Cellular ROS tolerance determines the effect of plumbagin on osteoclast differentiation.

Plumbagin is used in traditional medicine because of its anti-inflammatory and anti-microbial properties. As a naphthoquinone, plumbagin triggers the production of reactive oxygen species (ROS). In vitro cancer studies showed that plumbagin triggers apoptosis in cancer cells through ROS production. As cancer-mediated chronic inflammation can affect bone density, it was hypothesized that plumbagin might directly inhibit the formation of bone-resorbing osteoclasts. We previously showed that the effect of plumbagin on osteoclastogenesis differed between bone marrow-derived macrophages and the macrophage cell line RAW 264.7. Although RAW 264.7 macrophages are able to initiate the gene program required for osteoclastogenesis, only primary macrophages successfully differentiate into osteoclasts. Here, we show that RAW 264.7 cells are more sensitive toward plumbagin-induced apoptosis. In the presence of plumbagin and the cytokine RANKL, which triggers ROS production to drive osteoclastogenesis, RAW 264.7 macrophages produce increased amounts of ROS and die. Addition of the ROS scavenger N-acetyl cysteine prevented cell death, linking the failure to differentiate to increased ROS levels. RAW 264.7 cells show reduced expression of genes protective against oxidative stress, while primary macrophages have a higher tolerance toward ROS. Our data suggest that it is indispensable to consider cell (line)-intrinsic properties when studying phytochemicals.

Mechanistic insight of interleukin-9 induced osteoclastogenesis.

Interleukin (IL)-9 is an emerging player in the pathogenesis of various chronic inflammatory diseases including bone disorders like rheumatoid arthritis (RA) and psoriatic arthritis. Recently, IL-9 was shown to enhance the osteoclast formation and their function in RA. However, the mechanisms by which IL-9 influences osteoclastogenesis are not known. Therefore, in this study we aimed to unravel the direct and indirect ways by which IL-9 can influence osteoclast formation. We used mouse bone marrow precursor cells for checking the effect of IL-9 on osteoclast differentiation and its function. Next, IL-9 induced signalling pathway were checked in the process of osteoclastogenesis. T cells play an important role in enhancing osteoclastogenesis in inflammatory conditions. We used splenic T cells to understand the impact of IL-9 on the functions of T effector (Teff) and regulatory T (Treg) cells. Furthermore, the effect of IL-9 mediated modulation of the T cell response on osteoclasts was checked using a coculture model of T cells with osteoclast precursors. We showed that IL-9 enhanced osteoclast formation and its function. We found that IL-9 activates STAT3, P38 MAPK, ERK1/2, NFκB and we hypothesize that it mediates the effect on osteoclastogenesis by accelerating mitochondrial biogenesis. Additionally, IL-9 was observed to facilitate the functions of pro-osteoclastogenic IL-17 producing T cells, but inhibits the function of anti-osteoclastogenic Treg cells. Our observations suggest that IL-9 can influence osteoclastogenesis directly by modulating the signalling cascade in the precursor cells; indirectly by enhancing IL-17 producing T cells and by reducing the functions of Treg cells.

Negative impact of Interleukin-9 on synovial regulatory T cells in rheumatoid arthritis.

In Rheumatoid Arthritis (RA), regulatory T cells (Tregs) have been found to be enriched in the synovial fluid. Despite their accumulation, they are unable to suppress synovial inflammation. Recently, we showed the synovial enrichment of interleukin-9 (IL-9) producing helper T cells and its positive correlation with disease activity. Therefore, we investigated the impact of IL-9 on synovial Tregs in RA. Here, we confirmed high synovial Tregs in RA patients, however these cells were functionally impaired in terms of suppressive cytokine production (IL-10 and TGF-ß). Abrogating IL-9/ IL-9 receptor interaction could restore the suppressive cytokine production of synovial Tregs and reduce the synovial inflammatory T cells producing IFN-γ, TNF-α, IL-17. However, blocking these inflammatory cytokines failed to show any effect on IL-9 producing T cells, highlighting IL-9's hierarchy in the inflammatory network. Thus, we propose that blocking IL-9 might dampen synovial inflammation by restoring Tregs function and inhibiting inflammatory T cells.

