Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions. (c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Nur77 regulates lipolysis in skeletal muscle cells - Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway

Skeletal muscle is a major mass peripheral tissue that accounts for similar to 40% of total body weight and 50% of energy expenditure and is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. Excessive caloric intake is sensed by the brain and induces beta-adrenergic receptor (beta-AR)- mediated adaptive thermogenesis. beta-AR null mice develop severe obesity on a high fat diet. However, the target gene(s), target tissues(s), and molecular mechanism involved remain obscure. We observed that 30 - 60 min of beta-AR agonist ( isoprenaline) treatment of C2C12 skeletal muscle cells strikingly activated (> 100-fold) the expression of the mRNA encoding the nuclear hormone receptor, Nur77. In contrast, the expression of other nuclear receptors that regulate lipid and carbohydrate metabolism was not induced. Stable transfection of Nur77-specific small interfering RNAs (siNur77) into skeletal muscle cells repressed endogenous Nur77 mRNA expression. Moreover, we observed attenuation of gene and protein expression associated with the regulation of energy expenditure and lipid homeostasis, for example AMP-activated protein kinase gamma 3, UCP3, CD36,adiponectin receptor 2, GLUT4, and caveolin-3. Attenuation of Nur77 expression resulted in decreased lipolysis. Finally, in concordance with the cell culture model, injection and electrotransfer of siNur77 into mouse tibialis cranialis muscle resulted in the repression of UCP3 mRNA expression. This study demonstrates regulatory cross-talk between the nuclear hormone receptor and beta-AR signaling pathways. Moreover, it suggests Nur77 modulates the expression of genes that are key regulators of skeletal muscle lipid and energy homeostasis. In conclusion, we speculate that Nur77 agonists would stimulate lipolysis and increase energy expenditure in skeletal muscle and suggest selective activators of Nur77 may have therapeutic utility in the treatment of obesity.

Rev-erb beta regulates the expression of genes involved in lipid absorption in skeletal muscle cells - Evidence for cross-talk between orphan nuclear receptors and myokines

Rev-erbbeta is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor-alpha (RORalpha). RORalpha has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Rev-erbbeta and RORalpha are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erbbeta has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erbbeta regulates lipid metabolism in skeletal muscle. This lean tissue accounts for > 30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erbbeta in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erbbeta decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (> 15-fold) in mRNA expression of interleukin-6, an exercise-induced myokine that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (> 20- fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erbbeta in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erbbeta may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth.