PubMed · 2451349
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Z Magić, D Pantelić. [Acute phase proteins].. https://pubmed.ncbi.nlm.nih.gov/2451349/
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The Ca2+-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: Galphai to inhibit the activity of adenylyl cyclase and activate ERK, Galphaq to stimulate phospholipase C and phospholipase A2, and Gbetagamma to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to Galpha12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known Galpha12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaRWT) or the nonfunctional mutant CaRR796W as a negative control, prelabeled these cells with [3H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [3H]phosphatidylethanol (PEt). The formation of [3H]PEt increased in a time-dependent manner in the cells that overexpress the CaRWT but not the CaRR796W. Treatment of the cells with C3 exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of Galpha12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate Galphai or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for Galphai and Galphaq, and a p115RhoGEF construct containing the RGS domain for Galpha12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaRWT inhibited extracellular Ca2+-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to Galpha12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of Galphai and Galphaq. This suggests that the CaR may regulate cytoskeleton via Galpha12/13, Rho, and PLD.
Lipoarabinomannans (LAMs) are glycolipids from the mycobacterial cell wall that exhibit various biological activities, including proinflammatory and anti-inflammatory responses. However, little is known about the properties of lipomannans (LMs), considered to be precursors of LAMs. In this study, we provide evidence that LMs purified from Mycobacterium chelonae and a clinical strain of Mycobacterium kansasii stimulated mRNA expression and secretion of TNF-alpha and IL-8 from human macrophage-like differentiated THP-1 cells. In contrast to LMs, LAMs were not able to induce a significant cytokine-inducing effect. The mechanism of activation by LMs was investigated using various Abs raised against surface receptors for multiple bacterial products. The presence of anti-CD14 or anti-Toll-like receptor 2 (TLR2) Abs profoundly affected production of TNF-alpha and IL-8, suggesting that both CD14 and TLR2 participate in the LM-mediated activation process. Furthermore, stimulation of cells was dependent on the presence of the LPS-binding protein, a plasma protein that transfers glycolipids to CD14. Chemical degradation of the arabinan domain of mannose-capped LAM from M. kansasii, which presented no cytokine-eliciting effect, restored the cytokine-inducing activity at a level similar to those of LMs. These results support the hypothesis that the presence of an arabinan in LAMs prevents the interaction of these glycolipids with TLR2/CD14 receptors. In addition, we found that phosphatidylinositol dimannosides isolated from M. kansasii did not induce cytokine secretion. This study suggests that LMs isolated from different mycobacterial species participate in the immunomodulation of the infected host and that the D-mannan core of this glycolipid is essential for this function.
Increasing evidence indicates that the expression of the human immunodeficiency virus-1 (HIV-1) Nef protein significantly influences the activation state of the host cell. Here we report that Nef specifically activates STAT3 in primary human monocyte-derived macrophages (MDM). This was demonstrated by both single-cycle infection experiments driven by Vesicular Stomatitis virus glycoprotein (VSV-G) pseudotyped HIV-1 and treatment with exogenous recombinant Nef. The analysis of the effects of Nef mutants revealed that domains of the C-terminal flexible loop interacting with the cell endocytotic machinery are involved in the STAT3 activation. In particular, our data suggest that the Nef-dependent STAT3 activation relies on the targeting of Nef to the late endosome/lysosome compartment. In addition, we found that Nef activates STAT3 through a mechanism mediated by the release of soluble factor(s), including MIP-1alpha, that requires de novo protein synthesis but appears independent from the activation of src tyrosine kinases. The results presented here support the idea that the first intervention of Nef in the intracellular signaling of monocyte-macrophages could generate, by means of the release of soluble factor(s), a secondary wave of activation that could be of a potential pathogenetic significance.