Staphylococci planktonic and biofilm environments differentially affect osteoclast formation.

INTRODUCTION: The pathophysiology of chronic implant-related bone infections is characterized by an increase in osteoclast numbers and enhanced bone resorption. Biofilms are a major reason for chronicity of such infections as the biofilm matrix protects bacteria against antibiotics and impairs the function of immune cells. Macrophages are osteoclast precursor cells and therefore linked to inflammation and bone destruction. OBJECTIVE AND METHOD: Investigations on the impact of biofilms on the ability of macrophages to form osteoclasts are yet missing and we, therefore, analyzed the effect of Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) planktonic and biofilm environments on osteoclastogenesis using RAW 264.7 cells and conditioned media (CM). RESULTS: Priming with the osteoclastogenic cytokine RANKL before CM addition enabled the cells to differentiate into osteoclasts. This effect was highest in SE planktonic or SA biofilm CM. Simultaneous stimulation with CM and RANKL, however, suppressed osteoclast formation and resulted in formation of inflammation-associated multinucleated giant cells (MGCs) which was most pronounced in SE planktonic CM. CONCLUSION: Our data indicate that the biofilm environment and its high lactate levels are not actively promoting osteoclastogenesis. Hence, the inflammatory immune response against planktonic bacterial factors through Toll-like receptors seems to be the central cause for the pathological osteoclast formation. Therefore, immune stimulation or approaches that aim at biofilm disruption need to consider that this might result in enhanced inflammation-mediated bone destruction.

The phytochemical plumbagin reciprocally modulates osteoblasts and osteoclasts.

Bone metabolism is essential for maintaining bone mineral density and bone strength through a balance between bone formation and bone resorption. Bone formation is associated with osteoblast activity whereas bone resorption is linked to osteoclast differentiation. Osteoblast progenitors give rise to the formation of mature osteoblasts whereas monocytes are the precursors for multi-nucleated osteoclasts. Chronic inflammation, auto-inflammation, hormonal changes or adiposity have the potential to disturb the balance between bone formation and bone loss. Several plant-derived components are described to modulate bone metabolism and alleviate osteoporosis by enhancing bone formation and inhibiting bone resorption. The plant-derived naphthoquinone plumbagin is a bioactive compound that can be isolated from the roots of the Plumbago genus. It has been used as traditional medicine for treating infectious diseases, rheumatoid arthritis and dermatological diseases. Reportedly, plumbagin exerts its biological activities primarily through induction of reactive oxygen species and triggers osteoblast-mediated bone formation. It is plausible that plumbagin's reciprocal actions - inhibiting or inducing death in osteoclasts but promoting survival or growth of osteoblasts - are a function of the synergy with bone-metabolizing hormones calcitonin, Parathormone and vitamin D. Herein, we develop a framework for plausible molecular modus operandi of plumbagin in bone metabolism.

Report of the 24th Meeting on Signal Transduction 2021.

The annual meeting "Signal Transduction-Receptors, Mediators and Genes" of the Signal Transduction Society (STS) is an interdisciplinary conference which is open to all scientists sharing a common interest in the elucidation of the signaling pathways mediating physiological or pathological processes in the health and disease of humans, animals, plants, fungi, prokaryotes, and protists. The 24th meeting on signal transduction was held from 15 to 17 November 2021 in Weimar, Germany. As usual, keynote presentations by invited scientists introduced the respective workshops, and were followed by speakers chosen from the submitted abstracts. A special workshop focused on "Target Identification and Interaction". Ample time was reserved for the discussion of the presented data during the workshops. Unfortunately, due to restrictions owing to the SARS-CoV-2 pandemic, the poster sessions-and thus intensive scientific discussions at the posters-were not possible. In this report, we provide a concise summary of the various workshops and further aspects of the scientific program.

Ras Isoforms from Lab Benches to Lives-What Are We Missing and How Far Are We?

