Katja Farhat

Heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum but does not lead to differential signaling.

TLR are primary triggers of the innate immune system by recognizing various microorganisms through conserved pathogen‐associated molecular patterns. TLR2 is the receptor for a functional recognition of bacterial lipopeptides (LP) and is up‐regulated during various disorders such as chronic obstructive pulmonary disease and sepsis. This receptor is unique in its ability to form heteromers with TLR1 or TLR6 to mediate intracellular signaling. According to the fatty acid pattern as well as the assembling of the polypeptide tail, LP can signal through TLR2 in a TLR1‐ or TLR6‐dependent manner. There are also di‐ and triacylated LP, which stimulate TLR1‐deficient cells and TLR6‐deficient cells. In this study, we investigated whether heterodimerization evolutionarily developed to broaden the ligand spectrum or to induce different immune responses. We analyzed the signal transduction pathways activated through the different TLR2 dimers using the three LP, palmitic acid (Pam)octanoic acid (Oct)2C‐(VPGVG)4VPGKG, fibroblast‐stimulating LP‐1, and Pam2C‐SK4. Dominant‐negative forms of signaling molecules, immunoblotting of MAPK, as well as microarray analysis indicate that all dimers use the same signaling cascade, leading to an identical pattern of gene activation. We conclude that heterodimerization of TLR2 with TLR1 or TLR6 evolutionarily developed to expand the ligand spectrum to enable the innate immune system to recognize the numerous, different structures of LP present in various pathogens. Thus, although mycoplasma and Gram‐positive and Gram‐negative bacteria may activate different TLR2 dimers, the development of different signal pathways in response to different LP does not seem to be of vital significance for the innate defense system.

The Stimulatory Gαs Protein Is Involved in Olfactory Signal Transduction in Drosophila

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology, constituting a key difference between the olfactory systems of insects and other animals. While heteromeric insect ORs form ligand-activated non-selective cation channels in recombinant expression systems, the evidence for an involvement of cyclic nucleotides and G-proteins in odor reception is inconsistent. We addressed this question in vivo by analyzing the role of G-proteins in olfactory signaling using electrophysiological recordings. We found that Gαs plays a crucial role for odorant induced signal transduction in OR83b expressing olfactory sensory neurons, but not in neurons expressing CO2 responsive proteins GR21a/GR63a. Moreover, signaling of Drosophila ORs involved Gαs also in a heterologous expression system. In agreement with these observations was the finding that elevated levels of cAMP result in increased firing rates, demonstrating the existence of a cAMP dependent excitatory signaling pathway in the sensory neurons. Together, we provide evidence that Gαs plays a role in the OR mediated signaling cascade in Drosophila.

Unfavourable consequences of chronic cardiac HIF-1α stabilization

AIMS The hypoxia-inducible factor-1 (HIF-1) is the master modulator of hypoxic gene expression. The effects of chronically stabilized cardiac HIF-1α and its role in the diseased heart are not precisely known. The aims of this study were as follows: (i) to elucidate consequences of HIF-1α stabilization in the heart; (ii) to analyse long-term effects of HIF-1α stabilization with ageing and the ability of the HIF-1α overexpressing hearts to respond to increased mechanical load; and (iii) to analyse HIF-1α protein levels in failing heart samples. METHODS AND RESULTS In a cardiac-specific HIF-1α transgenic mouse model, constitutive expression of HIF-1α leads to changes in capillary area and shifts the cardiac metabolism towards glycolysis with a net increase in glucose uptake. Furthermore, Ca(2+) handling is altered, with increased Ca(2)(+) transients and faster intracellular [Ca(2+)] decline. These changes are associated with decreased expression of sarcoplasmic/endoplasmic reticulum calcium ATPase 2a but elevated phosphorylation of phospholamban. HIF-1α transgenic mice subjected to transverse aortic constriction exhibited profound cardiac decompensation. Moreover, cardiomyopathy was also seen in ageing transgenic mice. In parallel, we found an increased stabilization of HIF-1α in heart samples of patients with end-stage heart failure. CONCLUSION Changes induced with transgenic cardiac HIF-1α possibly mediate beneficial effects in the short term; however, with increased mechanical load and ageing they become detrimental for cardiac function. Together with the finding of increased HIF-1α protein levels in samples from human patients with cardiomyopathy, these data indicate that chronic HIF-1α stabilization drives autonomous pathways that add to disease progression.

