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== (A, B) Raw264.7 macrophages expressing EGFP (A) or EGFP-Rab39a (B) were treated with LPS and immunostained with anti-LC3 antibody. residues from 34thto 41stin Rab39a were indispensable for this interaction. These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages. == Introduction == Rab GTPases localize to specific subcellular organelles and regulate various membrane trafficking [1,2]. The roles of Rab GTPases in endcytosis and exocytosis have been well studied and their functions in phagocytosis and autophagy are also being elucidated [3-6]. Recently, we screened and identified LDC1267 Rab GTPases that regulate phagosome maturation in macrophages [7]. One of these Rab GTPases, Rab39a was involved in phagosomal acidification [7]. Rab39a has been also demonstrated to be involved in the regulation of Caspase-1 activity [8] and the differentiation of neuron cells [9]. These results together indicate that Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Toll-like receptors (TLRs) are pattern recognition receptors to detect infection by recognizing the conserved pathogen-associated molecular patterns such as lipopolysaccharide (LPS) and trigger innate immune responses to defend invading LDC1267 microbes [10,11]. However, if these innate immune responses are dysregulated, it can lead to adverse systemic disorder, sepsis syndrome. Sepsis syndrome is mainly caused by excess inflammatory cytokines secreted from monocytes/macrophages [12]. New immunotherapeutic agents that modulate immune responses have been developing to control sepsis syndrome [13]. In macrophages, the stimulation by LPS or other TLR ligands induces a unique lysosomal degradation pathway for cytoplasmic materials termed autophagy LDC1267 [14-19]. Autophagy induced by LPS contributes to control the inflammatory immune response because protein degradation by autophagy regulates the secretion of inflammatory cytokines [20]. Autophagy pathway is also triggered by invasion of intracellular pathogen and contributes to the protection of host cells [21]. In this study, we characterized the functions of Rab39a in membrane trafficking, phagocytosis and autophagy induction in macrophages. We found that Rab39a interacts with class III Rabbit polyclonal to ITM2C phosphatidylinositol 3-kinase (PI3K) and regulates autophagy induced by various TLR stimulations. These results imply the possibility that Rab39a is a potent target molecule in the clinical therapy for the sepsis syndrome. == Materials and Methods == == Ethics statement == Animal experiments in this study were approved by the Hamamatsu University School of Medicine Animal Care Committees at the Center Animal Care facility (permit number: 2012074). Mice were sacrificed by cervical dislocation and all efforts were made to minimize suffering. == Cells == Raw264.7 and HEK293T cells were obtained from ATCC and maintained in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 25 LDC1267 g/ml penicillin G and 25 g/ml streptomycin. Bone marrow-derived macrophages (BMM) were differentiated from bone marrow cells of C57BL/6 mice by culturing in DMEM supplemented with 30% L929-conditional medium, 10% FBS and above antibiotics. == Plasmid construction == Construction of the plasmid for EGFP-Rab39a was described previously [7]. PCR for Beclin1, Vps34, Atg14L, Rab39b or Bcl2 was carried out using random-primed cDNA derived from human peripheral blood mononuclear cells (Clonetech) as a template and the primer sets listed inTable S1. For UVRAG, PCR was carried out using pCI-HA-UVRAG [22] as a template. PCR products were inserted into pCMV-Myc (Clonetech) or pEGFP-C1 (Clonetech). pEGFP-Rab39a_M1 or pEGFP-Rab39b_M1 was generated by PCR using primers LDC1267 listed inTable S1and pEGFP-Rab39a or pEGFP-Rab39b as a template, respectively. The resulting PCR products were incubated with T4 DNA ligase andDpnI, followed by the transformation. Transfection of Raw264.7 or HEK293T with plasmid was performed using an MP-100 electroporator (Digital Bio Technology) or X-tremeGENE 9 (Roche), respectively, according to the manufacturers instructions. == Antibody == Rat anti-mouse LAMP2 monoclonal antibody (SouthernBiotech), rabbit anti-LC3 polyclonal antibody (MBL), mouse anti-tubulin monoclonal antibody (Sigma-Aldrich), rabbit anti-p62 antibody (MBL), mouse anti-ubiquitin antibody (MBL), rabbit anti-Beclin1.