The Effects of the Levosimendan Metabolites OR-1855 and OR-1896 on Endothelial Pro-Inflammatory Responses.

The calcium sensitizer levosimendan is used for the treatment of acute decompensated heart failure. A small portion (4-7%) of levosimendan is metabolized to the pharmacologically active metabolite OR-1896 via the inactive intermediate OR-1855. In addition, levosimendan has been shown to exert positive effects on the endothelium in vitro antagonizing vascular dysfunction and inflammation. However, the function of the levosimendan metabolites within this context is still unknown. In this study, we thus investigated the impact of the metabolites OR-1896 and OR-1855 on endothelial inflammatory processes in vitro. We observed a reduction of IL-1ß-dependent endothelial adhesion molecule ICAM-1 and VCAM-1 as well as interleukin (IL) -6 expression upon levosimendan treatment but not after treatment with OR-1855 or OR-1896, as assessed by western blotting, flow cytometry, and qRT-PCR. Instead, the metabolites impaired IL-1ß-induced ROS formation via inactivation of the MAPK p38, ERK1/2, and JNK. Our results suggest that the levosimendan metabolites OR-1896 and OR-1855 have certain anti-inflammatory properties, partly other than levosimendan. Importantly, they additionally show that the intermediate metabolite OR-1855 does, in fact, have pharmacological effects in the endothelium. This is interesting, as the metabolites are responsible for the long-term therapeutic effects of levosimendan, and heart failure is associated with vascular dysfunction and inflammation.

A Functional Network Driven by MicroRNA-125a Regulates Monocyte Trafficking in Acute Inflammation.

During the onset of acute inflammation, rapid trafficking of leukocytes is essential to mount appropriate immune responses towards an inflammatory insult. Monocytes are especially indispensable for counteracting the inflammatory stimulus, neutralising the noxa and reconstituting tissue homeostasis. Thus, monocyte trafficking to the inflammatory sites needs to be precisely orchestrated. In this study, we identify a regulatory network driven by miR-125a that affects monocyte adhesion and chemotaxis by the direct targeting of two adhesion molecules, i.e., junction adhesion molecule A (JAM-A), junction adhesion molecule-like (JAM-L) and the chemotaxis-mediating chemokine receptor CCR2. By investigating monocytes isolated from patients undergoing cardiac surgery, we found that acute yet sterile inflammation reduces miR-125a levels, concomitantly enhancing the expression of JAM-A, JAM-L and CCR2. In contrast, TLR-4-specific stimulation with the pathogen-associated molecular pattern (PAMP) LPS, usually present within the perivascular inflamed area, resulted in dramatically induced levels of miR-125a with concomitant repression of JAM-A, JAM-L and CCR2 as early as 3.5 h. Our study identifies miR-125a as an important regulator of monocyte trafficking and shows that the phenotype of human monocytes is strongly influenced by this miRNA, depending on the type of inflammatory stimulus.

Cell-Crossing Functional Network Driven by microRNA-125a Regulates Endothelial Permeability and Monocyte Trafficking in Acute Inflammation.

Opening of the endothelial barrier and targeted infiltration of leukocytes into the affected tissue are hallmarks of the inflammatory response. The molecular mechanisms regulating these processes are still widely elusive. In this study, we elucidate a novel regulatory network, in which miR-125a acts as a central hub that regulates and synchronizes both endothelial barrier permeability and monocyte migration. We found that inflammatory stimulation of endothelial cells induces miR-125a expression, which consecutively inhibits a regulatory network consisting of the two adhesion molecules VE-Cadherin (CDH5) and Claudin-5 (CLDN5), two regulatory tyrosine phosphatases (PTPN1, PPP1CA) and the transcription factor ETS1 eventually leading to the opening of the endothelial barrier. Moreover, under the influence of miR-125a, endothelial expression of the chemokine CCL2, the most predominant ligand for the monocytic chemokine receptor CCR2, was strongly enhanced. In monocytes, on the other hand, we detected markedly repressed expression levels of miR-125a upon inflammatory stimulation. This induced a forced expression of its direct target gene CCR2, entailing a strongly enhanced monocyte chemotaxis. Collectively, cell-type-specific differential expression of miR-125a forms a synergistic functional network controlling monocyte trafficking across the endothelial barrier towards the site of inflammation. In addition to the known mechanism of miRNAs being shuttled between cells via extracellular vesicles, our study uncovers a novel dimension of miRNA function: One miRNA, although disparately regulated in the cells involved, directs a biologic process in a synergistic and mutually reinforcing manner. These findings provide important new insights into the regulation of the inflammatory cascade and may be of great use for future clinical applications.

