TRPM7 Kinase Controls Calcium Responses in Arterial Thrombosis and Stroke in Mice

Objective: TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic alpha-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg2+ and Ca2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown. Approach and Results: Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice (Trpm7(R/R)) causes a marked signaling defect in platelets. Trpm7(R/R) platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C gamma 2 and beta 3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo. Conclusions: Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca2+ entry in platelets. The protection of Trpm7(R/R) mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target

Cigarette smoke inhibits LPS-induced FABP5 expression by preventing c-Jun binding to the FABP5 promoter.

Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD) with repeated and sustained infections linked to disease pathogenesis and exacerbations. The airway epithelium constitutes the first line of host defense against infection and is known to be impaired in COPD. We have previously identified Fatty Acid Binding Protein 5 (FABP5) as an important anti-inflammatory player during respiratory infections and showed that overexpression of FABP5 in primary airway epithelial cells protects against bacterial infection and inflammation. While cigarette smoke down regulates FABP5 expression, its mechanism remains unknown. In this report, we have identified three putative c-Jun binding sites on the FABP5 promoter and show that cigarette smoke inhibits the binding of c-Jun to its consensus sequence and prevents LPS-induced FABP5 expression. Using chromatin immunoprecipitation, we have determined that c-Jun binds the FABP5 promoter when stimulated with LPS but the presence of cigarette smoke greatly reduces this binding. Furthermore, cigarette smoke or a mutation in the c-Jun binding site inhibits LPS-induced FABP5 promoter activity. These data demonstrate that cigarette smoke interferes with FABP5 expression in response to bacterial infection. Thus, functional activation of FABP5 may be a new therapeutic strategy when treating COPD patients suffering from exacerbations

CS inhibits c-Jun activity in human lung epithelial BEAS-2B cells.

<p>Cells were treated with 2% CSE for 24 hours and then stimulated with 1 μg/ml LPS for the indicated time points. <b>A.</b> Phosphorylation of c-Jun at Ser63 and total c-Jun were determined by Western blot. <b>B.</b> Phosphorylation of c-Jun at Ser73 and total c-Jun were determined by Western blot. <b>C.</b> Ratio of total c-Jun and GAPDH densitometry determined by Western blot. <b>D.</b> Activation of c-Jun transcription factor was determined using an ELISA-based TransAM c-Jun activation assay. Data are representative of 6 independent experiments. * p<0.05, ** p<0.01 compared to no CSE and no LPS.</p

CS inhibits c-Jun binding to the FABP5 promoter in human lung epithelial BEAS-2B cells.

<p><b>A.</b> Schematic of the three putative c-Jun binding sites on the <i>FABP5</i> promoter region (not drawn in scale). <b>B.</b> Without and <b>C.</b> with 2% CSE for 24 hours. Cells were stimulated with LPS (1 μg/ml) for 1 hour and chromatin immunoprecipitation assays were performed using anti-c-Jun or anti-rabbit Ig-G antibodies. PCR was performed using primers specific to the proximal c-Jun consensus site (-495) within the <i>FABP5</i> gene promoter. Data are representative of 3 independent experiments.</p

CS inhibits <i>FABP5</i> promoter activity in human lung epithelial BEAS-2B cells.

<p><b>A.</b> Schematic overview of the wild type and mutant c-Jun binding site on the FABP5 promoter at position -495. <b>B.</b> Dual luciferase assay following 2% CSE and/or 1 μg/ml LPS treatment of the wild type <i>FABP5</i> promoter or the promoter mutated at position -495 in the c-Jun binding site. Data are representative of 3 independent experiments.</p

Impact of fatty acid binding protein 5-deficiency on COPD exacerbations and cigarette smoke-induced inflammatory response to bacterial infection

Abstract Background Although cigarette smoking (CS) is by far the most important risk factor of chronic obstructive pulmonary disease (COPD), repeated and sustained infections are clearly linked to disease pathogenesis and are responsible for acute inflammatory flares (i.e. COPD exacerbations). We have previously identified Fatty Acid Binding Protein 5 (FABP5) as an important anti-inflammatory protein in primary airway epithelial cells. Results In this study we found decreased FABP5 mRNA and protein levels in peripheral blood mononuclear cells (PBMCs) of COPD patients, especially among those who reported episodes of COPD exacerbations. Using wildtype (WT) and FABP5−/− mice, we examined the effects of FABP5 on CS and infection-induced inflammatory responses. Similarly to what we saw in airway epithelial cells, infection increased FABP5 expression while CS decreased FABP5 expression in mouse lung tissues. CS-exposed and P. aeruginosa-infected FABP5−/− mice had significantly increased inflammation as shown by increased lung histopathological score, cell infiltration and inflammatory cytokine levels. Restoration of FABP5 in alveolar macrophages using a lentiviral approach attenuated the CS- and bacteria-induced pulmonary inflammation. And finally, while P. aeruginosa infection increased PPARγ activity, CS or FABP5 knockdown greatly reduced PPARγ activity. Conclusions These findings support a model in which CS-induced FABP5 inhibition contributes to increased inflammation in COPD exacerbations. It is interesting to speculate that the increased inflammation is a result of decreased PPARγ activity