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  • Our study expands our current

    2022-08-12

    Our study expands our current knowledge of Ras-like small GTPase molecules involved in TLR-mediated responses in macrophages. We found that steady-state levels of Rap2a mRNA and protein undergo alterations after stimuli with TLR agonists. Although statistically significant, the effect of LPS-induced early reduction on Rap2a mRNA levels in macrophages is modest, not achieving a reduction of more than 2-fold, which could be interpreted as of questionable biological importance. However, the level of reduction in gene DCC-2036 does not exclude the relevance of the observed effect, since alterations in the mRNA levels in macrophages used in this study is very consistent among the different agonists tested. More important, we found that the protein levels of Rap2a in cell extracts from macrophages treated for extended time intervals with LPS or Pam3CSK4 agonists did not correlate straight with the mRNA levels, being intriguing and also challenging to search for the mechanisms involved. One possible explanation for these interesting findings is that TLR2 or TLR4 pathways may regulate Rap2a protein levels at the posttranscriptional events during macrophage activation. These observations suggest that perturbations in Rap2a mRNA levels caused in response to treatment with TLR agonists in macrophages may lead to critical biological changes in the translation of its own mRNA. An interesting study has found similar findings to our observations by demonstrating that Gfi1 mRNA levels (an important molecule implicated in myeloid differentiation) decrease during monocyte differentiation; however, that protein levels increase significantly due to lower proteasomal degradation (Marteijn et al., 2007). Several important regulators of NF-κB activity and immune system signaling pathway have their expression modulated in inflammation so they may play their functions. The expression of A20 protein is rapidly induced after activation of NF-κB (Verstrepen et al., 2010). SIRPα and also RasGRP3 are highly abundant in macrophages, and their levels are significantly reduced following treatment with inflammatory TLR agonist. This indicates that a well-controlled and tightly decrease for some molecules might be critical for macrophage activation (Kong et al., 2007; Tang et al., 2014). Interestingly, levels of active Rap2a in RAW264.7 macrophages following PMA treatment are higher than the observed for LPS stimuli. Rap1 is a PMA-activated molecule in different cell types, such as platelets and lymphocytes (Franke et al., 2000; Eppler et al., 2017). PMA is a PKC activator, a kinase involved in signal transduction of several pathways related to cell growth and proliferation (Wu-Zhang and Newton, 2013), indicating that further studies on Rap2 proteins in PMA-activated pathways may be promising and reveal new implications for these GTPases. It is not clearly defined to date the degree of substrate specificity of GEFs for the Rap1a/b and Rap2a/b/c isoforms in cellular systems or in vivo, as many GEFs have been proposed. Given the numerous GEFs and their partner GTPase, a systematic study will be thus required to determine the GEF and/or GEFs that regulate Rap2a activation in macrophages in response to TLR stimulation. RasGEF1b in HEK293 cells can directly activate Rap2a, and reduced active Rap2a is seen in BMDMs lacking RasGEF1b upon LPS stimulation. Biochemical approaches indicated that RasGEF1b acts as GEF specific to Rap2a (Yaman et al., 2009). Therefore, it is very likely that additional GEFs will act on the activation of Rap2a induced by LPS or contributes with RasGEF1b. EPAC2 was recently demonstrated as Rap2a activator in neuroendocrine cells (Emery et al., 2017), and could be a putative GEF to be investigated in Rap2a-related inflammatory pathways. Ras small GTPases have been implicated in the production of molecules related to the immune system, most of them related to leukocyte motility and adhesion and chemokine production (Bokoch, 2005; Yu et al., 2015). To our knowledge, our work is pioneer by demonstrating the interference of Rap2a protein in the production of inflammatory mediators and NF-κB activity in macrophages. Thus, the relevance of our findings provokes further investigation by examining the function of Rap2a with additional TLR agonists.