Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1

Lysophosphatidic acid (LPA) is an agonist for peroxisome proliferator activated receptor-γ (PPARγ). Although glycerol-3-phosphate acyltransferase-1 (GPAT1) esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO) cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA) or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.     

Type I interferon-mediated pathway interacts with peroxisome proliferator activated receptor-γ (PPAR-γ): At the cross-road of pancreatic cancer cell proliferation

Pancreatic adenocarcinoma remains an unresolved therapeutic challenge because of its intrinsically refractoriness to both chemo- and radiotherapy due to the complexity of signaling and the activation of survival pathways in cancer cells. Recent studies have demonstrated that the combination of some drugs, targeting most of aberrant pathways crucial for the survival of pancreatic cancer cells may be a valid antitumor strategy for this cancer. Type I interferons (IFNs) may have a role in the pathogenesis and progression of pancreatic adenocarcinoma, but the limit of their clinical use is due to the activation of tumor resistance mechanisms, including JAK-2/STAT-3 pathway. Moreover, aberrant constitutive activation of STAT-3 proteins has been frequently detected in pancreatic adenocarcinoma. The selective targeting of these cell survival cascades could be a promising strategy in order to enhance the antitumor effects of type I IFNs. The activation of peroxisome proliferator-activated receptor γ (PPAR-γ), on the other hand, has a suppressive activity on STAT-3. In fact, PPAR-γ agonists negatively modulate STAT-3 through direct and/or indirect mechanisms in several normal and cancer models. This review provides an overview on the current knowledge about the molecular mechanisms and antitumor activity of these two promising classes of drugs for pancreatic cancer therapy. Finally, the synergistic antiproliferative activity of combined IFN-β and troglitazone treatment on pancreatic cancer cell lines, evaluated in vitro, and the consequent potential clinical applications will be discussed.     

Mechanisms of divergent effects of activated peroxisome proliferator-activated receptor-γ on mitochondrial citrate carrier expression in 3T3-L1 fibroblasts and mature adipocytes

The citrate carrier (CIC), a nuclear-encoded protein located in the mitochondrial inner membrane, plays an important metabolic role in the transport of acetyl-CoA from the mitochondrion to the cytosol in the form of citrate for fatty acid and cholesterol synthesis. Citrate has been reported to be essential for fibroblast differentiation into fat cells. Because peroxisome proliferator-activated receptor-gamma (PPARγ) is known to be one of the master regulators of adipogenesis, we aimed to study the regulation of CIC by the PPARγ ligand rosiglitazone (BRL) in 3T3-L1 fibroblasts and in adipocytes. We demonstrated that BRL up-regulated CIC mRNA and protein levels in fibroblasts, while it did not elicit any effects in mature adipocytes. The enhancement of CIC levels upon BRL treatment was reversed using the PPARγ antagonist GW9662, addressing how this effect was mediated by PPARγ. Functional experiments using a reporter gene containing rat CIC promoter showed that BRL enhanced CIC promoter activity. Mutagenesis studies, electrophoretic-mobility-shift assay and chromatin-immunoprecipitation analysis revealed that upon BRL treatment, PPARγ and Sp1 are recruited on the Sp1-containing region within the CIC promoter, leading to an increase in CIC expression. In addition, mithramycin, a specific inhibitor for Sp1-DNA binding activity, abolished the PPARγ-mediated up-regulation of CIC in fibroblasts. The stimulatory effects of BRL disappeared in mature adipocytes in which PPARγ/Sp1 complex recruited SMRT corepressor to the Sp1 site of the CIC promoter. Taken together, our results contribute to clarify the molecular mechanisms by which PPARγ regulates CIC expression during the differentiation stages of fibroblasts into mature adipocytes.     

