TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle.

Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca(2+) signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca(2+) entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP-BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20ng/ml, 48h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10µM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca(2+) (EGTA; 1mM) or intracellular Ca(2+) (BAPTA; 5µM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca(2+) influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca(2+) and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.

NHERF-2 maintains endothelial homeostasis.

The Na(+)/H(+) exchanger regulatory factor-2 (NHERF-2) is an integral component of almost all endothelial cells (ECs), yet its endothelial function is not known. Here, we found that NHERF-2, is a key regulator of endothelial homeostasis because NHERF-2-silenced ECs proliferate at a much higher rate even in the absence of mitogens such as VEGF compared with control ECs. We further show that the hyperproliferation phenotype of NHERF-2-silenced EC is because of an accelerated cell cycle that is probably caused by a combination of the following factors: increased cytoplasmic calcium, increased expression of c-Myc, increased expression of cyclin D1, and reduced expression of p27. Using an experimental mouse model of human hemangioma, we found that the endothelial neoplasms derived from NHERF-2-silenced cells were much larger in volume than those derived from control cells. Thus, NHERF-2 is a negative regulator of endothelial proliferation and may have important roles in endothelial homeostasis and vascular modeling.

Dopamine inhibits pulmonary edema through the VEGF-VEGFR2 axis in a murine model of acute lung injury.

The neurotransmitter dopamine and its dopamine receptor D2 (D2DR) agonists are known to inhibit vascular permeability factor/vascular endothelial growth factor (VEGF)-mediated angiogenesis and vascular permeability. Lung injury is a clinical syndrome associated with increased microvascular permeability. However, the effects of dopamine on pulmonary edema, a phenomenon critical to the pathophysiology of both acute and chronic lung injuries, have yet to be established. Therefore, we sought to determine the potential therapeutic effects of dopamine in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Compared with sham-treated controls, pretreatment with dopamine (50 mg/kg body wt) ameliorated LPS-mediated edema formation and lowered myeloperoxidase activity, a measure of neutrophil infiltration. Moreover, dopamine significantly increased survival rates of LPS-treated mice, from 0-75%. Mechanistically, we found that dopamine acts through the VEGF-VEGFR2 axis to reduce pulmonary edema, as dopamine pretreatment in LPS-treated mice resulted in decreased serum VEGF, VEGFR2 phosphorylation, and endothelial nitric oxide synthase phosphorylation. We used D2DR knockout mice to confirm that dopamine acts through D2DR to block vascular permeability in our lung injury model. As expected, a D2DR agonist failed to reduce pulmonary edema in D2DR(-/-) mice. Taken together, our results suggest that dopamine acts through D2DR to inhibit pulmonary edema-associated vascular permeability, which is mediated through VEGF-VEGFR2 signaling and conveys protective effects in an ALI model.

Targeting GIPC/synectin in pancreatic cancer inhibits tumor growth.

PURPOSE: Various studies have shown the importance of the GAIP interacting protein, COOH-terminus (GIPC, also known as Synectin) as a central adaptor molecule in different signaling pathways and as an important mediator of receptor stability. GIPC/Synectin is associated with different growth-promoting receptors such as insulin-like growth factor receptor I (IGF-IR) and integrins. These interactions were mediated through its PDZ domain. GIPC/Synectin has been shown to be overexpressed in pancreatic and breast cancer. The goal of this study was to show the importance of GIPC/Synectin in pancreatic cancer growth and to evaluate a possible therapeutic strategy by using a GIPC-PDZ domain inhibitor. Furthermore, the effect of targeting GIPC on the IGF-I receptor as one of its associated receptors was tested. EXPERIMENTAL DESIGN: The in vivo effects of GIPC/Synectin knockdown were studied after lentiviral transduction of luciferase-expressing pancreatic cancer cells with short hairpin RNA against GIPC/Synectin. Additionally, a GIPC-PDZ--targeting peptide was designed. This peptide was tested for its influence on pancreatic cancer growth in vitro and in vivo. RESULTS: Knockdown of GIPC/Synectin led to a significant inhibition of pancreatic adenocarcinoma growth in an orthotopic mouse model. Additionally, a cell-permeable GIPC-PDZ inhibitor was able to block tumor growth significantly without showing toxicity in a mouse model. Targeting GIPC was accompanied by a significant reduction in IGF-IR expression in pancreatic cancer cells. CONCLUSIONS: Our findings show that targeting GIPC/Synectin and its PDZ domain inhibits pancreatic carcinoma growth and is a potential strategy for therapeutic intervention of pancreatic cancer.

Analysis of ErbB receptors in pulmonary carcinoid tumors.

