1. Planar Cell Polarity protein network, which controls ciliogenesis and cilia function, is altered in human Cystic Fibrosis Bronchial Epithelial cells through response to endoplasmic reticulum stress Sabrina Noel, PhD Louvain centre for Toxicology and Applied Pharmacology Université Catholique de Louvain Brussels, Belgium

2. Mucociliary Clearance is defective in CF Frizzell & Pilewski, Nat Med. 2004 Cilia Mucus hydration Sneezing Cough Mucus composition Mucociliary clearance (MCC) is regulated by several mechanisms MCC is impaired in CF lung No structural abnormalities of cilia Cilia beating is absent from polarized CF-HBE cultures Disorganization of cilia function secondary to inflammation was shown in COPD, asthma and chronic bronchitis models Higher goblet cells (mucus) to ciliated cells ratio in CF airways (epithelial mucous metaplasia) Impairment of cilia activity at the epithelium level ???

3. Planar Cell Polarity (PCP) network PCP is a protein network expressed in various epithelial cell types PCP controls the orientation of specialized structures within the plane of the epithelial sheet Planar Cell Polarity (PCP) has been described in mammal lung epithelial cells as a crucial mechanism controlling ciliogenesis and cilia function (Vladar EK et al, Curr Biol 2012) Transgenic Mice invalidated for or overexpressing PCP genes suffered from diverse lung malfunction (MCC defect, ventilation failure…) (Tissir et al., Nat. neurosci. 2005 - Dean CH, Organogesis 2011) Scales of fish Feathers of bird Fur of mammals Sensory hairs on fruit fly wings

4. CELSR : Cadherin EGF lag Seven Pass G-type Receptor Vangl : Van Gogh-like FZD : Frizzled Pk : Prickle Dvl : Dishevelled INVS : Inversin INTU : Inturn Basal bodies Cilia Planar Cell polarity network regulates ciliogenesis and cilia function Localized at the intercellular junction Asymmetric distribution Transmembrane (core) proteins + intracellular effectors

5. Workplan Which PCP genes isoforms are expressed in HBEs ? Does CFTR genotype affect PCP genes expression ? Does rescuing F508del-CFTR trafficking defects modulate PCP gene expression ? Does ER stress inducers/inhibitors modulate PCP gene expression ?

6. Several PCP genes are expressed in HBEs Expression of isoforms of PCP core and effectors genes (real-time RT-PCR arrays) in HBEs cell lines

7. Several PCP genes are expressed in HBEs Expression of isoforms of PCP core and effectors genes (real-time RT-PCR arrays) in HBEs cell lines CELSR Vangl Prickle Frizzled Effectors genes und.

8. Low expression of CELSR3 is associated with F508del mutation Effect of CFTR genotype on PCP genes expression  CFBE41o- (F508del-CFTR)  16HBE-AS3 (CFTR -/-) CELSR3 expression was strongly down-regulated in CFBE41o- No change in CELSR3 expression in 16HBE-AS3 CELSR3 expression was strongly down-regulated in CF (homoz. F508del) primary HBEs

9. Rescue of F508del trafficking by small molecules is not associated with increased expression of CELSR3 VRT-325 and 4-PBA: Chemical chaperones Promote folding 4PBA suppresses ER stress (by enhancing ER folding capacity) 10µM, 24h except 4-PBA 5mM, 24h

10. ER stress inducer Tunicamycin causes decrease in CELSR3 expression No treatment Tunicamycin 16HBE14o-

11. ER stress inhibitor 4-PBA enhances expression of CELSR3 16HBE14o- CFBE41o-

12. Conclusions Expression of several genes controlling Planar Cell Polarity network is deregulated in CF-HBEs ER stress is associated with low expression of CELSR3 in bronchial epithelial cells Rescuing F508del-CFTR trafficking by small molecules do not systematically translate into increase in expression of CELSR3 suggesting that alleviating ER stress may be a key factor PCP network represents a novel ER stress dependent-pathway that could be critical for MCC in CF lung disease

13. Acknowledgments Our group: Wendy Delbart Barbara Dhooghe Mathilde Beka Pr Teresinha Leal Switch lab, Vrije Instituut for Biotechnologies, Leuven, Belgium Dr B Schmidt CFTR Modulator Chemical Compound program sponsored by CFFT Pr Bob Bridges Kim Hankin