not only produce less TGF-β1, but also respond less well to TGF-β to provide for inflammatory response.     

Secretion of IL-1β triggered by dynasore in murine peritoneal macrophages

The interaction of lipopolysaccharide-primed murine peritoneal macrophages with ivermectin, an antiparasite drug which potentiates P2X(4) receptors and dynasore which inhibits the GTPase activity of dynamin, a protein contributing to the internalization of plasma membrane proteins, was tested. Murine peritoneal macrophages express P2X(4) receptors which are mostly intracellular. In cells from P2X(7)-knockout mice (KO mice), 10 μm adenosine triphosphate (ATP) provoked a transient increase of the intracellular concentration of calcium. Ivermectin had no effect by itself but potentiated the increase of the intracellular concentration of calcium by ATP. The combination of ATP plus ivermectin also decreased the intracellular concentration of potassium and promoted the secretion of IL-1β. Concentrations of dynasore above 50?μm affected the integrity of mitochondria (MTT test) and of the plasma membrane (release of lactate dehydrogenase, LDH). At a 10 μm concentration, dynasore had no effect on the responses to ATP and on the internalization of P2X(4) receptors. By itself dynasore promoted the release of potassium and the secretion of IL-1β after activation of caspase-1. In conclusion, our results confirm that ivermectin potentiates the responses coupled to P2X(4) receptors probably by interaction with an allosteric site. We also show that this potentiation triggers the release of IL-1β by macrophages. As opposed to ivermectin, dynasore has no effect on P2X(4) receptors. This drug triggers a potassium efflux via a mechanism which does not involve purinergic receptors and generates, in consequence, the activation of caspase-1 and the secretion of IL-1β.     

Heterogeneity in 5′-nucleotidase activity of mouse peritoneal macrophages

Summary After stimulation of the mouse peritoneal cavity with newborn calf serum (NBCS), four types of monocyte and macrophage were distinguished on the basis of peroxidase (PO) patterns. Cytochemically, these cells showed strong heterogeneity in 5-nucleotidase (5N) activity. Monocytes and monocyte-derived macrophages with PO activity in granules lacked 5N activity. Resident macrophages (with PO activity in RER and nuclear envelope) generally had significant 5N activity on the plasma membrane, the pattern showing close correlation with the biochemical findings. The group of PO-negative macrophages comprised both 5N-negative and 5N-positive cells. These findings suggest two possibilities, i.e., that monocytes (5N-)transform via PO-negative cells (5N-/+) into resident macrophages (5N+), or that the monocytes and monocyte-derived macrophages and the resident macrophages represent separate lineages. The fourth type of macrophage, the exudate-resident cell (with PO activity both in granules and in the RER and nuclear envelope), occurred only in low numbers and very late after NBCS stimulation, and is therefore considered not to be a transitional cell between monocytes and resident macrophages.     

PPARγ stabilizes HO-1 mRNA in monocytes/macrophages which affects IFN-β expression

NADPH oxidase activation in either RAW264.7 cells or peritoneal macrophages (PM) derived from PPARγ wild-type mice increased reactive oxygen species (ROS) formation, caused PPARγ activation, heme oxygenase-1 (HO-1) induction, and concomitant IFN-β expression. In macrophages transduced with a dominant negative (d/n) mutant of PPARγ (RAW264.7 AF2) as well as PPARγ negative PM derived from Mac-PPARγ-KO mice, NADPH oxidase-dependent IFN-β expression was attenuated. As the underlying mechanism, we noted decreased HO-1 mRNA stability in RAW264.7 AF2 cells as well as PPARγ negative PM, compared to either parent RAW264.7 cells or wild-type PM. Assuming mRNA stabilization of HO-1 by PPARγ we transfected macrophages with a HO-1 3′-UTR reporter construct. The PPARγ agonist rosiglitazone significantly up-regulated luciferase expression in RAW264.7 cells, while it remained unaltered in RAW264.7 AF2 macrophages. Deletion of each of two AU-rich elements in the 3′-UTR HO-1 decreased luciferase activity in RAW264.7 cells. Using LPS as a NADPH oxidase activator, PM from Mac-PPARγ-KO mice showed a decreased HO-1 mRNA half-life in vitro and in vivo compared to PPARγ wild-type mice. These data identified a so far unappreciated role of PPARγ in stabilizing HO-1 mRNA, thus, contributing to the expression of the HO-1 target gene IFN-β.     