PURPOSE: This study aimed to investigate the expression of the ErbB family of receptor tyrosine kinases in pulmonary typical carcinoid and atypical carcinoid tumors and to understand the role of epidermal growth factor receptor (EGFR) signaling in pulmonary carcinoid tumor proliferation. EXPERIMENTAL DESIGN: Surgically resected typical carcinoid (n = 24) and atypical carcinoid (n = 7) tumor tissues were analyzed by immunohistochemical staining for EGFR, ErbB2, ErbB3, and ErbB4. Sequencing of tumor DNA of exons 18 to 21 of the EGFR gene and the KRAS gene was carried out. Biochemical analysis of lung carcinoid cell lines was used to investigate EGFR signal transduction and response to erlotinib inhibition. RESULTS: The analysis showed that 45.8% of typical carcinoid and 28.6% of atypical carcinoid tumors express EGFR, 100% of the tumors lack expression of ErbB2, and 100% have moderate to intense staining for ErbB3 and ErbB4. Sequencing of tumor DNA of exons 18 to 21 of the EGFR gene revealed the absence of tyrosine kinase domain mutations in these tumors. Instead, 80.6% tumors harbored a synonymous single nucleotide polymorphism in exon 20. Because EGFR and KRAS mutations tend not to be present at the same time, we sequenced the KRAS gene from pulmonary carcinoid tumor DNA and found that 100% were wild-type. Using a lung carcinoid cell line that expresses EGFR, we found that erlotinib reduced proliferation by inhibiting EGFR signal transduction. CONCLUSIONS: Our findings suggest clinical potential for the use of EGFR inhibitors in the treatment of patients with pulmonary carcinoid tumors, particularly for patients with EGFR-positive pulmonary carcinoid tumors not amenable to surgical resection.

Biochemical requirements for PCBCK1 kinase activity, the Pneumocystis carinii MEKK involved in cell wall integrity.

Fungal cell wall assembly is a complicated process involving multiple enzymes and coordinated signaling pathways. The cell wall integrity MAPK pathway acts to stabilize the fungal cell wall during conditions of elevated temperature by regulation of glucan synthesis. The upstream kinase, BCK1, is a critical component of this pathway. Pneumonia is a significant cause of death from the fungal opportunistic pathogen Pneumocystis in immunocompromised states, especially with HIV infection. We have previously shown that PCBCK1 functions in the cell wall integrity pathway in yeast as a functional protein kinase. Kinases have specific requirements for enzymatic function which have not been investigated in fungi. Here we examine the biochemical requirements for PCBCK1 kinase activity expressed in Saccharomyces cerevisiae bck1Delta yeast. PCBCK1 requires 10 mM MgCl(2), pH 6, temperature 30 degrees C, and 10 microM ATP for kinase activity. Interference of the Pneumocystis cell wall integrity pathway is an attractive target for drug development since glucan synthesis machinery is not present in humans.

Expression analysis of PCSTE3, a putative pheromone receptor from the lung pathogenic fungus Pneumocystis carinii.

The fungal pathogen Pneumocystis carinii remains the most prevalent opportunistic infection in patients infected with HIV. Fungal pheromone receptors are seven transmembrane domain G-protein-coupled receptors which are expressed on specific mating types, and have ligand-binding extracellular domains for specific pheromones from cells of the opposite mating type. We have cloned and characterized PCSTE3 from P. carinii, which encodes a seven transmembrane domain protein orthologous to the Saccharomyces cerevisiae pheromone receptor Ste3. We detect PCSTE3 by indirect immunofluorescence using antibodies designed to extracellular domains of the receptor in yeast expressing the protein. Using a downstream Fus1-lacZ reporter gene, we determined that PCSTE3 does not recognize a- or alpha-factor pheromones as ligands for the receptor. We isolated P. carinii life cycle stages and examined PCSTE3 expression by immunofluorescence microscopy and flow cytometry, and found PCSTE3 expression exclusively on a population of trophic forms. PCSTE3 receptor expression was not found on cysts.

Complementation and characterization of the Pneumocystis carinii MAPK, PCM.

Mitogen-activated protein kinase (MAPK) pathways transfer environmental signals into intracellular events such as proliferation and differentiation. Fungi utilize a specific pheromone-induced MAPK pathway to regulate conjugation, formation of an ascus, and entry into meiosis. We have previously identified a MAPK, PCM, from the fungal opportunist Pneumocystis, responsible for causing severe pneumonia in patients with AIDS. In order to gain insight into the function of PCM, we expressed it in Saccharomyces cerevisiae deficient in pheromone signaling and tested activation and inhibition of this MAPK pathway. PCM restored pheromone signaling in S. cerevisiae fus3Delta kss1Delta mutants with alpha-factor pheromone (six-fold increase) and was not activated by osmotic stress. Signaling through this pathway decreased 2.5-fold with 10 microM U0126, and was unaffected with SB203580. We evaluated the conditions for native PCM kinase activity isolated from Pneumocystis carinii organisms and found that 0.1 mM MgCl2, pH 6.5, temperature 30-35 degrees C, and 10 microM ATP were optimal. The activity of PCM is significantly elevated in P. carinii trophic forms compared to cysts, implicating a role for PCM in the life cycle transition of P. carinii from trophic forms to cysts.