Lipid synthesis in macrophages during inflammation in vivo: Effect of agonists of peroxisome proliferator activated receptors α and γ and of retinoid X receptors

The effects of peroxisome proliferator activated receptors α and γ (PPAR-α and PPAR-γ) and retinoid X receptor (RXR) agonists upon synthesis and accumulation of lipids in murine C57B1 macrophages during inflammation induced by injection of zymosan and Escherichia coli lipopolysaccharide (LPS) have been studied. It is significant that intraperitoneal injection of zymosan (50 mg/kg) or LPS (0.1 mg/kg) in mice led to a dramatic increase of [¹⁴C]oleate incorporation into cholesteryl esters and triglycerides and [¹⁴C]acetate incorporation into cholesterol and fatty acids in peritoneal macrophages. Lipid synthesis reached its maximum rate 18–24 h after injection and was decreased 5–7 days later to control level after LPS injection or was still heightened after zymosan injection. In macrophages obtained in acute phase of inflammation (24 h), degradation of ¹²⁵I-labeled native low density lipoprotein (NLDL) was 4-fold increased and degradation of ¹²⁵I-labeled acetylated LDL (AcLDL) was 2–3-fold decreased. Addition of NLDL (50 μg/ml) or AcLDL (25 μg/ml) into the incubation medium of activated macrophages induced 9–14-and 1.25-fold increase of cholesteryl ester synthesis, respectively, compared with control. Addition of NLDL and AcLDL into the incubation medium completely inhibited cholesterol synthesis in control macrophages but had only slightly effect on cholesterol synthesis in activated macrophages. Injection of RXR, PPAR-α, or PPAR-γ agonists—9-cis-retinoic acid (5 mg/kg), bezafibrate (10 mg/kg), or rosiglitazone (10 mg/kg), respectively—30 min before zymosan or LPS injection led to significant decrease of lipid synthesis. Ten hour preincubation of activated in vivo macrophages with the abovementioned agonists (5 μM) decreased cholesteryl ester synthesis induced by NLDL and AcLDL addition into the cell cultivation medium. The data suggest that RXR, PPAR-α, or PPAR-γ agonists inhibited lipid synthesis and induction of cholesteryl ester synthesis in inflammatory macrophages caused by capture of native or modified LDL. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 364–374.     

Different skeletal effects of the peroxisome proliferator activated receptor (PPAR)α agonist fenofibrate and the PPARγ agonist pioglitazone

Background

All the peroxisome proliferator activated receptors (PPARs) are found to be expressed in bone cells. The PPARγ agonist rosiglitazone has been shown to decrease bone mass in mice and thiazolidinediones (TZDs) have recently been found to increase bone loss and fracture risk in humans treated for type 2 diabetes mellitus. The aim of the study was to examine the effect of the PPARα agonist fenofibrate (FENO) and the PPARγ agonist pioglitazone (PIO) on bone in intact female rats.

Methods

Rats were given methylcellulose (vehicle), fenofibrate or pioglitazone (35 mg/kg body weight/day) by gavage for 4 months. BMC, BMD, and body composition were measured by DXA. Histomorphometry and biomechanical testing of excised femurs were performed. Effects of the compounds on bone cells were studied.

Results

The FENO group had higher femoral BMD and smaller medullary area at the distal femur; while trabecular bone volume was similar to controls. Whole body BMD, BMC, and trabecular bone volume were lower, while medullary area was increased in PIO rats compared to controls. Ultimate bending moment and energy absorption of the femoral shafts were reduced in the PIO group, while similar to controls in the FENO group. Plasma osteocalcin was higher in the FENO group than in the other groups. FENO stimulated proliferation and differentiation of, and OPG release from, the preosteoblast cell line MC3T3-E1.

Conclusion

We show opposite skeletal effects of PPARα and γ agonists in intact female rats. FENO resulted in significantly higher femoral BMD and lower medullary area, while PIO induced bone loss and impairment of the mechanical strength. This represents a novel effect of PPARα activation.     

