Olaf Pinkenburg

Increased Vascular Smooth Muscle Contractility in TRPC6−/− Mice

ABSTRACT Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6 − / − smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6 −/− smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.

Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

Regional alveolar hypoxia causes local vasoconstriction in the lung, shifting blood flow from hypoxic to normoxic areas, thereby maintaining gas exchange. This mechanism is known as hypoxic pulmonary vasoconstriction (HPV). Disturbances in HPV can cause life-threatening hypoxemia whereas chronic hypoxia triggers lung vascular remodeling and pulmonary hypertension. The signaling cascade of this vitally important mechanism is still unresolved. Using transient receptor potential channel 6 (TRPC6)-deficient mice, we show that this channel is a key regulator of acute HPV as this regulatory mechanism was absent in TRPC6−/− mice whereas the pulmonary vasoconstrictor response to the thromboxane mimetic U46619 was unchanged. Accordingly, induction of regional hypoventilation resulted in severe arterial hypoxemia in TRPC6−/− but not in WT mice. This effect was mirrored by a lack of hypoxia-induced cation influx and currents in smooth-muscle cells from precapillary pulmonary arteries (PASMC) of TRPC6−/− mice. In both WT and TRPC6−/− PASMC hypoxia caused diacylglycerol (DAG) accumulation. DAG seems to exert its action via TRPC6, as DAG kinase inhibition provoked a cation influx only in WT but not in TRPC6−/− PASMC. Notably, chronic hypoxia-induced pulmonary hypertension was independent of TRPC6 activity. We conclude that TRPC6 plays a unique and indispensable role in acute hypoxic pulmonary vasoconstriction. Manipulation of TRPC6 function may thus offer a therapeutic strategy for the control of pulmonary hemodynamics and gas exchange.

Pressure-induced and store-operated cation influx in vascular smooth muscle cells is independent of TRPC1

Among the classical transient receptor potential (TRPC) subfamily, TRPC1 is described as a mechanosensitive and store-operated channel proposed to be activated by hypoosmotic cell swelling and positive pipette pressure as well as regulated by the filling status of intracellular Ca2+ stores. However, evidence for a physiological role of TRPC1 may most compellingly be obtained by the analysis of a TRPC1-deficient mouse model. Therefore, we have developed and analyzed TRPC1−/− mice. Pressure-induced constriction of cerebral arteries was not impaired in TRPC1−/− mice. Smooth muscle cells from cerebral arteries activated by hypoosmotic swelling and positive pipette pressure showed no significant differences in cation currents compared to wild-type cells. Moreover, smooth muscle cells of TRPC1−/− mice isolated from thoracic aortas and cerebral arteries showed no change in store-operated cation influx induced by thapsigargin, inositol-1,4,5 trisphosphate, and cyclopiazonic acid compared to cells from wild-type mice. In contrast to these results, small interference RNAs decreasing the expression of stromal interaction molecule 1 (STIM1) inhibited thapsigargin-induced store-operated cation influx, demonstrating that STIM1 and TRPC1 are mutually independent. These findings also imply that, as opposed to current concepts, TRPC1 is not an obligatory component of store-operated and stretch-activated ion channel complexes in vascular smooth muscle cells.

Microbial DNA Induces a Host Defense Reaction of Human Respiratory Epithelial Cells

Epithelial cells represent the initial site of bacterial colonization in the respiratory tract. TLR9 has been identified in B cells and CD 123+ dendritic cells and found to be involved in the recognition of microbial DNA. It was the aim of the study to investigate the role of TLR9 in the host defense reactions of the respiratory epithelium. Respiratory epithelial cell lines (IHAEo−, Calu-3) or fully differentiated primary human cells as air-liquid interface cultures were stimulated with bacterial DNA or synthetic oligonucleotides containing CpG motifs (CpG oligodeoxynucleotides). Expression of TLR9, cytokines, and human β-defensin 2 was determined by quantitative RT-PCR or by ELISA. We found that TLR9 is expressed by respiratory epithelial cell lines and fully differentiated primary epithelial cells at low levels. Stimulation of the above-mentioned cells with bacterial DNA or CpG oligodeoxynucleotide resulted in an inflammatory reaction characterized by a dose-dependent up-regulation of cytokines (IL-6, IL-8) and human β-defensin 2. Up-regulation of NF-κB in epithelial cells in response to the CpG motif containing DNA was inhibited by overexpression of a dominant negative form of MyD88. These results provide clear evidence that the human respiratory epithelium is capable of detecting microbial DNA by TLR9. The respiratory epithelium has an important function in triggering innate immune responses and therefore represents an interesting target for anti-inflammatory therapy.