The central protein in the oncogenic circuitry is the Ras GTPase that has been under intense scrutiny for the last four decades. From its discovery as a viral oncogene and its non-oncogenic contribution to crucial cellular functioning, an elaborate genetic, structural, and functional map of Ras is being created for its therapeutic targeting. Despite decades of research, there still exist lacunae in our understanding of Ras. The complexity of the Ras functioning is further exemplified by the fact that the three canonical Ras genes encode for four protein isoforms (H-Ras, K-Ras4A, K-Ras4B, and N-Ras). Contrary to the initial assessment that the H-, K-, and N-Ras isoforms are functionally similar, emerging data are uncovering crucial differences between them. These Ras isoforms exhibit not only cell-type and context-dependent functions but also activator and effector specificities on activation by the same receptor. Preferential localization of H-, K-, and N-Ras in different microdomains of the plasma membrane and cellular organelles like Golgi, endoplasmic reticulum, mitochondria, and endosome adds a new dimension to isoform-specific signaling and diverse functions. Herein, we review isoform-specific properties of Ras GTPase and highlight the importance of considering these towards generating effective isoform-specific therapies in the future.

Plumbagin, a Biomolecule with (Anti)Osteoclastic Properties.

Plumbagin is a plant-derived naphthoquinone that is widely used in traditional Asian medicine due to its anti-inflammatory and anti-microbial properties. Additionally, plumbagin is cytotoxic for cancer cells due to its ability to trigger reactive oxygen species (ROS) formation and subsequent apoptosis. Since it was reported that plumbagin may inhibit the differentiation of bone resorbing osteoclasts in cancer-related models, we wanted to elucidate whether plumbagin interferes with cytokine-induced osteoclastogenesis. Using C57BL/6 mice, we unexpectedly found that plumbagin treatment enhanced osteoclast formation and that this effect was most pronounced when cells were pre-treated for 24 h with plumbagin before subsequent M-CSF/RANKL stimulation. Plumbagin caused a fast induction of NFATc1 signalling and mTOR-dependent activation of p70S6 kinase which resulted in the initiation of protein translation. In line with this finding, we observed an increase in RANK surface expression after Plumbagin stimulation that enhanced the responsiveness for subsequent RANKL treatment. However, in Balb/c mice and Balb/c-derived RAW264.7 macrophages, these findings could not be corroborated and osteoclastogenesis was inhibited. Our results suggest that the effects of plumbagin depend on the model system used and can therefore either trigger or inhibit osteoclast formation.

Report of the 23rd Meeting on Signal Transduction 2019-Trends in Cancer and Infection.

The annual meeting "Signal Transduction-Receptors, Mediators and Genes" of the Signal Transduction Society (STS) is an interdisciplinary conference open to all scientists sharing the common interest in elucidating the signalling pathways underlying the physiological or pathological processes in health and disease of humans, animals, plants, fungi, prokaryotes and protists. The 23rd meeting on signal transduction was held from 4-6 November 2019 in Weimar, Germany, and focused on "Trends in Cancer and Infection". As usual, keynote presentations by invited scientists introduced the respective workshops and were followed by speakers chosen from the submitted abstracts. Ample time had been reserved for discussion of the presented data during the workshops. In this report, we provide a concise summary of the various workshops and further aspects of the scientific program.

Report of the Signal Transduction Society Meeting 2018-Signaling: From Past to Future.

The annual meeting "Signal Transduction-Receptors, Mediators, and Genes" of the Signal Transduction Society (STS) is an interdisciplinary conference open to all scientists sharing the common interest in elucidating signaling pathways in physiological or pathological processes in humans, animals, plants, fungi, prokaryotes, and protists. On the occasion of the 20th anniversary of the STS, the 22nd joint meeting took place in Weimar from 5⁻7 November 2018. With the focus topic "Signaling: From Past to Future" the evolution of the multifaceted research concerning signal transduction since foundation of the society was highlighted. Invited keynote speakers introduced the respective workshop topics and were followed by numerous speakers selected from the submitted abstracts. All presentations were lively discussed during the workshops. Here, we provide a concise summary of the various workshops and further aspects of the scientific program.

An Update on Interleukin-9: From Its Cellular Source and Signal Transduction to Its Role in Immunopathogenesis.