Dehydrozingerone ameliorates Lipopolysaccharide induced acute respiratory distress syndrome by inhibiting cytokine storm,oxidative stress via modulating the MAPK/NF-κB pathway

BackgroundInflammation-mediated lung injury is a major cause of health problems in many countries and has been the leading cause of morbidity/mortality in intensive care units. In the current COVID-19 pandemic, the majority of the patients experienced serious pneumonia resulting from inflammation (Acute respiratory distress syndrome/ARDS). Pathogenic infections cause cytokine release syndrome (CRS) by hyperactivation of immune cells, which in turn release excessive cytokines causing ARDS. Currently, there are no standard therapies for viral, bacterial or pathogen-mediated CRS.PurposeThis study aimed to investigate and validate the protective effects of Dehydrozingerone (DHZ) against LPS induced lung cell injury by in-vitro and in-vivo models and to gain insights into the molecular mechanisms that mediate these therapeutic effects.MethodsThe therapeutic activity of DHZ was determined in in-vitro models by pre-treating the cells with DHZ and exposed to LPS to stimulate the inflammatory cascade of events. We analysed the effect of DHZ on LPS induced inflammatory cytokines, chemokines and cell damage markers expression/levels using various cell lines. We performed gene expression, ELISA, and western blot analysis to elucidate the effect of DHZ on inflammation and its modulation of MAPK and NF-κB pathways. Further, the prophylactic and therapeutic effect of DHZ was evaluated against the LPS induced ARDS model in rats.ResultsDHZ significantly (p < 0.01) attenuated the LPS induced ROS, inflammatory cytokine, chemokine gene expression and protein release in macrophages. Similarly, DHZ treatment protected the lung epithelial and endothelial cells by mitigating the LPS induced inflammatory events in a dose-dependent manner. In vivo analysis showed that DHZ treatment significantly (p < 0.001) mitigated the LPS induced ARDS pathophysiology of increase in the inflammatory cells in BALF, inflammatory cytokine and chemokines in lung tissues. LPS stimulated neutrophil-mediated events, apoptosis, alveolar wall thickening and alveolar inflammation were profoundly reduced by DHZ treatment in a rat model.ConclusionThis study demonstrates for the first time that DHZ has the potential to ameliorate LPS induced ARDS by inhibiting cytokine storm and oxidative through modulating the MAPK and NF-κB pathways. This data provides pre-clinical support to develop DHZ as a potential therapeutic agent against ARDS.     

Effect of phosphatidylinositol 3-kinase-γ inhibitor CAY10505 in hypertension, and its associated vascular endothelium dysfunction in rats

This study has been designed to investigate the role of phosphatidyl-inositol 3-kinase-γ (PI3Kγ) in deoxycorticosterone acetate salt (DOCA) hypertension induced vascular endothelium dysfunction. Wistar rats were uninephrectomised and DOCA (40 mg·(kg body mass)(-1), subcutaneous injection) was administered twice weekly for 6 weeks to produce hypertension. Rats with mean arterial blood pressure ≥ 140 mm Hg (1 mm Hg = 133.322 Pa) were selected as hypertensive. Vascular endothelium dysfunction was assessed in terms of attenuation of acetylcholine-induced endothelium-dependent relaxation (isolated aortic ring preparation), decrease in serum nitrate and (or) nitrite level, as well as reduced level of glutathione and disruption of integrity of vascular endothelium (histopathology). Five weeks of DOCA administration were followed by 7 days of daily administration of PI3Kγ inhibitor (5-[[5-(4-fluorophenyl)-2-furanyl]methylene]-2,4-thiazolidinedione (CAY10505), 0.6 mg·kg(-1), per os (p.o.)), atorvastatin (30 mg·kg(-1), p.o.), and losartan (25 mg·kg(-1), p.o.) (positive control of hypertension), which significantly improved acetylcholine-induced endothelium dependent relaxation, serum nitrate and (or) nitrite level, glutathione level, and the vascular endothelial lining in hypertensive rats.Therefore, it may be concluded that CAY10505, a specific inhibitor of PI3Kγ, improves hypertension-associated vascular endothelial dysfunction. Thus, inhibition of PI3Kγ might be a useful approach in the therapeutics of vascular endothelium dysfunction.     