Loss of epithelial hypoxia-inducible factor prolyl hydroxylase 2 accelerates skin wound healing in mice.

ABSTRACT Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.

Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.

Prolyl-4-hydroxylase domain 3 (PHD3) is a critical terminator for cell survival of macrophages under stress conditions

On a molecular level, cells sense changes in oxygen availability through the PHDs, which regulate the protein stability of the α‐subunit of the transcription factor HIF. Especially, PHD3 has been additionally associated with apoptotic cell death. We hypothesized that PHD3 plays a role in cell‐fate decisions in macrophages. Therefore, myeloid‐specific PHD3−/− mice were created and analyzed. PHD3−/− BMDM showed no altered HIF‐1α or HIF‐2α stabilization or increased HIF target gene expression in normoxia or hypoxia. Macrophage M1 and M2 polarization was unchanged likewise. Compared with macrophages from WT littermates, PHD3−/− BMDM exhibited a significant reduction in TUNEL‐positive cells after serum withdrawal or treatment with stauro and SNAP. Under the same conditions, PHD3−/− BMDM also showed less Annexin V staining, which is representative for membrane disruption, and indicated a reduced early apoptosis. In an unbiased transcriptome screen, we found that Angptl2 expression was reduced in PHD3−/− BMDM under stress conditions. Addition of rAngptl2 rescued the antiapoptotic phenotype, demonstrating that it is involved in the PHD3‐mediated response toward apoptotic stimuli in macrophages.

Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1α in Cofilin Regulation and Cytoplasmic Actin Distribution

Cells can adapt to hypoxia by various mechanisms. Yet, hypoxia-induced effects on the cytoskeleton-based cell architecture and functions are largely unknown. Here we present a comprehensive analysis of the architecture and function of L929 fibroblasts under hypoxic conditions (1% O2). Cells cultivated in hypoxia showed striking morphological differences as compared to cells cultivated under normoxic conditions (20% O2). These changes include an enlargement of cell area and volume, increased numbers of focal contacts and loss of cell polarization. Furthermore the β- and γ-actin distribution is greatly altered. These hypoxic adjustments are associated with enhanced cell spreading and a decline of cell motility in wound closure and single cell motility assays. As the hypoxia-inducible factor-1α (HIF-1α) is stabilised in hypoxia and plays a pivotal role in the transcriptional response to changes in oxygen availability we used an shRNA-approach to examine the role of HIF-1α in cytoskeleton-related architecture and functions. We show that the observed increase in cell area, actin filament rearrangement, decrease of single cell migration in hypoxia and the maintenance of p-cofilin levels is dependent on HIF-1α stabilisation.

Regulators of G-protein signalling are modulated by bacterial lipopeptides and lipopolysaccharide.

Regulators of G‐protein signalling accelerate the GTPase activity of Gα subunits, driving G proteins in their inactive GDP‐bound form. This property defines them as GTPase activating proteins. Here the effect of different Toll‐like receptor agonists on RGS1 and RGS2 expression in murine bone marrow‐derived macrophages and J774 cells was analysed. After stimulation with TLR2/1 or TLR2/6 lipopeptide ligands and the TLR4/MD2 ligand lipopolysaccharide, microarray analyses show only modulation of RGS1 and RGS2 among all the regulators of G‐protein signalling tested. Real‐time PCR confirmed modulation of RGS1 and RGS2. In contrast to RGS2, which was always downregulated, RGS1 mRNA was upregulated during the first 30 min after stimulation, followed by downregulation. Similar results were also found in the murine macrophage cell line J774. The ligand for intracellular TLR9 modulates RGS1 and RGS2 in a similar manner. However, the TLR3 ligand poly(I:C) permanently upregulates RGS1 and RGS2 expression indicating a different modulation by the MyD88‐ and TRIF‐signalling pathway. This was confirmed using MyD88−/− and TRIF−/− bone marrow‐derived macrophages. Modulation of RGS1 and RGS2 by Toll‐like receptor ligands plays an important role during inflammatory and immunological reactions after bacterial and viral infection.

