Receptor-mediated activation of CFTR via prostaglandin signaling pathways in the airway

Cystic fibrosis (CF) is a genetic disease caused by mutations of the gene encoding a cAMP-activated Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR modulator therapies consist of small-molecule drugs that rescue mutant CFTR. Regimens of single or combinations of CFTR modulators still rely on endogenous levels of cAMP to regulate CFTR activity. We investigated CFTR activation by the natural mediator prostaglandin E2 (PGE2) and lubiprostone and tested the hypothesis that receptor-mediated CFTR activators can be used in combination with currently available CFTR modulators to increase function of mutant CFTR. Primary cultured airway epithelia were assayed in Ussing chambers. Experimental CFTR activators and established CFTR modulators were applied for 24 h and/or acutely and analyzed for their effect on CFTR activity as measured by changes in short-circuit current (ISC). In non-CF airway epithelia, acute application of lubiprostone and PGE2 activated CFTR to levels comparable to forskolin. Pre-treatment (24 h) with antagonists to prostaglandin receptors EP2 and EP4 abolished the ability of lubiprostone to acutely activate CFTR. In F508del homozygous airway epithelia pre-treated with the triple combination of elexacaftor, tezacaftor, and ivacaftor (ELEXA/TEZ/IVA; i.e., Trikafta), acute application of lubiprostone was able to maximally activate CFTR. Prolonged (24 h) co-treatment of F508del homozygous epithelia with ELEXA/TEZ/IVA and lubiprostone increased acute CFTR activation by ~60% compared to treatment with ELEXA/TEZ/IVA alone. This work establishes the feasibility of targeting prostaglandin receptors to activate CFTR on the airway epithelia and demonstrates that co-treatment with lubiprostone can further restore modulator-rescued CFTR.

Gαs directly drives PDZ-RhoGEF signaling to Cdc42

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα13. Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gαs (GαsQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by Gs-coupled receptors, but not by those coupled to Gi or Gq, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gαs-binding region, PRG-linker. Active Gαs interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with GαsQ227L's ability to guide PRG's interaction with Cdc42. Endogenous Gs-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.

Prostaglandin receptors induce urothelial tumourigenesis as well as bladder cancer progression and cisplatin resistance presumably via modulating PTEN expression,

Background: We investigated the role of prostaglandin receptors (e.g. prostaglandin E2 receptor 2 (EP2), EP4) and the efficacy ofcelecoxib in urothelial tumourigenesis and cancer progression.Methods: We performed immunohistochemistry in bladder cancer (BC) tissue microarrays, in vitro transformation assay in anormal urothelial SVHUC line, and western blot/reverse transcription–polymerase chain reaction/cell growth assays in BC lines.Results: EP2/EP4 expression was elevated in BCs compared with non-neoplastic urothelial tissues and in BCs from those whowere resistant to cisplatin-based neoadjuvant chemotherapy. Strong positivity of EP2/EP4 in non-muscle-invasive tumours orpositivity of EP2/EP4 in muscle-invasive tumours strongly correlated with disease progression or disease-specific mortality,respectively. In SVHUC cells, exposure to a chemical carcinogen 3-methylcholanthrene considerably increased and decreased theexpression of EP2/EP4 and phosphatase and tensin homologue (PTEN), respectively. Treatment with selective EP2/EP4 antagonistor celecoxib also resulted in prevention in 3-methylcholanthrene-induced neoplastic transformation of SVHUC cells. In BC lines,EP2/EP4 antagonists and celecoxib effectively inhibited cell viability and migration, as well as augmented PTEN expression.Furthermore, these drugs enhanced the cytotoxic activity of cisplatin in BC cells. EP2/EP4 and PTEN were also elevated andreduced, respectively, in cisplatin-resistant BC sublines.Conclusions: EP2/EP4 activation correlates with induction of urothelial cancer initiation and outgrowth, as well aschemoresistance, presumably via downregulating PTEN expression.