Katharina F. Kubatzky

Modulation of host cell gene expression through activation of STAT transcription factors by Pasteurella multocida toxin.

The Pasteurella multocida toxin (PMT) is highly mitogenic and has potential carcinogenic properties. PMT causes porcine atrophic rhinitis that is characterized by bone resorption and loss of nasal turbinates, but experimental nasal infection also leads to excess proliferation of bladder epithelial cells. PMT acts intracellularly and activates phospholipase C-linked signals and MAPK pathways via the heterotrimeric Gαq and Gα12/13 proteins. We found that PMT induces activation of STAT proteins, and we identified STAT1, STAT3, and STAT5 as new targets of PMT-induced Gαq signaling. Inhibition of Janus kinases completely abolished STAT activation. PMT-dependent STAT phosphorylation remained constitutive for at least 18 h. PMT caused down-regulation of the expression of the suppressor of cytokine signaling-3, indicating a novel mechanism to maintain activation of STATs. Moreover, stimulation of Swiss 3T3 cells with PMT increased transcription of the cancer-associated STAT-dependent gene cyclooxygenase-2. Because constitutive activation of STATs has been found in a number of cancers, our findings offer a new mechanism for a carcinogenic role of PMT.

Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation

Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation.

Pasteurella multocida toxin is a potent activator of anti‐apoptotic signalling pathways

Toxigenic Pasteurella multocida strains produce a 146 kDa protein toxin (PMT) that due to its high mitogenic activity is thought to possess carcinogenic properties. PMT affects several signal transduction pathways related to cancer by constitutively stimulating heterotrimeric G proteins. Downstream of Gαq, Gα13 and Gαi, the toxin activates the small GTPase RhoA, MAP kinases and signal transducer and activator of transcription (STAT) proteins. PMT also stimulates Gβγ signalling and activates phosphoinositide 3‐kinase (PI3K)‐related pathways, which play a crucial role in proliferation and apoptosis. We show that treatment of HEK293 cells with PMT inhibits staurosporine‐mediated apoptosis through PI3K‐dependent phosphorylation of Akt and constitutive expression of Pim‐1 kinase. Simultaneous activation of these survival kinases allows the activation of pro‐survival pathways, such as GSK3β, Mcl‐1, Bcl‐xL and Bcl‐2, as well as the downregulation of apoptotic signals by Bax or Puma. Only the combined inhibition of Akt and Pim reverses the PMT‐induced protection from staurosporine‐induced apoptosis. In addition, we show that apoptosis induced by tumour chemotherapeutic agents is blocked by PMT in human cancer cell lines. Our data indicate that PMT is a highly potent anti‐apoptotic agent, which supports the view of a carcinogenic potential of the toxin.

Pasteurella multocida toxin-stimulated osteoclast differentiation is B cell dependent.

ABSTRACT Pasteurella multocida is a Gram-negative bacillus that infects a number of wild and domestic animals, causing respiratory diseases. Toxigenic Pasteurella multocida strains produce a protein toxin (PMT) that leads to atrophic rhinitis in swine due to enhanced osteoclastogenesis and the inhibition of osteoblast function. We show that PMT-induced osteoclastogenesis is promoted by an as-yet-uncharacterized B-cell population. The toxin, however, is not acting at the level of hematopoietic stem cells, since purified CD117+ cells from murine hematopoietic progenitor cells cultivated with PMT did not mature into osteoclasts. The early macrophages contained within this cell population (CD117+/CD11b+) did not further differentiate into osteoclasts but survived and were able to phagocytose. Within the CD117− population, however, we detected PMT-induced generation of a B220+/CD19+ and B220+/IgM+ B-cell population that was able to take up fluorescently labeled PMT. Using purified B-cell and macrophage populations, we show that these B cells are needed to efficiently generate osteoclasts from macrophages. Cells of the immune system are thought to affect osteoclast formation and function by secreting cytokines and growth factors. We show here that PMT-stimulated B cells produce elevated levels of the osteoclastogenic factors interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, and receptor activator of nuclear factor receptor ligand (RANKL) compared to B cells generated through incubation with IL-7. These results suggest that the osteoclastic properties characteristic for PMT may result from a cross talk between bone cells and lymphoid cells and that B cells might be an important target of Pasteurella multocida.

Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

BackgroundLipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins Gαq, Gα13 and Gαi independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement.ResultsHere we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gαi-triggered signalling as well as by Gβγ-dependent activation of PI3kinase and JNK.Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gβγ-mediated activation of PI3kinase and JNK activation.ConclusionsOn the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host’s immune response.

