Intracellular membrane trafficking in Osteoclasts The role of direct Rab protein – Rac 1 interactions in the targeting of intracellular vesicles

Osteoclasts are cells responsible for bone resorption. These cells undergo extensive membrane re-organization during their polarization for bone resorption and form four distinct membrane domains, namely the ruffled border, the basolateral membrane, the sealing zone and the functional secretory domain. The endocytic/biosynthetic pathway and transcytotic route(s) are important for the resorption process, since the endocytic/biosynthetic pathway brings the specific vesicles to the ruffled border whereas the transcytotic flow is believed to transport the degraded bone matrix away from the resorption lacuna to the functional secretory domain. In the present study, we found a new transcytotic route from the functional secretory domain to the ruffled border, which may compensate membrane loss from the ruffled border during the resorption process. We also found that lipid rafts are essential for the ruffled border-targeted late endosomal pathways. A small GTP-binding protein, Rab7, has earlier been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts it is involved in the formation of the ruffled border, which displays several features of late endosomal membranes. Here we discovered a new Rab7-interacting protein, Rac1, which is another small GTP-binding protein and binds to the GTP-form of Rab7 in vitro. We demonstrated further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, this colocalization is mainly perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, we suggest that the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments, thus enabling endosomal vesicles to switch tracks from microtubules to microfilaments before their fusion to the ruffled border. We then studied the role of Rab-Rac1 interaction in the slow recycling pathway. We revealed that Rac1 also binds directly to Rab11 and to some other but not all Rab-proteins, suggesting that Rab-Rac1 interaction could be a general regulatory mechanism to direct the intracellular vesicles from microtubule mediated transport to actin filament mediated transport and vice versa. On the basis of our results we thus propose a new hypothesis for these GTPases in the regulation of intracellular membrane flow.

The expression of HLA-B27 modulates intracellular signaling in human monocytic macrophages

Reactive arthritis (ReA) is an inflammatory joint disease triggered by certain bacterial infections e.g. gastroenteritis caused by Salmonella. ReA is strongly associated to HLA-B27. However, the mechanism behind this association is unknown but it is suggested that the bacteria or bacterial compartments persist in the body. In this study, it was investigated whether the intracellular signaling is altered in HLA-B27- transfected U937 monocytic macrophages. Moreover, the contribution of HLA–B27 heavy chain (HC) misfolding was of interest. The study revealed that p38 activity plays a crucial role in controlling intracellular Salmonella Enteritidis in U937 cells. The replication of intracellular bacteria was dependent on p38 kinase and the activity of p38 was dysregulated in HLA-B27- transfected cells expressing misfolding heavy chains (HCs). Also the double-stranded RNA -dependent kinase (PKR) that modifies p38 signaling was overexpressed and hypophosphorylated upon infection and lipopolysaccharide stimulation. The expression of CCAAT enhancer binding protein beta (C/EBPβ) was found to be increased after infection and stimulation. Increased amount of full length human antigen R (HuR), disturbed HuR cleavage and reduced dependence on PKR after infection were observed. All the findings were linked to HLA-B27 HCs containing misfoldingassociated glutamic acid 45 (Glu45) at the peptide binding groove. The results indicate that the expression of HLA-B27 modulates the intracellular environment of U937 monocytic macrophages by altering signaling. This phenomenon is at least partially associated to the HLA-B27 misfolding. These observations offer a novel explanation how HLA-B27 may modulate inflammatory response induced by ReA-triggering bacteria.

Cardiac Imaging in the Diagnosis of Coronary Artery Disease: The Value of Quantitative Imaging of Myocardial Perfusion and Deformation

Atherosclerosis is a chronic and progressive disease of the vasculature. Increasing coronary atherosclerosis can lead to obstructive coronary artery disease (CAD) or myocardial infarction. Computed tomography angiography (CTA) allows noninvasive assessment of coronary anatomy and quantitation of atherosclerotic burden. Myocardial blood flow (MBF) can be accurately measured in absolute terms (mL/g/min) by positron emission tomography (PET) with [15O] H O as a radiotracer. We studied the coronary microvascular dysfunction as a risk factor for future coronary calcification in healthy young men by measuring the coronary flow reserve (CFR) which is the ratio between resting and hyperemic MBF. Impaired vasodilator function was not linked with accelerated atherosclerosis 11 years later. Currently, there is a global interest in quantitative PET perfusion imaging. We established optimal thresholds of [15O] H O PET perfusion for diagnosis of CAD (hyperemic MBF of 2.3 mL/g/min and CFR of 2.5) in the first multicenter study of this type (Turku, Amsterdam and Uppsala). In myocardial bridging a segment of the coronary artery travels inside the myocardium and can be seen as intramural course (CTA) or systolic compression (invasive coronary angiography). Myocardial bridging is frequently linked with proximal atherosclerotic plaques. We used quantitative [15O] H O PET perfusion to evaluate the hemodynamic effects of myocardial bridging. Myocardial bridging was not associated with decreased absolute MBF or increased atherosclerotic burden. Speckle tracking allows quantitative echocardiographic imaging of myocardial deformation. Speckle tracking during dobutamine stress echocardiography was feasible and comparable to subjective wall motion analysis in the diagnosis of CAD. In addition, it correctly risk stratified patients with multivessel disease and extensive ischemia.