Jakub Siednienko

The cellular receptors of exogenous RNA

One of the key determinants of survival for organisms is proper recognition of exogenous and endogenous nucleic acids. Therefore, high eukaryotes developed a number of receptors that allow for discrimination between friend or foe DNA and RNA. Appearance of exogenous RNA in cytoplasm provides a signal of danger and triggers cellular responses that facilitate eradication of a pathogen. Recognition of exogenous RNA is additionally complicated by fact that large amount of endogenous RNA is present in cytoplasm Thus, number of different receptors, found in eukaryotic cells, is able to recognize that nucleic acid. First group of those receptors consist endosomal Toll like receptors, namely TLR3, TLR7, TLR8 and TLR13. Those receptors recognize RNA released from pathogens that enter the cell by endocytosis. The second group includes cytoplasmic sensors like PKR and the family of RLRs comprised of RIG-I, MDA5 and LGP2. Cytoplasmic receptors recognize RNA from pathogens invading the cell by non-endocytic pathway. In both cases binding of RNA by its receptors results in activation of the signalling cascades that lead to the production of interferon and other cytokines.

Sp1 mediates phorbol ester (PMA)-induced expression of membrane-bound guanylyl cyclase GC-A in human monocytic cells

Cyclic guanosine monophosphate (cGMP) is synthesized by two types of enzymes: particulate (membrane-bound) guanylyl cyclases (pGCs) and soluble (cytosolic) guanylyl cyclases (sGCs). sGCs are primarily activated by binding of nitric oxide to their prosthetic heme group while pGCs are activated by binding of peptide ligands to their extracellular domains. One of them, pGC type A (GC-A) is activated by atrial and brain natriuretic peptides (ANP and BNP, respectively). Human monocytes isolated from peripheral blood mononuclear cells have been found to display sGC expression without concomitant expression of GC-A. However, GC-A activity appears in monocytes under certain conditions but a molecular mechanism of GC-A expression is still poorly understood. In this report we show that phorbol ester (PMA) induces transcription of a gene encoding GC-A in human monocytic THP-1 cells. Moreover, we find that PMA-treated THP-1 cells raise cGMP content following treatment with ANP. Studies using pharmacological inhibitors of protein kinases suggest involvement of protein kinase C (PKC), mitogen extracellular kinases (MEK1/2), and extracellular signal-regulated kinases (ERK1/2) in PMA-induced expression of the GC-A encoding gene in THP-1 cells. Finally, we show that PMA stimulates binding of Sp1 transcription factor to GC-rich DNA sequences and mithramycin A (a selective Sp1 inhibitor) inhibits expression of the GC-A mRNA in PMA-treated THP-1 cells. Taken together, our findings suggest that the PMA-stimulated PKC and MEK/ERK signaling pathways induce Sp1-mediated transcription of the GC-A encoding gene in human monocytic THP-1 cells.

[Multilevel maturation of Toll-like receptor 9].

Toll-like receptors (TLRs) are essential elements of the innate immune response. TLRs induce expression of inflammatory cytokines or interferons after recognition of microbial or viral structures called pathogen-associated molecular patterns (PAMPs). Two different groups of TLRs can be distinguished: TLRs residing in the plasma membrane or in the endosomal compartment. TLRs localized in endosomes act as sensors for nucleic acids. TLR9, which recognizes unmethylated CpG, belongs to endosomal TLRs. The proper ligand detection by TLR9 depends on its specific subcellular localization and maturation. TLR9 delivery to the endosomes is mediated by two distinct proteins, UNC93B1 and AP-2, and post-early endosome distribution is determined by AP-3. TLR9 localized in the endosome is cleaved by at least two classes of proteases, AEP and cathepsins, which generate the mature form of receptor. Functional C-terminal form of TLR9 is capable of recognition of CpG and activation of signal pathways. Ligand binding to TLR9 causes conformational changes in the structure of this receptor which facilitates recruitment of MyD88 adaptor protein and activation of two distinct cytokine-inducing pathways: IRF-7- and NF-κB-dependent. The specific structure of the synthetic ligand (CpG-A or CpG-B) determines activation of certain transcription factors. Recognition of multimeric CpG-A results in IRF-7-dependent induction of type I interferon production. Monomeric CpG-B activates NF-κB-dependent induction of proinflammatory cytokines, in particular TNFα and IL-6.

Activation of NF-κB by nitric oxide/cGMP in human blood CD14+ cells

Nuclear factor κB (NF-κB) is a common transcription factor regulating expression of multiple genes relevant for immune reactions. It may by activated by numerous stimuli and a number of data have indicated a relationship between its activity and intracellular concentration of cGMP. Increased levels of the nucleotide have been shown to affect expression of TNFα, IL-1, IL-6 and NOS2 in various immune cells. Particularly, cGMP has been shown to affect several important functions of cells belonging to a monocyte/macrophage lineage. Recently, we reported that in freshly isolated human peripheral blood mononuclear cells (PBMC) the NF-κB activity was modulated through a cGMP-dependent pathway. The aim of this study was to determine whether and how cGMP may affect an activity of NF-κB in human CD14 monocytes. They express soluble guanylyl cyclase (sGC) as well as cGMP-dependent protein kinase I (PKG I). We show that in this cells nitric oxide (NO) stimulates the NF-κB activity at low and inhibits it at high doses. This effect is mimicked by membrane-permeable analogues of cGMP. Using specific inhibitors, we also show that stimulatory effect of NO/cGMP on the NF-κB activity is mediated by PKG I. This observation is additionally supported by the fact that stimulatory effect of NO disappears in cultured cells lacking PKG I. Therefore, our results show that at least some reported effects of NO/cGMP on monocytic cells directly involve PKG I and NF-κB.