Interleukin-9 (IL-9) is a pleiotropic cytokine and was primarily studied in the context of T helper 2 (TH2)-associated immuno-pathological conditions such as asthma and parasitic infections. There was a paradigm shift in the biology of IL-9 after the recent discovery of TH9 cells, a new subtype of TH cells which secrete IL-9 in copious amounts. This has resulted in renewed interest in this cytokine, which was neglected since discovery because it was considered it to be just another TH2 cytokine. Recent studies have shown that it has multiple cellular sources and is critically involved in the immune-pathogenesis of inflammatory diseases and in guarding immune tolerance. In this review, we will discuss its discovery, gene organization, cellular sources, and signaling pathways. Especially, we will give an update on the recent development regarding its relevance in the immune pathogenesis of human diseases.

Conceptual Evolution of Cell Signaling.

During the last 100 years, cell signaling has evolved into a common mechanism for most physiological processes across systems. Although the majority of cell signaling principles were initially derived from hormonal studies, its exponential growth has been supported by interdisciplinary inputs, e.g., from physics, chemistry, mathematics, statistics, and computational fields. As a result, cell signaling has grown out of scope for any general review. Here, we review how the messages are transferred from the first messenger (the ligand) to the receptor, and then decoded with the help of cascades of second messengers (kinases, phosphatases, GTPases, ions, and small molecules such as cAMP, cGMP, diacylglycerol, etc.). The message is thus relayed from the membrane to the nucleus where gene expression ns, subsequent translations, and protein targeting to the cell membrane and other organelles are triggered. Although there are limited numbers of intracellular messengers, the specificity of the response profiles to the ligands is generated by the involvement of a combination of selected intracellular signaling intermediates. Other crucial parameters in cell signaling are its directionality and distribution of signaling strengths in different pathways that may crosstalk to adjust the amplitude and quality of the final effector output. Finally, we have reflected upon its possible developments during the coming years.

From macrophage to osteoclast - How metabolism determines function and activity.

Osteoclasts are specialised cells that resorb bone and develop from the monocyte/macrophage lineage. While there is a wealth of information on the regulation of macrophage function through metabolic activity, the connection between osteoclast differentiation and metabolism is less well understood. Recent data show that mitochondria participate in switching macrophages from an inflammatory phenotype towards differentiation into osteoclasts. Additionally, it was found that reactive oxygen species (ROS) actively take place in osteoclast differentiation by acting as secondary signalling molecules. Bone resorption is an energy demanding process and differentiating osteoclasts triggers the biogenesis of mitochondria. In addition, the activity of specific OXPHOS components of macrophages and osteoclasts is differentially regulated. This review summarises our knowledge on macrophage-mediated inflammation, its impact on a cell's metabolic activity and its effect on osteoclast differentiation.

Report of the Signal Transduction Society Meeting 2017-Metabolism in Health and Disease.

The annual "Joint Meeting Signal Transduction-Receptors, Mediators and Genes" of the Signal Transduction Society (STS) aims to be an interdisciplinary forum for researchers who share a common interest in deciphering signal transduction pathways in normal or transformed cells, in health and disease, in humans and animal models, or in plants or bacteria. The special focus of the 21st annual Joint Meeting, which took place from 8-10 November 2017 in Weimar, was the topic "Metabolism in Health and Disease" and covered multiple aspects of this highly exciting and fast developing research field. Invited keynote speakers introduced the impact of metabolism on tumor immunology, immune cell signaling, and posttranslational modifications in three specific workshops to the audience. Various other aspects of signal transduction were intensively discussed in five additional workshops. Here, we give an overview of the various workshops and further aspects of the scientific program.

Analysis of the interplay between all-trans retinoic acid and histone deacetylase inhibitors in leukemic cells.

The treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) induces granulocytic differentiation. This process renders APL cells resistant to cytotoxic chemotherapies. Epigenetic regulators of the histone deacetylases (HDACs) family, which comprise four classes (I-IV), critically control the development and progression of APL. We set out to clarify the parameters that determine the interaction between ATRA and histone deacetylase inhibitors (HDACi). Our assays included drugs against class I HDACs (MS-275, VPA, and FK228), pan-HDACi (LBH589, SAHA), and the novel HDAC6-selective compound Marbostat-100. We demonstrate that ATRA protects APL cells from cytotoxic effects of SAHA, MS-275, and Marbostat-100. However, LBH589 and FK228, which have a superior substrate-inhibitor dissociation constant (Ki) for the class I deacetylases HDAC1, 2, 3, are resistant against ATRA-dependent cytoprotective effects. We further show that HDACi evoke DNA damage, measured as induction of phosphorylated histone H2AX and by the comet assay. The ability of ATRA to protect APL cells from the induction of p-H2AX by HDACi is a readout for the cytoprotective effects of ATRA. Moreover, ATRA increases the fraction of cells in the G1 phase, together with an accumulation of the cyclin-dependent kinase inhibitor p21 and a reduced expression of thymidylate synthase (TdS). In contrast, the ATRA-dependent activation of the transcription factors STAT1, NF-κB, and C/EBP hardly influences the responses of APL cells to HDACi. We conclude that the affinity of HDACi for class I HDACs determines whether such drugs can kill naïve and maturated APL cells.

Pasteurella multocida toxin- induced osteoclastogenesis requires mTOR activation.

BACKGROUND: Pasteurella multocida toxin (PMT) is a potent inducer of osteoclast formation. Pigs suffering from an infection with toxigenic Pasteurella multocida strains develop atrophic rhinitis characterised by a loss of turbinate bones and conchae. However, on the molecular level the process of bone loss remains largely uncharacterised. RESULTS: Recently it was found that PMT activates the serine/threonine kinase mammalian target of rapamycin (mTOR) in fibroblasts. Using RAW264.7 macrophages, we investigated the role of the mTOR complex 1 (mTORC1) in PMT-mediated osteoclast formation. PMT induces the differentiation of RAW264.7 macrophages into multinucleated, tartrate resistant acid phosphatase (TRAP) positive osteoclasts that are capable to resorb bone. In the presence of the mTORC1 inhibitor rapamycin, PMT was significantly less able to induce the formation of TRAP-positive osteoclasts. Accordingly, the resulting resorption of bone was strongly reduced. A major target of mTOR is the 70 kDa ribosomal protein S6 kinase 1 (p70 S6K1). Activated p70 S6K1 decreases the expression of programmed cell death protein 4 (PDCD4), a negative transcriptional regulator of osteoclastogenesis, at the protein and gene level. Ultimately this results in the activation of c-Jun, a component of the activator protein 1 (AP-1) complex, which is a major transcription factor for the induction of osteoclast-specific genes. We now demonstrate that c-Jun and its downstream target, the osteoclast-specific bone degrading protease cathepsin K, are upregulated upon PMT treatment in an mTOR-dependent manner. CONCLUSIONS: Activation of mTOR signalling plays a central role in the formation of osteoclasts through the bacterial toxin PMT. On the molecular level, PMT-induced activation of mTOR leads to down regulation of PDCD4, a known repressor of AP-1 complex, culminating in the activation of c-Jun, an essential transcription factor for triggering osteoclastogenesis.

Toxin-induced RhoA activity mediates CCL1-triggered signal transducers and activators of transcription protein signaling.

RhoA is reportedly involved in signal transducers and activators of transcription (STAT)-dependent transcription. However, the pathway connecting the GTPase and STAT signaling has not been characterized. Here, we made use of bacterial toxins, which directly activate Rho GTPases to analyze this pathway. Cytotoxic necrotizing factors (CNFs) are produced by pathogenic Escherichia coli strains and by Yersinia pseudotuberculosis. They activate small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. We show that RhoA activation leads to phosphorylation and activation of STAT3 and identify signal proteins involved in this pathway. RhoA-dependent STAT3 stimulation requires ROCK and Jun kinase activation as well as AP1-induced protein synthesis. The secretion of one or more factors activates the JAK-STAT pathway in an auto/paracrine manner. We identify CCL1/I-309 as an essential cytokine, which is produced and secreted upon RhoA activation and which is able to activate STAT3-dependent signaling pathways.

Pasteurella multocida and immune cells.