Granzyme A Produces Bioactive IL-1β through a Nonapoptotic Inflammasome-Independent Pathway

Bacterial components are recognized by the immune system through activation of the inflammasome, eventually causing processing of the proinflammatory cytokine interleukin-1? (IL-1?), a pleiotropic cytokine and one of the most important mediators of inflammation, through the protease caspase-1. Synthesis of the precursor protein and processing into its bioactive form are tightly regulated, given that disturbed control of IL-1? release can cause severe autoinflammatory diseases or contribute to cancer development. We show that the bacterial Pasteurella multocida toxin (PMT) triggers Il1b gene transcription in macrophages independently of Toll-like receptor signaling through RhoA/Rho-kinase-mediated NF-?? activation. Furthermore, PMT mediates signal transducer and activator of transcription (STAT) protein-controlled granzyme A (a serine protease) expression in macrophages. The exocytosed granzyme A enters target cells and mediates IL-1? maturation independently of caspase-1 and without inducing cytotoxicity. These findings show that macrophages can induce an IL-1?-initiated immune response independently of inflammasome activity.

Pasteurella multocida Toxin-induced Pim-1 expression disrupts suppressor of cytokine signalling (SOCS)-1 activity.

Pasteurella multocida Toxin (PMT) is a mitogenic protein toxin that manipulates signal transduction cascades of mammalian host cells and upregulates Janus kinase (JAK) and signal transducers of transcription (STAT) activity. Here we show that in the presence of PMT, increased levels of suppressors of cytokine signalling‐1 (SOCS‐1) proteins significantly enhance STAT activity. This occurs via PMT‐induced expression of the serine/threonine kinase Pim‐1 and subsequent threonine phosphorylation of SOCS‐1. The ability of SOCS‐1 to act as an E3 ubiquitin ligase is regulated by its phosphorylation status. Thus, the tyrosine kinase JAK2 cannot be marked for proteasomal degradation by threonine phosphorylated SOCS‐1. Consequently, the expression levels of JAK2 are increased, eventually leading to hyperactivity of JAK2 and its target, the transcription factor STAT3. Eventually this causes increased anchorage‐independent cell growth that correlates with the expression levels of SOCS‐1. Interestingly, endogenous SOCS‐1 production after Toll‐like receptor activation also causes STAT3 hyperactivation. Thus we hypothesize that P. multocida Toxin alters host cell signalling using mechanisms that have so far only been known to be employed by oncogenic viral kinases to avoid host immune defence mechanisms.

The haematopoietic GTPase RhoH modulates IL3 signalling through regulation of STAT activity and IL3 receptor expression.

BackgroundRhoH is a constitutively active member of the family of Rho GTPases. Its expression is restricted to the haematopoietic lineage, where it serves as a positive regulator for T cell selection and mast cell function and as a negative regulator for growth-related functions in other lineages. Here, we examined the activation of signal transducer and activator of transcription (STAT) proteins in response to stimulation with interleukin 3 (IL3).ResultsUsing the murine IL3-dependent cell line BaF3 we investigated the influence of RhoH protein expression levels on IL3-mediated cellular responses. RhoH overexpressing cells showed lower sensitivity to IL3 and decreased STAT5 activation. SiRNA-mediated repression of RhoH gene expression led to an increase in proliferation and STAT5 activity which correlated with an increased number of IL3 receptor α chain molecules, also known as CD123, expressed at the cell surface. Interestingly, these findings could be reproduced using human THP-1 cells as a model system for acute myeloid leukaemia, where low RhoH levels are known to be an unfavourable prognostic marker. Overexpression of RhoH on the other hand caused an induction of STAT1 activity and western blot analysis revealed that activated STAT1 is phosphorylated on Tyr701. STAT1 is known to induce apoptosis or cell cycle arrest and we detected an upregulation of cyclin-dependent kinase inhibitors (CDKI) p21Cip1 and p27Kip1 in RhoH overexpressing BaF3 cells.ConclusionsWe propose that RhoH functions as a negative regulator for IL3-induced signals through modulation of the JAK-STAT pathway. High levels of RhoH allow the IL3-dependent activation of STAT1 causing decreased proliferation through upregulation of p21Cip1 and p27Kip1. Low RhoH levels on the other hand led to an upregulation of IL3-dependent cell growth, STAT5 activity and an increase of CD123 surface expression, linking RhoH to a CD123/STAT5 phenotype that has been described in AML patients.

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 hosts 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 hosts immune system.

Toxin-induced RhoA Activity Mediates CCL1-triggered Signal Transducers and Activators of Transcription Protein Signaling

Background: Rho GTPases are involved in STAT-dependent transcription. However, the connecting signaling pathways are unknown. Results: Activated RhoA induces STAT3 phosphorylation via ROCK, JNK, CCL1 synthesis, and secretion. Conclusion: Auto/paracrine cytokine signaling is a link between RhoA and STAT3. Significance: The knowledge about the connection between RhoA and STAT signaling is crucial for understanding several diseases, especially cancer. 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.