Abstract
Suppressor of cytokine signaling (SOCS) 3 is a critical negative regulator of cytokine signaling and is induced by Mycobacterium bovis Bacille Calmette-Guérin (M. bovis BCG) in mouse macrophages. However, little is known about the early receptor proximal signaling mechanisms underlying mycobacteria-mediated induction of SOCS3. We demonstrate here for the first time that M. bovis BCG up-regulates NOTCH1 and activates the NOTCH1 signaling pathway, leading to the expression of SOCS3. We show that perturbing Notch signaling in infected macrophages results in the marked reduction in the expression of SOCS3. Furthermore, enforced expression of the Notch1 intracellular domain in RAW 264.7 macrophages induces the expression of SOCS3, which can be further potentiated by M. bovis BCG. The perturbation of Toll-like receptor (TLR) 2 signaling resulted in marked reduction in SOCS3 levels and expression of the NOTCH1 target gene, Hes1. The down-regulation of MyD88 resulted in a significant decrease in SOCS3 expression, implicating the role of the TLR2-MyD88 axis in M. bovis BCG-triggered signaling. However, the SOCS3 inducing ability of M. bovis BCG remains unaltered also upon infection of macrophages from TLR4-defective C3H/HeJ mice. More importantly, signaling perturbation data suggest the involvement of cross-talk among members of the phosphoinositide 3-kinase and mitogen-activated protein kinase cascades with NOTCH1 signaling in SOCS3 expression. Furthermore, SOCS3 expression requires the NOTCH1-mediated recruitment of Suppressor of Hairless (CSL) and nuclear factor-kappaB to the Socs3 promoter. Overall, these results implicate NOTCH1 signaling during inducible expression of SOCS3 following infection of macrophages with an intracellular bacillus-like M. bovis BCG.
Highlights
Wide, with one-third of the world’s population is estimated to be infected with this microorganism [1, 2]
It has been suggested that M. bovis BCGtriggered SOCS3 and SOCS1 proteins leads to the inhibition of IFN-␥-stimulated JAK/STAT signaling in macrophages [26]
We show for the first time that infection of macrophages with M. bovis BCG activates NOTCH1 signaling events that leads to activation of SOCS3
Summary
Peritoneal macrophages were left uninfected or infected with M. bovis BCG for 6 h as in the case of NF-B or 12 h for CSL/ RBP-Jk. The cells were fixed with 1.42% formaldehyde for 15 min at room temperature followed by inactivation of formaldehyde with addition of 125 mM glycine. Electrophoretic Mobility Shift Assay (EMSA)—Mouse peritoneal macrophages were infected with M. bovis BCG for the indicated time points and gently lysed in buffer containing Nonidet P-40. Nuclear proteins (5 g) were incubated for 30 min at 25 °C with 32P-labeled NF-B or CSL/RBP-Jk probe in binding buffer for NF-B, 10 mM Tris-Cl, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 10 mg/ml bovine serum albumin, 1% Nonidet P-40, 5% glycerol, and 1 g of poly(dI-dC) and for CSL/RBP-Jk, 20 mM HEPES, pH 7.9, 60 mM KCl, 1.33 mM MgCl2, 1 mM dithiothreitol, 4% glycerol, and 1 g of poly(dI-dC), and separated by electrophoresis through 6% Tris borate-EDTA gels. Graphpad Prism 3.0 software (Graphpad Software) was used for all the statistical analysis
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