TNFα and IL-1β are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

A growing body of evidence recently suggests that glial cell activation plays an important role in several neurodegenerative diseases and neuropathic pain. Microglia in the central nervous system express toll-like receptor 4 (TLR4) that is traditionally accepted as the primary receptor of lipopolysaccharide (LPS). LPS activates TLR4 signaling pathways to induce the production of proinflammatory molecules. In the present studies, we verified the LPS signaling pathways using cultured highly aggressively proliferating immortalized (HAPI) microglial cells. We found that HAPI cells treated with LPS upregulated the expression of TLR4, phospho-JNK (pJNK) and phospho-NF-κB (pNF-κB), TNFα and IL-1β. Silencing TLR4 with siRNA reduced the expression of pJNK, TNFα and IL-1β, but not pNF-κB in the cells. Inhibition of JNK with SP600125 (a JNK inhibitor) decreased the expression of TNFα and IL-1β. Unexpectedly, we found that inhibition of Nod1 with ML130 significantly reduced the expression of pNF-κB. Inhibition of NF-κB also reduced the expression of TNFα and IL-1β. Nod1 ligand, DAP induced the upregulation of pNF-κB which was blocked by Nod1 inhibitor. These data indicate that LPS-induced pJNK is TLR4-dependent, and that pNF-κB is Nod1-dependent in HAPI cells treated with LPS. Either TLR4-JNK or Nod1-NF-κB pathways is involved in the expression of TNFα and IL-1β.     

β-defensin-3 negatively regulates TLR4–HMGB1 axis mediated HLA-G expression in IL-1β treated glioma cells

The non-classical HLA class I antigen HLA-G contributes to immune escape mechanisms in glioblastoma multiforme (GBM). We have previously shown that IL-1β–HIF-1α axis connects inflammatory and oncogenic pathways in GBM. In this study, we investigated the role of IL-1β induced inflammation in regulating HLA-G expression. IL-1β increased HLA-G and Toll like receptor 4 (TLR4) expression in a HIF-1α dependent manner. Inhibition of TLR4 signaling abrogated IL-1β induced HLA-G. IL-1β increased HMGB1 expression and its interaction with TLR4. Inhibition of HMGB1 inhibited TLR4 and vice versa suggesting the existence of HMGB1–TLR4 axis in glioma cells. Interestingly, HMGB1 inhibition prevented IL-1β induced HLA-G expression. Elevated levels of HMGB1 and β-defensin 3 were observed in GBM tumors. Importantly, β-defensin-3 prevented IL-1β induced HLA-G, TLR4, HMGB1 expression and release of pro-inflammatory mediators. Our studies indicate that β-defensin-3 abrogates IL-1β induced HLA-G expression by negatively affecting key molecules associated with its regulation.     

Tumor-produced, active interleukin-1β regulates gene expression in carcinoma-associated fibroblasts

Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial–mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1β (IL1-β) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-β expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-β processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-β. IL1-β signaling was investigated by western blot and immunocytochemistry. IL1-β-regulated genes were analyzed by real-time qPCR.SCC-25 cells produced 16 kD active IL1-β, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NFκBα. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-β reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-β-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-β in the tumor cells leads to IL1-β-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression.     

17β-estradiol downregulates angiotensin-II-induced endothelin-1 gene expression in rat aortic smooth muscle cells

It is well documented that 17-estradiol (E₂) exerts a cardiovascular protective effect. A possible role of E₂ in the regulation of endothelin-1 (ET-1) production has been reported. However, the complex mechanisms by which E₂ inhibits ET-1 expression are not completely understood. The aims of this study were to examine whether E₂ may alter angiotensin II (Ang II)-induced cell proliferation and ET-1 gene expression and to identify the putative underlying signaling pathways in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with E₂, then stimulated with Ang II, and [³H]thymidine incorporation and ET-1 gene expression were examined. The effect of E₂ on Ang-II-induced extracellular signal-regulated kinase (ERK) phosphorylation was tested to elucidate the intracellular mechanism of E₂ in proliferation and ET-1 gene expression. Ang II increased DNA synthesis which was inhibited with E₂ (1–100 nM). E₂, but not 17-estradiol, inhibited the Ang-II-induced ET-1 gene expression as revealed by Northern blotting and promoter activity assay. This effect was prevented by coincubation with the estrogen receptor antagonist ICl 182,780 (1 µM). E₂ also inhibited Ang-II-increased intracellular reactive oxygen species (ROS) as measured by a redox-sensitive fluorescent dye, 2,7-dichlorofluorescin diacetate, and ERK phosphorylation. Furthermore, E₂ and antioxidants, such as N-acetyl cysteine and diphenylene iodonium, decreased Ang-II-induced cell proliferation, ET-1 promoter activity, ET-1 mRNA, ERK phosphorylation, and activator protein-1-mediated reporter activity. In summary, our results suggest that E₂ inhibits Ang-II-induced cell proliferation and ET-1 gene expression, partially by interfering with the ERK pathway via attenuation of ROS generation. Thus, this study provides important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of estrogen on the cardiovascular system.     

