Increased expression of miR146a dysregulates TLR2-induced HBD2 in airway epithelial cells from patients with COPD

Background Airway epithelial cells from patients with COPD show suboptimal innate immune responses to nontypeable Haemophilus influenzae (NTHi) and Toll-like receptor (TLR)2 ligands despite expressing TLR2 similar to normal airway epithelial cells, but the underlying mechanisms are poorly understood. Methods Normal or COPD mucociliary-differentiated airway epithelial cells were treated with TLR2 agonists or infected with NTHi and expression of β-defensin (HBD)2 was examined. Interleukin-1 receptor-associated kinase (IRAK)-1 and microRNA (miR)146a were genetically inhibited in normal and COPD airway epithelial cell cultures, respectively, and HBD2 responses to TLR2 ligands were determined. IRAK-1 expression in lung sections was determined by immunofluorescence microscopy. Results Compared to normal, COPD airway epithelial cell cultures showed impaired expression of HBD2 in response to TLR2 agonists or NTHi infection. Apical secretions from TLR2 agonist-treated normal, but not COPD, airway epithelial cells efficiently killed NTHi. Knockdown of HBD2 significantly reduced NTHi killing by apical secretions of normal airway epithelial cells. Compared to normal, COPD cells showed significantly reduced expression of IRAK-1 and this was associated with increased expression of miR146a. Inhibition of miR146a increased the expression of IRAK-1, improved the expression of HBD2 in response to TLR2 agonists in COPD cells and enhanced the killing of bacteria by apical secretions obtained from TLR2 agonist-treated COPD cells. Bronchial epithelium of COPD patients showed reduced expression of IRAK-1. Conclusions These results suggest that reduced levels of IRAK-1 due to increased expression of miR146a may contribute to impaired expression of TLR2-induced HBD2 in COPD airway epithelial cells

Biochemical characterization of spleen tyrosine kinase (SYK) isoforms in platelets

Alternate splicing is among the regulatory mechanisms imparting functional diversity in proteins. Studying protein isoforms generated through alternative splicing is therefore critical for understanding protein functions in many biological systems. Spleen tyrosine kinase (Syk) plays an essential role in ITAM/hemITAM signaling in many cell types, including platelets. However, the spectrum of Syk isoforms expressed in platelets has not been characterized. Syk has been shown to have a full-length long isoform SykL and a shorter SykS lacking 23 amino acid residues within its interdomain B. Furthermore, putative isoforms lacking another 23 amino acid-long sequence or a combination of the two deletions have been postulated to exist. In this report, we demonstrate that mouse platelets express full-length SykL and the previously described shorter isoform SykS, but lack other shorter isoforms, whereas human platelets express predominantly SykL. These results both indicate a possible role of alternative Syk splicing in the regulation of receptor signaling in mouse platelets and a difference between signaling regulation in mouse and human platelets

Phosphorylation of (Ser 291) in the linker insert of Syk negatively regulates ITAM signaling in platelets

Receptor-induced tyrosine phosphorylation of spleen tyrosine kinase (Syk) has been studied extensively in hematopoietic cells. Metabolic mapping and high-resolution mass spectrometry, however, indicate that one of the most frequently detected phosphorylation sites encompassed S297 (S291 in mice) located within the linker B region of Syk. It has been reported that Protein kinase C (PKC) phosphorylates Syk S297, thus influencing Syk activity. However, conflicting studies suggest that this phosphorylation enhances as well as reduces Syk activity. To clarify the function of this site, we generated Syk S291A knock-in mice. We used platelets as a model system as they possess Glycoprotein VI (GPVI), a receptor containing an immunoreceptor tyrosine-based activation motif (ITAM) which transduces signals through Syk. Our analysis of the homozygous mice indicated that the knock-in platelets express only one isoform of Syk, while the wild-type expresses two isoforms at 69 and 66 kDa. When the GPVI receptor was activated with collagen-related peptide (CRP), we observed an increase in functional responses and phosphorylations in Syk S291A platelets. This potentiation did not occur with AYPGKF or 2-MeSADP, although they also activate PKC isoforms. Although there was potentiation of platelet functional responses, there was no difference in tail bleeding times. However, the time to occlusion in the FeCl3 injury model was enhanced. These data indicate that the effects of Syk S291 phosphorylation represent a significant outcome on platelet activation and signaling in vitro but also reveals its multifaceted nature demonstrated by the differential effects on physiological responses in vivo