The effect of combined regulation of the expression of peroxisome proliferator-activated receptor-γ and calcitonin gene-related peptide on alcohol-induced adipogenic differentiation of bone marrow mesenchymal stem cells

Studies have shown that alcohol can upregulate the expression of peroxisome proliferator-activated receptor-γ (PPARγ) gene in bone marrow mesenchymal stem cells (BMSCs). High expression of PPARγ can promote adipogenic differentiation of BMSCs, and reduce their osteogenic differentiation. Abnormal proliferation of adipocytes and fatty accumulation in osteocytes can result in high intraosseous pressure and disturbance of blood circulation in the femoral head, which induces osteonecrosis of the femoral head (ONFH). Downregulation of PPARγ is efficient in inhibiting adipogenesis and maintaining osteogenesis of BMSCs, which might potentially reduce the incidence of ONFH. Calcitonin gene-related peptide (CGRP) is a neuropeptide gene which has been closely associated with bone regeneration. In this study, we aimed to observe the effect of combined regulation of the expression of PPARγ and CGRP genes on alcohol-induced adipogenic differentiation of BMSCs. Our results demonstrated that simultaneous downregulation of PPARγ and upregulation of CGRP was efficient in suppressing adipogenic differentiation of BMSCs and promoting their osteogenic differentiation. These findings might enlighten a novel approach for the prevention of ONFH.     

Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcγR pathway

Public health measures successfully contained outbreaks of the severe acute respiratory syndrome coronavirus (SARS-CoV) infection. However, the precursor of the SARS-CoV remains in its natural bat reservoir, and reemergence of a human-adapted SARS-like coronavirus remains a plausible public health concern. Vaccination is a major strategy for containing resurgence of SARS in humans, and a number of vaccine candidates have been tested in experimental animal models. We previously reported that antibody elicited by a SARS-CoV vaccine candidate based on recombinant full-length Spike-protein trimers potentiated infection of human B cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents. These observations prompted us to investigate the mechanisms underlying antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro. We demonstrate here that anti-Spike immune serum, while inhibiting viral entry in a permissive cell line, potentiated infection of immune cells by SARS-CoV Spike-pseudotyped lentiviral particles, as well as replication-competent SARS coronavirus. Antibody-mediated infection was dependent on Fcγ receptor II but did not use the endosomal/lysosomal pathway utilized by angiotensin I converting enzyme 2 (ACE2), the accepted receptor for SARS-CoV. This suggests that ADE of SARS-CoV utilizes a novel cell entry mechanism into immune cells. Different SARS vaccine candidates elicit sera that differ in their capacity to induce ADE in immune cells despite their comparable potency to neutralize infection in ACE2-bearing cells. Our results suggest a novel mechanism by which SARS-CoV can enter target cells and illustrate the potential pitfalls associated with immunization against it. These findings should prompt further investigations into SARS pathogenesis.     

Effect of surfactant pluronic F-68 on CHO cell growth, metabolism, production, and glycosylation of human recombinant IFN-γ in mild operating conditions

The control of glycosylation to satisfy regulatory requirements and quality consistency of recombinant proteins produced by different processes has become an important issue. With two N-glycosylation sites, γ-interferon (IFN-γ) can be seen as a prototype of a recombinant therapeutic glycoprotein for this purpose. The effect of the nonionic surfactant Pluronic F-68 (PF-68) on cell growth and death was investigated, as well as production and glycosylation of recombinant IFN-γ produced by a CHO cell line that was maintained in a rich protein-free medium in the absence or presence of low agitation. Under these conditions, a dose-dependent effect of PF-68 (0-0.1%) was shown not only to significantly enhance growth but also to reduce cell lysis. Interestingly, supplementing the culture medium with PF-68 led to increased IFN-γ production as a result of both higher cell densities and a higher specific production rate of IFN-γ. If cells were grown with agitation, lack of PF-68 in the culture medium decreased the fraction of the fully glycosylated IFN-γ glycoform (2N) from 80% to 65-70% during the initial period. This effect appeared to be due to a lag phase in cell growth observed during this period. Finally, a global kinetic study of CHO cell metabolism indicated higher efficiency in the utilization of the two major carbon substrates when cultures were supplemented with PF-68. Therefore, these results highlight the importance of understanding how media surfactant can affect cell growth as well as cell death and the product quality of a recombinant glycoprotein expressed in CHO cell cultures.     