Resveratrol induces insulin gene expression in mouse pancreatic α-cells

Background

Type 1 and type 2 diabetes are characterized by loss of β-cells; therefore, β-cell regeneration has become one of the primary approaches to diabetes therapy. Resveratrol, a naturally occurring polyphenolic compound, has been shown to improve glycaemic control in diabetic patients, but its action on pancreatic α-cells is not well understood.

Findings

Using mouse α-cells (αTC9), we showed that resveratrol induces expression of pancreatic β-cell genes such as Pdx1 and Ins2 in a SirT1-dependent manner. The mRNA and protein levels of insulin were further increased by histone deacetylase (HDAC) inhibition.

Conclusion

In summary, we provide new mechanistic insight into the anti-diabetic action of resveratrol through its ability to express β-cell genes in α-cells.

     

Association of IL1β and IL4 gene polymorphisms with nasal polyps in a Polish population

Imbalance between proinflammatory and anti-inflammatory cytokines may regulate the inflammatory reaction in the nasal polyps. Polymorphisms in the regulatory regions of the cytokines genes may influence their expression. The aim of this study was to investigate the relationship between an IL-1β and IL-4 promoter polymorphisms and nasal polyps. The C-511T promoter polymorphism of the IL-1β gene and C-590T promoter polymorphism of the IL-4 gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis in 208 Polish patients with nasal polyps and 200 healthy Polish subjects. The risk of susceptibility to NP was significantly higher in patients with NP who had ?511 T/T genotype of IL1β than in controls (OR 3.07; 95 % CI 1.18–7.99). No statistically significant differences were found between NP patients and the control group with regard to genotype distribution and allele frequencies of C/T polymorphism of IL4 gene. Our study demonstrated that the TT genotype for C-511T mutation associated with the risk of developing NP in a Polish population.     

β1-Adrenergic receptor downregulates the expression of cyclooxygenase-2

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the generation of prostanoids, and is thus one of the key players in the inflammatory process. Contrary to the constitutively expressed isoform COX-1, the expression of COX-2 is rapidly and transiently upregulated following pathological stimuli but little is known about pathways that mediate its degradation. Here we show that co-expression of COX-2 together with the β₁ adrenergic receptor (β₁AR) specifically lowers the expression of COX-2 in a dose-dependent manner. We further find that stimulation of the receptor for prolonged periods of time does not reverse the β₁AR-induced decrease in COX-2, suggesting that this effect does not occur via classical β₁-mediated signaling pathways. Rather we find that the half-life of COX-2 is significantly decreased in the presence of β₁AR and that inhibition of the proteasome reverses the effect of the receptor on COX-2. Together these findings ascribe a new role for β₁AR in the downregulation of COX-2.     

OxLDL up-regulates Niemann-Pick type C1 expression through ERK1/2/COX-2/PPARα-signaling pathway in macrophages

The Niemann-Pick type C1 (NPC1) is located mainly in the membranes of the late endosome/lysosome and controls the intracellular cholesterol trafficking from the late endosome/lysosome to the plasma membrane. It has been reported that oxidized low-density lipoprotein (oxLDL) can up-regulate NPC1 expression. However, the detailed mechanisms are not fully understood. In this study, we investigated the effect of oxLDL stimulation on NPC1 expression in THP-1 macrophages. Our results showed that oxLDL up-regulated NPC1 expression at both mRNA and protein levels in a dose-dependent and time-dependent manner. In addition, oxLDL also induced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Treatment with oxLDL significantly increased cyclooxygenase-2 (COX-2) mRNA and protein expression in the macrophages, and these increases were suppressed by the ERK1/2 inhibitor PD98059 or ERK1/2 small interfering RNA (siRNA) treatment. OxLDL up-regulated the expression of peroxisome proliferator-activated receptor α (PPARα) at the mRNA and protein levels, which could be abolished by COX-2 siRNA or COX-2 inhibitor NS398 treatment in these macrophages. OxLDL dramatically elevated cellular cholesterol efflux, which was abrogated by inhibiting ERK1/2 and/or COX-2. In addition, oxLDL-induced NPC1 expression and cellular cholesterol efflux were reversed by PPARα siRNA or GW6471, an antagonist of PPARα. Taken together, these results provide the evidence that oxLDL can up-regulate the expression of the NPC1 through ERK1/2/COX-2/PPARα-signaling pathway in macrophages.