Effects of prostaglandin receptors EP2 and FP on the alterations of the trabecular meshwork alterations : implications in glaucoma

Le glaucome est défini par une dégénérescence du nerf optique dont le principal facteur de risque est l’hypertension oculaire due à des altérations du tissu trabéculaire. Les traitements incluent des agonistes du récepteur FP, les agonistes du récepteur EP2 pouvant également avoir des effets bénéfiques.Au cours de cette thèse, un modèle de cellules trabéculaires primaires humaines a été défini et les effets du latanoprost, un agoniste FP, et du butaprost, un agoniste EP2, ont été étudiés, d'une part, sur la survie des cellules trabéculaires, et d’autre part, sur la transition myofibroblastique. Il a été montré que l’activation du récepteur EP2 permet de protéger les cellules trabéculaires d’un stress du réticulum endoplasmique par une diminution de l’accumulation de p53 qui résulte en l’inhibition de l’expression de Puma. Enfin, le butaprost entraîne l’augmentation de l’expression de Bcl-2 et la phosphorylation de Bad qui participent à l’inhibition de l’apoptose.D’autre part, le latanoprost induit une contraction des cellules trabéculaires tandis que le butaprost inhibe la contraction induite par le TGF-B2. En revanche, les deux agonistes inhibent la déposition du collagène.En conclusion, indépendamment de leur effet hypotenseur connu, le latanoprost favoriserait l’acquisition par les cellules trabéculaires d’un phénotype contractile et l’activation du récepteur EP2 pourrait limiter le développement de la dysfonction trabéculaire en protégeant de la mort cellulaire et en favorisant une relaxation. Ces résultats suggèrent que la stimulation du récepteur EP2 pourrait limiter le développement du glaucome et serait plus favorable que les agonistes du récepteur FP.Glaucoma is defined as an optic neuropathy whose main risk factor is ocular hypertension due to alterations of the trabecular meshwork (TM). The first-line therapies for glaucoma are agonists of the FP receptor. Agonists of the EP2 receptor could also present beneficial effects.During the thesis project, a model of primary human TM cells has been defined and the effects of latanoprost, an FP agonist, and butaprost, an EP2 agonist, have been studied on, firstly, the survival of TM cells and, secondly, on the myofibroblast transition.We have shown that activation of the EP2 receptor protects TM cells from an endoplasmic reticulum stress by a decreased accumulation of p53 which results in the inhibition of Puma transcription. Finally, butaprost mediates an increased expression of Bcl-2 and an elevation of Bad phosphorylation which contribute to protection against TM cell death.Moreover, latanoprost induces TM cell contraction while butaprost inhibits TGF-B2-dependent contraction. On the other hand, both agonists inhibit collagen.In conclusion, independently of their hypotensive effects, latanoprost would favor the acquisition by TM cells of a contractile phenotype while stimulation of EP2 receptor could limit TM dysfunction by protecting against TM cell death and relaxing the tissue. These results suggest that activation of EP2 receptor could slow down or inhibit the course of glaucoma progression and would be more favorable than the FP agonists currently used

Prostaglandin EP2 receptor signaling protects human trabecular meshwork cells from apoptosis induced by ER stress through down-regulation of p53

International audienceE-prostanoid receptor subtype 2 (EP2) agonists are currently under clinical development as hypotensive agents for the treatment of ocular hypertension. However, the effects of EP2 receptor agonists on trabecular meshwork (TM) alterations leading to primary open-angle glaucoma (POAG) are still unknown. Here, we evaluated whether EP2 receptor activation exhibits protective functions on TM cell death induced by endoplasmic reticulum (ER) stress. We show that the EP2 receptor agonist butaprost protects TM cell death mediated by the ER stress inducer tunicamycin through a cyclic AMP (cAMP)-dependent mechanism, but independent of the classical cAMP sensors, protein kinase A and exchange proteins activated by cAMP. The ER stress-induced intrinsic apoptosis inhibited by the EP2 receptor agonist was correlated with a decreased accumulation of the cellular stress sensor p53. In addition, p53 down-regulation was associated with inhibition of its transcriptional activity, which led to decreased expression of the pro-apoptotic p53-upregulated modulator of apoptosis (PUMA). The stabilization of p53 by nutlin-3a abolished butaprost-mediated cell death protection. In conclusion, we showed that EP2 receptor activation protects against ER stress-dependent mitochondrial apoptosis through down-regulation of p53. The specific inhibition of this pathway could reduce TM alterations observed in POAG patients

NBEAL2 deficiency in humans leads to low CTLA-4 expression in activated conventional T cells

Abstract Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking-down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in the T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4-immunoglobulin therapy in patients with GPS and autoimmune disease