The mitochondrial thioredoxin reductase system (TrxR2) in vascular endothelium controls peroxynitrite levels and tissue integrity.

The mitochondrial thioredoxin/peroxiredoxin system encompasses NADPH, thioredoxin reductase 2 (TrxR2), thioredoxin 2, and peroxiredoxins 3 and 5 (Prx3 and Prx5) and is crucial to regulate cell redox homeostasis via the efficient catabolism of peroxides (TrxR2 and Trxrd2 refer to the mitochondrial thioredoxin reductase protein and gene, respectively). Here, we report that endothelial TrxR2 controls both the steady-state concentration of peroxynitrite, the product of the reaction of superoxide radical and nitric oxide, and the integrity of the vascular system. Mice with endothelial deletion of the Trxrd2 gene develop increased vascular stiffness and hypertrophy of the vascular wall. Furthermore, they suffer from renal abnormalities, including thickening of the Bowman's capsule, glomerulosclerosis, and functional alterations. Mechanistically, we show that loss of Trxrd2 results in enhanced peroxynitrite steady-state levels in both vascular endothelial cells and vessels by using a highly sensitive redox probe, fluorescein-boronate. High steady-state peroxynitrite levels were further found to coincide with elevated protein tyrosine nitration in renal tissue and a substantial change of the redox state of Prx3 toward the oxidized protein, even though glutaredoxin 2 (Grx2) expression increased in parallel. Additional studies using a mitochondria-specific fluorescence probe (MitoPY1) in vessels revealed that enhanced peroxynitrite levels are indeed generated in mitochondria. Treatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin [Mn(III)TMPyP], a peroxynitrite-decomposition catalyst, blunted intravascular formation of peroxynitrite. Our data provide compelling evidence for a yet-unrecognized role of TrxR2 in balancing the nitric oxide/peroxynitrite ratio in endothelial cells in vivo and thus establish a link between enhanced mitochondrial peroxynitrite and disruption of vascular integrity.

Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2.

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

The Role of Connexin 43 and Pannexin 1 During Acute Inflammation.

During acute inflammation, the recruitment of leukocytes from the blood stream into the inflamed tissue is a well-described mechanism encompassing the interaction of endothelial cells with leukocytes allowing leukocytes to reach the site of tissue injury or infection where they can fulfill their function such as phagocytosis. This process requires a fine-tuned regulation of a plethora of signaling cascades, which are still incompletely understood. Here, connexin 43 (Cx43) and pannexin 1 (Panx1) are known to be pivotal for the correct communication of endothelial cells with leukocytes. Pharmacological as well as genetic approaches provide evidence that endothelial Cx43-hemichannels and Panx1-channels release signaling molecules including ATP and thereby regulate vessel function and permeability as well as the recruitment of leukocytes during acute inflammation. Furthermore, Cx43 hemichannels and Panx1-channels in leukocytes release signaling molecules and can mediate the activation and function of leukocytes in an autocrine manner. The focus of the present review is to summarize the current knowledge of the role of Cx43 and Panx1 in endothelial cells and leukocytes in the vasculature during acute inflammation and to discuss relevant molecular mechanisms regulating Cx43 and Panx1 function.

Structure and Emerging Functions of LRCH Proteins in Leukocyte Biology.

Actin-dependent leukocyte trafficking and activation are critical for immune surveillance under steady state conditions and during disease states. Proper immune surveillance is of utmost importance in mammalian homeostasis and it ensures the defense against pathogen intruders, but it also guarantees tissue integrity through the continuous removal of dying cells or the elimination of tumor cells. On the cellular level, these processes depend on the precise reorganization of the actin cytoskeleton orchestrating, e.g., cell polarization, migration, and vesicular dynamics in leukocytes. The fine-tuning of the actin cytoskeleton is achieved by a multiplicity of actin-binding proteins inducing, e.g., the organization of the actin cytoskeleton or linking the cytoskeleton to membranes and their receptors. More than a decade ago, the family of leucine-rich repeat (LRR) and calponin homology (CH) domain-containing (LRCH) proteins has been identified as cytoskeletal regulators. The LRR domains are important for protein-protein interactions and the CH domains mediate actin binding. LRR and CH domains are frequently found in many proteins, but strikingly the simultaneous expression of both domains in one protein only occurs in the LRCH protein family. To date, one LRCH protein has been described in drosophila and four LRCH proteins have been identified in the murine and the human system. The function of LRCH proteins is still under investigation. Recently, LRCH proteins have emerged as novel players in leukocyte function. In this review, we summarize our current understanding of LRCH proteins with a special emphasis on their function in leukocyte biology.

