G protein-coupled estrogen receptor inhibits the P2Y receptor-mediated Ca(2+) signaling pathway in human airway epithelia.

P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (E2) receptor, may play an anti-inflammatory role in this process. We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca(2+) signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca(2+)]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5'-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca(2+) were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca(2+) mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.

P2Y6 receptor-mediated proinflammatory signaling in human bronchial epithelia.

P2Y receptors are expressed in virtually all epithelia and are responsible for the control of fluid and electrolyte transport. In asthmatic inflammation, the bronchial epithelia are damaged by eosinophil-derived, highly toxic cationic proteins, such as major basic protein (MBP). Consequently, extracellular nucleotides are released into the extracellular space from airway epithelial cells, and act in an autocrine or paracrine fashion to regulate immune functions. Our data show damage to the human bronchial epithelial cell line, 16HBE14o-, by poly-L-arginine-induced UDP release into the extracellular medium. Activation of P2Y6 receptor by its natural ligand, UDP, or its specific agonist, MRS 2693, led to the production of two proinflammatory cytokines, interleukin (IL)-6 and IL-8. This may have resulted from increased IL-6 and IL-8 mRNA expression, and activation of p38 and ERK1/2 MAPK, and NF-κB pathways. Our previous study demonstrated that UDP stimulated transepithelial Cl- secretion via both Ca2+- and cAMP-dependent pathways in 16HBE14o- epithelia. This was further confirmed in this study by simultaneous imaging of Ca2+ and cAMP levels in single cells using the Fura-2 fluorescence technique and a FRET-based approach, respectively. Moreover, the P2Y6 receptor-mediated production of IL-6 and IL-8 was found to be dependent on Ca2+, but not the cAMP/PKA pathway. Together, these studies show that nucleotides released during the airway inflammatory processes will activate P2Y6 receptors, which will lead to further release of inflammatory cytokines. The secretion of cytokines and the formation of such "cytokine networks" play an important role in sustaining the airway inflammatory disease.

Differential inhibitory effects of CysLT(1) receptor antagonists on P2Y(6) receptor-mediated signaling and ion transport in human bronchial epithelia.

BACKGROUND: Cysteinyl leukotriene (CysLT) is one of the proinflammatory mediators released by the bronchi during inflammation. CysLTs exert their biological effects via specific G-protein-coupled receptors. CysLT(1) receptor antagonists are available for clinical use for the treatment of asthma. Recently, crosstalk between CysLT(1) and P2Y(6) receptors has been delineated. P2Y receptors are expressed in apical and/or basolateral membranes of virtually all polarized epithelia to control the transport of fluid and electrolytes. Previous research suggests that CysLT(1) receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors. However, the detailed molecular mechanism underlying the inhibition remains unresolved. METHODOLOGY/PRINCIPAL FINDINGS: In this study, western blot analysis confirmed that both CysLT(1) and P2Y(6) receptors were expressed in the human bronchial epithelial cell line 16HBE14o-. All three CysLT(1) antagonists inhibited the uridine diphosphate (UDP)-evoked I(SC), but only montelukast inhibited the UDP-evoked [Ca(2+)](i) increase. In the presence of forskolin or 8-bromoadenosine 3'5' cyclic monophosphate (8-Br-cAMP), the UDP-induced I(SC) was potentiated but was reduced by pranlukast and zafirlukast but not montelukast. Pranlukast inhibited the UDP-evoked I(SC) potentiated by an Epac activator, 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2'-O-Me-cAMP), while montelukast and zafirlukast had no such effect. Pranlukast inhibited the real-time increase in cAMP changes activated by 8-CPT-2'-O-Me-cAMP as monitored by fluorescence resonance energy transfer imaging. Zafirlukast inhibited the UDP-induced I(SC) potentiated by N(6)-Phenyladenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-6-Phe-cAMP; a PKA activator) and UDP-activated PKA activity. CONCLUSIONS/SIGNIFICANCE: In summary, our data strongly suggest for the first time that in human airway epithelia, the three specific CysLT(1) receptor antagonists exert differential inhibitory effects on P2Y(6) receptor-coupled Ca(2+) signaling pathways and the potentiating effect on I(SC) mediated by cAMP and Epac, leading to the modulation of ion transport activities across the epithelia.

Polarized secretion of interleukin (IL)-6 and IL-8 by human airway epithelia 16HBE14o- cells in response to cationic polypeptide challenge.