The antimicrobial peptide LL-37 modulates the inflammatory and host defense response of human neutrophils

The human cathelicidin antimicrobial peptide acts as an effector molecule of the innate immune system with direct antimicrobial and immunomodulatory effects. The aim of this study was to test whether the cathelicidin LL‐37 modulates the response of neutrophils to microbial stimulation. Human neutrophils were exposed to LPS, Staphylococcus aureus and Pseudomonas aeruginosa subsequent to incubation with LL‐37 and cytokine release was measured by ELISA. The incubation with LL‐37 significantly decreased the release of proinflammatory cytokines from stimulated human neutrophils. ROS production of neutrophils was determined by a luminometric and a flow cytometry method. The peptide induced the production of ROS and the engulfment of bacteria into neutrophils. Peritoneal mouse neutrophils isolated from CRAMP‐deficient and WT animals were treated with LPS and TNF‐α in the supernatant was measured by ELISA. Antimicrobial activity of neutrophils was detected by incubating neutrophils isolated from CRAMP‐knockout and WT mice with bacteria. Neutrophils from CRAMP‐deficient mice released significantly more TNF‐α after bacterial stimulation and showed decreased antimicrobial activity as compared to cells from WT animals. In conclusion, LL‐37 modulates the response of neutrophils to bacterial activation. Cathelicidin controls the release of inflammatory mediators while increasing antimicrobial activity of neutrophils.

Glutathione Primes T Cell Metabolism for Inflammation

&NA; Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc‐deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin‐1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc‐dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T‐cell‐specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses. Graphical Abstract Figure. No caption available. HighlightsGlutathione (GSH) is not needed for early T cell activation but promotes T cell growthGSH supports mTOR and NFAT activity and drives glycolysis and glutaminolysisGclc‐derived GSH buffers ROS and regulates Myc‐dependent metabolic reprogrammingAblation of Gclc in T cells impairs inflammatory responses in vivo &NA; Upon activation, T cells adapt their metabolism to meet their increased bioenergetic and biosynthetic needs. Activated T cells produce ROS, which trigger the antioxidative GSH response to prevent cellular damage. Mak et al. report that the GSH pathway plays an unexpected role in metabolic integration during inflammatory T cell responses.

Effect of an Oxygen-Tolerant Bifurcating Butyryl Coenzyme A Dehydrogenase/Electron-Transferring Flavoprotein Complex from Clostridium difficile on Butyrate Production in Escherichia coli

The butyrogenic genes from Clostridium difficile DSM 1296(T) have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro. While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD(+)-oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.

G13-dependent Activation of MAPK by Thyrotropin

Stimulation of the thyrotropin receptor (TSHR) activates G proteins of all four subfamilies (Gs, Gi/o, Gq/11, and G12/13). Whereas Gs/cAMP-dependent cellular responses upon TSHR stimulation are well established, other signaling pathways are less characterized. We evaluated TSH-elicited cellular responses in human follicular thyroid carcinoma cells stably expressing the TSHR and in primary, nonneoplastic human thyrocytes. In these cellular models, stimulation with TSH caused activation of p44/42 MAPK and subsequent induction of c-Fos. MAPK stimulation occurred independently of Gs, Gi/o, and Gq/11 signaling. Dominant negative constructs of G12 or G13 as well as shRNA-mediated suppression of Gα12 or Gα13 revealed that MAPK activation was dependent on G13 but not on G12 signaling. Furthermore, G13-dependent transactivation of the epidermal growth factor receptor was necessary for MAPK activation in follicular carcinoma cells, whereas EGFR was not involved in MAPK activation in nonneoplastic primary thyrocytes. The use of bacterial inhibitors of monomeric GTPases revealed that MAPK activation proceeded independently of Rho proteins but was clostridial toxin B-sensitive, suggesting involvement of Cdc42 or Rac. Thus, our data shed new light on cAMP-independent TSHR signaling and identify the first G13-dependent TSHR signaling pathway in human thyrocytes.