The IL-1 Antagonist Anakinra Attenuates Glioblastoma Aggressiveness by Dampening Tumor-Associated Inflammation.

BACKGROUND: The recombinant IL-1 receptor antagonist anakinra-currently approved for the treatment of autoinflammatory diseases-blocks IL-1ß-mediated inflammatory signaling. As inflammation is a major driver of cancer, we hypothesized that anakinra might be able to mitigate glioblastoma (GBM) aggressiveness. METHODS: Primary GBM or T98G cells were incubated alone or with peripheral blood mononuclear cells (PBMCs) and were subsequently treated with IL-1ß and/or anakinra. T cells were obtained by magnetic bead isolation. Protein and mRNA expression were quantified by SDS-PAGE, qRT-PCR, and ELISA, respectively. Cell proliferation and apoptosis were analyzed via flow cytometry. Chemotaxis was studied via time-lapse microscopy. RESULTS: Upon IL-1ß stimulation, anakinra attenuated proinflammatory gene expression in both GBM cells and PBMCs, and mitigated tumor migration and proliferation. In a more lifelike model replacing IL-1ß stimulation by GBM-PBMC co-culture, sole presence of PBMCs proved sufficient to induce a proinflammatory phenotype in GBM cells with enhanced proliferation and migration rates and attenuated apoptosis. Anakinra antagonized these pro-tumorigenic effects and, moreover, reduced inflammatory signaling in T cells without compromising anti-tumor effector molecules. CONCLUSION: By dampening the inflammatory crosstalk between GBM and immune cells, anakinra mitigated GBM aggressiveness. Hence, counteracting IL-1ß-mediated inflammation might be a promising strategy to pursue.

The Phosphatase SHP-2 Activates HIF-1α in Wounds In Vivo by Inhibition of 26S Proteasome Activity.

Vascular remodeling and angiogenesis are required to improve the perfusion of ischemic tissues. The hypoxic environment, induced by ischemia, is a potent stimulus for hypoxia inducible factor 1α (HIF-1α) upregulation and activation, which induce pro-angiogenic gene expression. We previously showed that the tyrosine phosphatase SHP-2 drives hypoxia mediated HIF-1α upregulation via inhibition of the proteasomal pathway, resulting in revascularization of wounds in vivo. However, it is still unknown if SHP-2 mediates HIF-1α upregulation by affecting 26S proteasome activity and how the proteasome is regulated upon hypoxia. Using a reporter construct containing the oxygen-dependent degradation (ODD) domain of HIF-1α and a fluorogenic proteasome substrate in combination with SHP-2 mutant constructs, we show that SHP-2 inhibits the 26S proteasome activity in endothelial cells under hypoxic conditions in vitro via Src kinase/p38 mitogen-activated protein kinase (MAPK) signalling. Moreover, the simultaneous expression of constitutively active SHP-2 (E76A) and inactive SHP-2 (CS) in separate hypoxic wounds in the mice dorsal skin fold chamber by localized magnetic nanoparticle-assisted lentiviral transduction showed specific regulation of proteasome activity in vivo. Thus, we identified a new additional mechanism of SHP-2 mediated HIF-1α upregulation and proteasome activity, being functionally important for revascularization of wounds in vivo. SHP-2 may therefore constitute a potential novel therapeutic target for the induction of angiogenesis in ischemic vascular disease.

Molecular regulation of myoendothelial gap junctions.

Myoendothelial gap junctions are involved in the regulation of vascular tone. The major connexins described in the vascular system are Cx37, Cx40, Cx43, and Cx45 with all but Cx45 found in myoendothelial connections. Although many reports on post-translational modifications of these connexins are available, only few groups have investigated their role in controlling myoendothelial communication and signal propagation. In particular, myoendothelial gap junctions serve as essential feedback pathways between vascular smooth muscle cells and endothelial cells in the regulation of vessel responses. In conclusion, myoendothelial gap junctions coordinate and shift the overall response of vessels toward relaxation and consequently limit the constriction of vessels.