BACKGROUND: The airway epithelium participates in asthmatic inflammation in many ways. Target cells of the epithelium can respond to a variety of inflammatory mediators and cytokines. Damage to the surface epithelium occurs following the secretion of eosinophil-derived, highly toxic cationic proteins. Moreover, the surface epithelium itself is responsible for the synthesis and release of cytokines that cause the selective recruitment, retention, and accumulation of various inflammatory cells. To mimic the damage seen during asthmatic inflammation, the bronchial epithelium can be challenged with highly charged cationic polypeptides such as poly-L-arginine. METHODOLOGY/PRINCIPAL FINDINGS: In this study, human bronchial epithelial cells, 16HBE14o- cells, were "chemically injured" by exposing them to poly-l-arginine as a surrogate of the eosinophil cationic protein. Cytokine antibody array data showed that seven inflammatory mediators were elevated out of the 40 tested, including marked elevation in interleukin (IL)-6 and IL-8 secretion. IL-6 and IL-8 mRNA expression levels were elevated as measured with real-time PCR. Cell culture supernatants from apical and basolateral compartments were collected, and the IL-6 and IL-8 production was quantified with ELISA. IL-6 and IL-8 secretion by 16HBE14o- epithelia into the apical compartment was significantly higher than that from the basolateral compartment. Using specific inhibitors, the production of IL-6 and IL-8 was found to be dependent on p38 MAPK, ERK1/2 MAPK, and NF-kappaB pathways. CONCLUSIONS/SIGNIFICANCE: The results clearly demonstrate that damage to the bronchial epithelia by poly-L-arginine stimulates polarized IL-6 and IL-8 secretion. This apically directed secretion of cytokines may play an important role in orchestrating epithelial cell responses to inflammation.

Apical versus basolateral P2Y(6) receptor-mediated Cl(-) secretion in immortalized bronchial epithelia.

Apical and/or basolateral membranes of polarized epithelia express P2Y receptors, which regulate the transport of fluid and electrolytes. In the airway, P2Y receptors modulate Cl(-) secretion through the phospholipase C and calcium signaling pathways. Recent evidence suggests that P2Y(6) receptors are expressed in bronchial epithelium and coupled to the cAMP/protein kinase A (PKA) pathways. We examined P2Y receptor subtype expression, including P2Y(6,) and the effect of extracellular nucleotides on basal short-circuit current (I(SC)) and intracellular calcium concentration ([Ca(2+)](i)) in a human bronchial epithelial cell line (16HBE14o-). Real-time PCR demonstrated P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor expression and confirmed that transcript levels were not altered when cells were grown under varied conditions. It was determined that P2Y agonists (ATP, UTP, UDP) stimulated a concomitant increase in I(SC) and [Ca(2+)](i). Apical nucleotides stimulated an increase in [Ca(2+)](i) more efficiently than basolateral nucleotides; however, P2Y agonistic effects on I(SC) were greater when applied basolaterally. Since the P2Y(6) receptors differentially regulate apical and basolateral UDP-induced I(SC) and [Ca(2+)](i), we investigated membrane-resident P2Y(6) receptor functions using Cl(-) or K(+) channels blockers. Apical and basolateral UDP activation of I(SC) was inhibited by applying DIDS apically or TRAM-34 and clotrimazole basolaterally. Although both apical and basolateral UDP increased PKA activity, only apical UDP-induced I(SC) was sensitive to a CFTR inhibitor. These data demonstrate that P2Y agonists stimulate Ca(2+)-dependent Cl(-) secretion across human bronchial epithelia and that the cAMP/PKA pathway regulates apical but not basolateral P2Y(6) receptor-coupled ion transport in human bronchial epithelia.

Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance.

The primary alcohol/aldehyde dehydrogenase (coded by the aad gene) is responsible for butanol formation in Clostridium acetobutylicum. We complemented the non-sporulating, non-solvent-producing C. acetobutylicum M5 strain (which has lost the pSOL1 megaplasmid containing aad and the acetone-formation genes) with aad expressed from the phosphotransbutyrylase promoter and restored butanol production to wild type levels. Because no acetone was produced, no acids (acetate or butyrate) were re-assimilated leading to high butyrate but especially acetate levels. To counter acetate production, we examined thiolase overexpression in order reduce the acetyl-CoA pool and enhance the butyryl-CoA pool. We combined thiolase overexpression with aad overexpression aiming to also enhance butanol formation. While limiting the formation of acetate and ethanol, the butanol titers were not improved. We also generated acetate kinase (AK) and butyrate kinase (BK) knockout (KO) mutants of M5 using a modified protocol to increase the antibiotic-resistance gene expression. These strains exhibited greater than 60% reduction in acetate or butyrate formation, respectively. We complemented the AKKO M5 strain with aad overexpression, but could not successfully transform the BKKO M5 strain. The AKKO M5 strain overexpressing aad produced less acetate, but also less butanol compared to the M5 aad overexpression strain. These data suggest that loss of the pSOL1 megaplasmid renders cells resistant to changes in the two acid-formation pathways, and especially so for butyrate formation. We argue that the difficulty in generating high butanol producers without acetone and acid production is hindered by the inability to control the electron flow, which appears to be affected by unknown pSOL1 genes.