Pasteurella multocida was first discovered by Perroncito in 1878 and named after Louis Pasteur who first isolated and described this Gram-negative bacterium as the cause of fowl disease in 1880. Subsequently, P. multocida was also found to cause atrophic rhinitis in pigs, haemorrhagic septicaemia in cattle and respiratory diseases in many other animals. Among other factors such as lipopolysaccharide, outer membrane proteins and its capsule, the protein toxin (PMT) of P. multocida is an important virulence factor that determines the immunological response of the host's immune system. However, the exact molecular mechanisms taking place in cells of the innate and adaptive immune system are largely unknown for any of these virulence factors. Due to the obvious function of PMT on cells of the porcine skeletal system where it causes bone destruction, PMT was regarded as an osteolytic protein toxin. However, it remained unclear what the actual benefit for the bacteria would be. Recently, more attention was drawn to the osteoimmunological effects of PMT and the interplay between bone and immune cells. This review summarises the knowledge of effects of P. multocida virulence factors on the host's immune system.

Staphylococcus aureus planktonic but not biofilm environment induces an IFN-ß macrophage immune response via the STING/IRF3 pathway.

Chronic implant-related bone infections are a severe complication in orthopaedic surgery. Biofilm formation on the implant impairs the immune response, leading to bacterial persistence. In a previous study, we found that Staphylococcus aureus (SA) induced interferon regulatory factor 3 (IRF3) activation and Ifnb expression only in its planktonic form but not in the biofilm. The aim of this study was to clarify the role of the stimulator of interferon genes (STING) in this process. We treated RAW 264.7 macrophages with conditioned media (CM) generated from planktonic or biofilm cultured SA in combination with agonists or inhibitors of the cyclic GMP-AMP synthase (cGAS)/STING pathway. We further evaluated bacterial gene expression of planktonic and biofilm SA to identify potential mediators. STING inhibition resulted in the loss of IRF3 activation and Ifnb induction in SA planktonic CM, whereas STING activation induced an IRF3 dependent IFN-ß response in SA biofilm CM. The expression levels of virulence-associated genes decreased during biofilm formation, but genes associated with cyclic dinucleotide (CDN) synthesis did not correlate with Ifnb induction. We further observed that cGAS contributed to Ifnb induction by SA planktonic CM, although cGAS activation was not sufficient to induce Ifnb expression in SA biofilm CM. Our data indicate that the different degrees of virulence associated with SA planktonic and biofilm environments result in an altered induction of the IRF3 mediated IFN-ß response via the STING pathway. This finding suggests that the STING/IRF3/IFN-ß axis is a potential candidate as an immunotherapeutic target for implant-related bone infections.

Post-translational modulation of cell signalling through protein succinylation.

Cells need to adapt their activities to extra- and intracellular signalling cues. To translate a received extracellular signal, cells have specific receptors that transmit the signal to downstream proteins so that it can reach the nucleus to initiate or repress gene transcription. Post-translational modifications (PTMs) of proteins are reversible or irreversible chemical modifications that help to further modulate protein activity. The most commonly observed PTMs are the phosphorylation of serine, threonine, and tyrosine residues, followed by acetylation, glycosylation, and amidation. In addition to PTMs that involve the modification of a certain amino acid (phosphorylation, hydrophobic groups for membrane localisation, or chemical groups like acylation), or the conjugation of peptides (SUMOylation, NEDDylation), structural changes such as the formation of disulphide bridge, protein cleavage or splicing can also be classified as PTMs. Recently, it was discovered that metabolites from the tricarboxylic acid (TCA) cycle are not only intermediates that support cellular metabolism but can also modify lysine residues. This has been shown for acetate, succinate, and lactate, among others. Due to the importance of mitochondria for the overall fitness of organisms, the regulatory function of such PTMs is critical for protection from aging, neurodegeneration, or cardiovascular disease. Cancer cells and activated immune cells display a phenotype of accelerated metabolic activity known as the Warburg effect. This metabolic state is characterised by enhanced glycolysis, the use of the pentose phosphate pathway as well as a disruption of the TCA cycle, ultimately causing the accumulation of metabolites like citrate, succinate, and malate. Succinate can then serve as a signalling molecule by directly interacting with proteins, by binding to its G protein-coupled receptor 91 (GPR91) and by post-translationally modifying proteins through succinylation of lysine residues, respectively. This review is focus on the process of protein succinylation and its importance in health and disease.