Inactivation of the tyrosine phosphatase SHP-2 drives vascular dysfunction in Sepsis.

BACKGROUND: Sepsis, the most severe form of infection, involves endothelial dysfunction which contributes to organ failure. To improve therapeutic prospects, elucidation of molecular mechanisms underlying endothelial vascular failure is of essence. METHODS: Polymicrobial contamination induced sepsis mouse model and primary endothelial cells incubated with sepsis serum were used to study SHP-2 in sepsis-induced endothelial inflammation. SHP-2 activity was assessed by dephosphorylation of pNPP, ROS production was measured by DCF oxidation and protein interactions were assessed by proximity ligation assay. Vascular inflammation was studied in the mouse cremaster model and in an in vitro flow assay. FINDINGS: We identified ROS-dependent inactivation of the tyrosine phosphatase SHP-2 to be decisive for endothelial activation in sepsis. Using in vivo and in vitro sepsis models, we observed a significant reduction of endothelial SHP-2 activity, accompanied by enhanced adhesion molecule expression. The impaired SHP-2 activity was restored by ROS inhibitors and an IL-1 receptor antagonist. SHP-2 activity inversely correlated with the adhesive phenotype of endothelial cells exposed to IL-1ß as well as sepsis serum via p38 MAPK and NF-κB. In vivo, SHP-2 inhibition accelerated IL-1ß-induced leukocyte adhesion, extravasation and vascular permeability. Mechanistically, SHP-2 directly interacts with the IL-1R1 adaptor protein MyD88 via its tyrosine 257, resulting in reduced binding of p85/PI3-K to MyD88. INTERPRETATION: Our data show that SHP-2 inactivation by ROS in sepsis releases a protective break, resulting in endothelial activation. FUND: German Research Foundation, LMU Mentoring excellence and FöFoLe Programme, Verein zur Förderung von Wissenschaft und Forschung, German Ministry of Education and Research.

Connexins in cancer: bridging the gap to the clinic.

Gap junctions comprise arrays of intercellular channels formed by connexin proteins and provide for the direct communication between adjacent cells. This type of intercellular communication permits the coordination of cellular activities and plays key roles in the control of cell growth and differentiation and in the maintenance of tissue homoeostasis. After more than 50 years, deciphering the links among connexins, gap junctions and cancer, researchers are now beginning to translate this knowledge to the clinic. The emergence of new strategies for connexin targeting, combined with an improved understanding of the molecular bases underlying the dysregulation of connexins during cancer development, offers novel opportunities for clinical applications. However, different connexin isoforms have diverse channel-dependent and -independent functions that are tissue and stage specific. This can elicit both pro- and anti-tumorigenic effects that engender significant challenges in the path towards personalised medicine. Here, we review the current understanding of the role of connexins and gap junctions in cancer, with particular focus on the recent progress made in determining their prognostic and therapeutic potential.

PKA negatively modulates the migration enhancing effect of Connexin 43.

Connexin 43 (Cx43) expression is associated with an increased cell migration and related changes of the actin cytoskeleton (enhanced filopodia formation). These effects are mediated by the C-terminal cytoplasmic part of Cx43 in a channel-independent manner. Since this part has been shown to interact with a variety of proteins and has multiple phosphorylation sites we analyzed here a potential role of the protein kinase A (PKA) for the Cx43 mediated increase in cell migration. Mutation of the PKA-phosphorylation site (substitution of three serines by alanine or glycine) resulted in a further increase in cell motility compared to wild-type Cx43, but with a loss of directionality. Likewise, cell motility was enhanced by PKA inhibition only in Cx43 expressing cells, while reduced in the presence of the PKA activator forskolin. In contrast, cell motility remained unaffected by stimulation with forskolin in cells expressing Cx43 with the mutated PKA phosphorylation site (Cx43-PKA) as well as in Cx-deficient cells. Moreover, PKA activation resulted in increased binding of PKA and VASP to Cx43 associated with an enhanced phosphorylation of VASP, an important regulatory protein of cell polarity and directed migration. Functionally, we could confirm these results in endothelial cells endogenously expressing Cx43. A Tat-Cx43 peptide containing the PKA phosphorylation site abolished the PKA dependent reduction in endothelial cell migration. Our results indicate that PKA dependent phosphorylation of Cx43 modulates cell motility and plays a pivotal role in regulating directed cell migration.

An update on minding the gap in cancer.