The response of HEK293 cells transfected with bovine TLR2 to established pathogen-associated molecular patterns and to bacteria causing mastitis in cattle ☆

Toll-like receptors (TLRs) are key sensors of pathogen-associated molecular patterns (PAMPs). Their role in immunity is difficult to examine in species of veterinary interest, due to restricted access to the knockout technology and TLR-specific antibodies. An alternative approach is to generate cell lines transfected with various TLRs and to examine the recognition of PAMPs or relevant bacteria. In this report, we examined whether recognition of various PAMPs and mastitis-causing bacteria is achieved by transfection of recombinant bovine TLR2 (boTLR2). Therefore, human embryonic kidney (HEK) 293 cells were transfected by whole boTLR2. A clonal analysis of stably transfected cells disclosed variable recognition of several putative TLR2 agonists although expressing similar amounts of the transgene and endogenous TLR6. One clone (clone 25) reacted by copious interleukin-8 (IL-8) production to several stimulants of TLR2 such as di-palmitoylated cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam2), a biochemical preparation of lipoteichoic acid from Staphylococcus aureus, a commercial preparation of peptidoglycan from S. aureus, and heat-killed Listeria monocytogenes (HKLM). TLR2-dependent induction of IL-8 release was stronger in medium containing human serum albumin than in medium containing fetal calf serum. Clone 25 cells responded to high concentrations of S. aureus and to Escherichia coli causing mastitis, but not to Streptococcus uberis and to Streptococcus agalactiae which also cause mastitis. Stimulation by S. aureus was relatively weak when compared (i) with stimulation of the same cells by HKLM and PAMPs derived from S. aureus, (ii) with a clone stably transfected with TLR4 and MD-2 and stimulated by E. coli causing mastitis, and (iii) with interferon-gamma-costimulated bovine macrophages stimulated by S. aureus and S. agalactiae. Thus, clone 25 is suitable for studying the interaction of putative TLR2 agonists with bovine TLR2-transfected cells, provides a cell to search for TLR2-specific antibodies, and is a tool for studying the interaction of TLR2 with bacteria causing disease, e.g. mastitis, in cattle.

Identification of full length bovine TLR1 and functional characterization of lipopeptide recognition by bovine TLR2/1 heterodimer

Toll-like receptors (TLR) are highly conserved pattern recognition receptors of the innate immune system. Toll-like receptor 2 (TLR2) recognizes bacterial lipopeptides in a heterodimeric complex with TLR6 or TLR1, thereby discriminating between di- or triacylated lipopeptides, respectively. Previously, we found that HEK293 cells transfected with bovine TLR2 (boTLR2) were able to respond to diacylated lipopeptides but did not recognize triacylated lipopeptides, even after cotransfection with the so far published sequence of boTLR1. In this study we now could show that primary bovine cells were in general able to detect triacylated lipopetides. A closer investigation of the boTLR1 gene locus revealed an additional ATG 195 base pairs upstream from the published start codon. Its transcription would result in an N-terminus with high identity to human and murine TLR1 (huTLR1, muTLR1). Cloning and cotransfection of this longer boTLR1 with boTLR2 now resulted in the recognition of triacylated lipopeptides by HEK293 cells, thereby resembling the ex vivo observation. Analysis of the structure-activity relationship showed that the ester-bound acid chains of these lipopeptides need to consist of at least 12 carbon atoms to activate the bovine heterodimer showing similarity to the recognition by huTLR2/huTLR1. In contrast, HEK293 cell cotransfected with muTLR2 and muTLR1 could already be activated by lipopeptides with shorter fatty acids of only 6 carbon atoms. Thus, our data indicate that the additional N-terminal nucleotides belong to the full length and functionally active boTLR1 (boTLR1-fl) which participates in a species-specific recognition of bacterial lipopeptides.