NF κB Activation in Embryonic Endothelial Progenitor Cells Enhances Neovascularization via PSGL-1 Mediated Recruitment: Novel Role for LL37†‡§

Embryonal endothelial progenitor cells (eEPCs) are capable of inducing therapeutic angiogenesis in a chronic hind limb model. However, the proportion of eEPCs recruited to the ischemic tissue appears to be a limiting step for the induction of cell‐based therapeutic neovascularization. In the present study, we primed eEPCs with the human cathelicidin LL37 (hCAP‐18) ex vivo to selectively enhance the eEPC‐dependent gain of perfusion in vivo and elucidated the mechanism of action of LL37 on eEPCs. Seven days after femoral artery excision, 5 × 106 eEPCs (wt, wild type; p65t, transiently p65 transient; p65s, stable p65‐transfected; LL37‐eEPCs, LL37 peptide preincubated) were retroinfused into the anterior tibial vein. Recruitment of diI‐labeled eEPCs in the ischemic gastrocnemic muscle was investigated 2 days later, whereas collateral growth and perfusion score (obtained by fluorescent microspheres) were assessed at day 7 and day 35 and are given as percentage of day 7 level. Capillary/muscle fiber ratio in the ischemic lower limb was obtained at day 35. Embryonic EPC recruitment in vitro and in vivo was found elevated after LL37 and p65t pretreatment, but not in p65s‐eEPCs displaying increased IκBα or after LL37 in IκB‐DN overexpressing eEPCs. Using LL37‐ and p65t‐eEPCs, collateral growth (181 ± 10% and 165 ± 8%, respectively) surpassed that of wt‐eEPCs (135 ± 7%), increasing perfusion ratio (208 ± 20% and 210 ± 17% vs. 142 ± 12% in wt‐eEPCs, respectively), whereas p65s‐eEPCs exerted no additive effect (collateral growth 130 ± 8%; perfusion ratio 155 ± 15%). Moreover, p65t‐eEPC‐induced neovascularization was abrogated by blocking antibodies against E‐selectin and P‐selectin glycoprotein ligand‐1 (PSGL‐1). We conclude that NF κB activation by LL37 or transient p65‐transfection increases functionally relevant eEPC recruitment to ischemic muscle tissue via induction of PSGL‐1 and E‐selectin. STEM CELLS 2010;28:376–385

Critical Role of Gα12 and Gα13 for Human Small Cell Lung Cancer Cell Proliferation In vitro and Tumor Growth In vivo

Purpose: In small cell lung cancer cells (SCLC), various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins. Although the contribution of the Gq/11-phospholipase C-β cascade to mitogenic effects in SCLC cells is well established, the relevance of G12/13 signaling is still elusive. In other tumor entities, G12/13 activation promotes invasiveness without affecting cellular proliferation. Here, we investigate the role of G12/13-dependent signaling in SCLC. Experimental Design: We used small hairpin RNA–mediated targeting of Gα12, Gα13, or both in H69 and H209 cells and analyzed the effects of Gα12 and/or Gα13 knockdown on tumor cells in vitro, tumor growth in vivo, and mitogen-activated protein kinase (MAPK) activation. Results: Lentiviral expression of small hairpin RNAs resulted in robust and specific Gα12 and Gα13 knockdown as well as markedly inhibited proliferation, colony formation, and bradykinin-promoted stimulation of cell growth. Analyzing the activation status of all three major MAPK families revealed nonredundant functions of Gα12 and Gα13 in SCLC and a marked p42/p44 activation upon Gα12/Gα13 knockdown. In a s.c. tumor xenograft mouse model, Gα12 or Gα13 downregulation led to decreased tumor growth due to reduced tumor cell proliferation. More importantly, Gα12/Gα13 double knockdown completely abolished H69 tumorigenicity in mice. Conclusions: Gα12 and Gα13 exert a complex pattern of nonredundant effects in SCLC, and in contrast to other tumor types, SCLC cell proliferation in vitro and tumorigenicity in vivo critically depend on G12/13 signaling. Due to the complete abolishment of tumorgenicity in our study, RNAi-mediated double knockdown may provide a promising new avenue in SCLC treatment. Clin Cancer Res; 16(5); 1402–15