This article is a report of the "International Colloquium on Gap junctions: 50Years of Impact on Cancer" that was held 8-9 September 2016, at the Amphitheater "Pôle Biologie Santé" of the University of Poitiers (Poitiers, France). The colloquium was organized by M Mesnil (Université de Poitiers, Poitiers, France) and C Naus (University of British Columbia, Vancouver, Canada) to celebrate the 50th anniversary of the seminal work published in 1966 by Loewenstein and Kanno [Intercellular communication and the control of tissue growth: lack of communication between cancer cells, Nature, 116 (1966) 1248-1249] which initiated studies on the involvement of gap junctions in carcinogenesis. During the colloquium, 15 participants presented reviews or research updates in the field which are summarized below.

NO Augments Endothelial Reactivity by Reducing Myoendothelial Calcium Signal Spreading: A Novel Role for Cx37 (Connexin 37) and the Protein Tyrosine Phosphatase SHP-2.

OBJECTIVE: Because of its strategic position between endothelial and smooth muscle cells in microvessels, Cx37 (Connexin 37) plays an important role in myoendothelial gap junctional intercellular communication. We have shown before that NO inhibits gap junctional intercellular communication through gap junctions containing Cx37. However, the underlying mechanism is not yet identified. APPROACH AND RESULTS: Using channel-forming Cx37 mutants exhibiting partial deletions or amino acid exchanges in their C-terminal loops, we now show that the phosphorylation state of a tyrosine residue at position 332 (Y332) in the C-terminus of Cx37 controls the gap junction-dependent spread of calcium signals. Mass spectra revealed that NO protects Cx37 from dephosphorylation at Y332 by inhibition of the protein tyrosine phosphatase SHP-2. Functionally, the inhibition of gap junctional intercellular communication by NO decreased the spread of the calcium signal (induced by mechanical stimulation of individual endothelial cells) from endothelial to smooth muscle cells in intact vessels, while, at the same time, augmenting the calcium signal spreading within the endothelium. Consequently, preincubation of small resistance arteries with exogenous NO enhanced the endothelium-dependent dilator response to acetylcholine in spite of a pharmacological blockade of NO-dependent cGMP formation by the soluable guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). CONCLUSIONS: Our results identify a novel mechanism by which NO can increase the efficacy of calcium, rising vasoactive agonists in the microvascular endothelium.

HIF-1α Dependent Wound Healing Angiogenesis In Vivo Can Be Controlled by Site-Specific Lentiviral Magnetic Targeting of SHP-2.

Hypoxia promotes vascularization by stabilization and activation of the hypoxia inducible factor 1α (HIF-1α), which constitutes a target for angiogenic gene therapy. However, gene therapy is hampered by low gene delivery efficiency and non-specific side effects. Here, we developed a gene transfer technique based on magnetic targeting of magnetic nanoparticle-lentivirus (MNP-LV) complexes allowing site-directed gene delivery to individual wounds in the dorsal skin of mice. Using this technique, we were able to control HIF-1α dependent wound healing angiogenesis in vivo via site-specific modulation of the tyrosine phosphatase activity of SHP-2. We thus uncover a novel physiological role of SHP-2 in protecting HIF-1α from proteasomal degradation via a Src kinase dependent mechanism, resulting in HIF-1α DNA-binding and transcriptional activity in vitro and in vivo. Excitingly, using targeting of MNP-LV complexes, we achieved simultaneous expression of constitutively active as well as inactive SHP-2 mutant proteins in separate wounds in vivo and hereby specifically and locally controlled HIF-1α activity as well as the angiogenic wound healing response in vivo. Therefore, magnetically targeted lentiviral induced modulation of SHP-2 activity may be an attractive approach for controlling patho-physiological conditions relying on hypoxic vessel growth at specific sites.

Endothelial Dysfunction, and A Prothrombotic, Proinflammatory Phenotype Is Caused by Loss of Mitochondrial Thioredoxin Reductase in Endothelium.