Effect of 1,25 dihydroxyvitamin D3 on intracellular IFN-γ and TNF-α positive T cell subsets in pulmonary tuberculosis

We studied the immunomodulatory effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α cytokines in T cell subsets of pulmonary tuberculosis (PTB) patients (n = 22) and normal healthy subjects (n = 22). Peripheral blood mononuclear cells (PBMCs) were cultured with live Mycobacterium tuberculosis (MTB) with or without 1,25(OH)₂D₃ (10^?7 M) for 48 h. T cell subsets positive for IFN-γ and TNF-α were enumerated by flow cytometry and the culture supernatants were assayed for both the cytokines using ELISA. In both NHS and PTB patients, a significantly reduced percentage of IFN-γ and TNF-α expressing CD3+, CD3+CD4+ and CD3+CD8+ T cells were observed in cultures stimulated with live MTB and treated with 1,25(OH)₂D₃ compared to cultures without 1,25(OH)₂D₃ (NHS; CD3+ IFN-γ+: p < 0.0001; CD3+TNF-α +: p = 0.0292 and PTB; CD3+ IFN-γ+: p = 0.0292; CD3+ TNF-α +: p = 0.0028). The levels of IFN-γ and TNF-α in the culture supernatants of 1,25(OH)₂D₃ treated cultures were also found to be significantly decreased in both groups (NHS; IFN-γ: p = 0.0001; TNF-α: p < 0.0001) and (PTB; IFN-γ: p < 0.0001; TNF-α: p < 0.0001). A positive correlation was observed between IFN-γ and TNF-α expressing CD3+CD8+ T cells in MTB stimulated cultures treated with or without 1,25(OH)₂D₃ in NHS (p = 0.0001; p = 0.001, respectively) and PTB patients (p = 0.002; p = 0.005, respectively). The present study revealed the suppressive effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α by CD3+CD4+ and CD3+CD8+ T cells in pulmonary tuberculosis. This suppressive effect of 1,25(OH)₂D₃ on proinflammatory and Th1 cytokine positive cells might have a role in reducing inflammation at the site of infection.     

Sweroside promotes osteoblastic differentiation and mineralization via interaction of membrane estrogen receptor-α and GPR30 mediated p38 signalling pathway on MC3T3-E1 cells

BackgroundDipsaci Radix has been clinically used for thousands of years in China for strengthening muscles and bones. Sweroside is the major active iridoid glycoside isolated from Dipsaci Radix. It has been reported that sweroside can promote alkaline phosphatase (ALP) activity in both the human osteosarcoma cell line MG-63 and rat osteoblasts. However, the underlying mechanism involved in these osteoblastic processes is poorly understood.PurposeThis study aimed to characterize the bone protective effects of sweroside and to investigate the signaling pathway that is involved in its actions in MC3T3-E1 cells.MethodsCell proliferation, differentiation and mineralization were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, ALP test and Alizarin Red S staining, respectively. The concentration of sweroside in intracellular and extracellular fluids was determined by ultra-performance liquid chromatography coupled to triple quadrupole xevo-mass spectrometry (UPLC/TQ-XS-MS). Proteins associated with the osteoblastic signaling pathway were analysed by western blot and immunofluorescence methods.ResultsSweroside did not obviously affect the proliferation but significantly promoted the ALP activity and mineralization of MC3T3-E1 cells. The maximal absorption amount 0.465 ng/ml (1.3 × 10⁻⁹ M) of sweroside was extremely lower than the tested concentration of 358.340 ng/ml (10⁻⁶ M), indicating an extremely low absorption rate by MC3T3-E1 cells. Moreover, the ALP activity, the protein expression of ER-α and G protein-coupled receptor 30 (GPR30) induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15. In addition, sweroside also activated the phosphorylation of p38 kinase (p-p38), while the phosphorylation effects together with ALP and mineralization activities were completely blocked by a p38 antagonist, SB203580. Additionally, the phosphorylation of p38 induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15.ConclusionsThe present study indicated that sweroside, as a potential agent in treatment of osteoporosis, might exert beneficial effects on MC3T3-E1 cells by interaction with the membrane estrogen receptor-α and GPR30 that then activates the p38 signaling pathway. This is the first study to report the specific mechanism of the effects of sweroside on osteoblastic differentiation and mineralization of MC3T3-E1 cells.