OBJECTIVE: Although the investigation on the importance of mitochondria-derived reactive oxygen species (ROS) in endothelial function has been gaining momentum, little is known on the precise role of the individual components involved in the maintenance of a delicate ROS balance. Here we studied the impact of an ongoing dysregulated redox homeostasis by examining the effects of endothelial cell-specific deletion of murine thioredoxin reductase 2 (Txnrd2), a key enzyme of mitochondrial redox control. APPROACH AND RESULTS: We analyzed the impact of an inducible, endothelial cell-specific deletion of Txnrd2 on vascular remodeling in the adult mouse after femoral artery ligation. Laser Doppler analysis and histology revealed impaired angiogenesis and arteriogenesis. In addition, endothelial loss of Txnrd2 resulted in a prothrombotic, proinflammatory vascular phenotype, manifested as intravascular cellular deposits, as well as microthrombi. This phenotype was confirmed by an increased leukocyte response toward interleukin-1 in the mouse cremaster model. In vitro, we could confirm the attenuated angiogenesis measured in vivo, which was accompanied by increased ROS and an impaired mitochondrial membrane potential. Ex vivo analysis of femoral arteries revealed reduced flow-dependent vasodilation in endothelial cell Txnrd2-deficient mice. This endothelial dysfunction could be, at least partly, ascribed to inadequate nitric oxide signaling. CONCLUSIONS: We conclude that the maintenance of mitochondrial ROS via Txnrd2 in endothelial cells is necessary for an intact vascular homeostasis and remodeling and that Txnrd2 plays a vitally important role in balancing mitochondrial ROS production in the endothelium.

Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision.

Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H2S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H2S.

Cx43 increases serum induced filopodia formation via activation of p21-activated protein kinase 1.

In a previous study we could show that connexin 43 (Cx43) expression increased the migration of cells in a channel-independent manner involving the MAPK p38. We analyzed here the mechanism by which Cx43 enhanced p38 activation and migration related changes of the actin cytoskeleton. HeLa cells were used as a model system for the controlled expression of Cx43 and truncated Cx43 proteins. The expression of Cx43 altered the actin cytoskeleton organization in response to serum stimulation. Cx43 expressing HeLa cells had significantly more filopodial protrusions per cell than empty-vector transfected control cells. The expression of the channel incompetent carboxyl tail of Cx43 was sufficient to enhance the filopodia formation whereas the N-terminal, channel-building part, had no such effect. The enhanced filopodia formation was p38 dependent since the p38 blocker SB203580 significantly diminished it. Immunoprecipitation revealed an interaction of the upstream regulator of p38, p21-activated protein kinase 1 (PAK1), with Cx43 resulting in an enhanced phosphorylation of PAK1. Moreover, p38 activation, filopodia formation and cell migration were significantly reduced by blocking the PAK1 activity with its pharmacological inhibitor, IPA-3. The p38 target Hsp27, which favors the actin polymerization in its phosphorylated form, was significantly more phosphorylated characterizing it as a potential candidate molecule to enhance the serum-induced actin polymerization in Cx43 expressing cells. Our results provide a novel mechanism by which Cx43 can modify actin cytoskeletal dynamics and may thereby enhance cell migration.

Knockout of mitochondrial thioredoxin reductase stabilizes prolyl hydroxylase 2 and inhibits tumor growth and tumor-derived angiogenesis.

AIMS: Mitochondrial thioredoxin reductase (Txnrd2) is a central player in the control of mitochondrial hydrogen peroxide (H2O2) abundance by serving as a direct electron donor to the thioredoxin-peroxiredoxin axis. In this study, we investigated the impact of targeted disruption of Txnrd2 on tumor growth. RESULTS: Tumor cells with a Txnrd2 deficiency failed to activate hypoxia-inducible factor-1α (Hif-1α) signaling; it rather caused PHD2 accumulation, Hif-1α degradation and decreased vascular endothelial growth factor (VEGF) levels, ultimately leading to reduced tumor growth and tumor vascularization. Increased c-Jun NH2-terminal Kinase (JNK) activation proved to be the molecular link between the loss of Txnrd2, an altered mitochondrial redox balance with compensatory upregulation of glutaredoxin-2, and elevated PHD2 expression. INNOVATION: Our data provide compelling evidence for a yet-unrecognized mitochondrial Txnrd-driven, regulatory mechanism that ultimately prevents cellular Hif-1α accumulation. In addition, simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems was used as an efficient therapeutic approach in hindering tumor growth. CONCLUSION: This work demonstrates an unexpected regulatory link between mitochondrial Txnrd and the JNK-PHD2-Hif-1α axis, which highlights how the loss of Txnrd2 and the resulting altered mitochondrial redox balance impairs tumor growth as well as tumor-related angiogenesis. Furthermore, it opens a new avenue for a therapeutic approach to hinder tumor growth by the simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems.