Interestingly, the same SNPs also lead to protection from asthma upon microbial exposure (51), suggesting that this locus is very environment-dependent and might be regulated by epigenetic modifications (53). None of the studies TG-02 (SB1317) addressing the relationship between ORMDL3 and asthma have so far studied experimental respiratory viral infections. systemic ceramide levels, but genetically interfering with expression does not result in altered experimental asthma. mRNA is also upregulated in murine asthma models, driven by ovalbumin (OVA), house dust mite (HDM) or (12,13). However, studies addressing the functional role of ORMDL3 in asthma generated conflicting conclusions. Both transgenic overexpression as well as genetic deficiency of can enhance key asthma features, whereas one study showed that deficiency suppressed only bronchial hyperreactivity (BHR) (14C16). Given the multitude of genetic association studies in humans, the currently prevailing hypothesis is still that ORMDL3 overexpression has a causal role in asthma development or progression. The molecular mechanism by which ORMDL3 contributes to asthma is still a matter of intense debate (6,8). ORMDL3 is member of an evolutionary conserved family of endoplasmic reticulum (ER)-residing proteins, and has two paralogues in vertebrates, ORMDL1 and ORMDL2, that have not been associated with asthma (17). In yeast, the ORM homologues are described as regulators of sphingolipid synthesis by controlling the activity of the rate limiting enzyme serine palmitoyl transferase (SPT) (18C23). In mammals however, ORMDLs lack the N-terminal phosphorylation site that is crucial for SPT regulation in yeast. Mammalian SPT activity seems to be affected only when all ORMDL paralogues are overexpressed or downregulated simultaneously (17,24C26), making it unlikely that SNPs in only influence asthma by SPT inhibition. As an ER-resident protein, ORMDL3 has also been described to affect calcium metabolism and the unfolded protein response, influencing cytokine secretion by structural or immune cells (6,12,27C29). However, most molecular studies on ORMDL3 Rabbit polyclonal to WWOX were performed and have led to contradictory results due to the use of different cell lines and distinct approaches to measure total sphingolipid synthesis and to control expression. Furthermore, many studies were performed on epithelial cells, macrophages, mast cells and eosinophils (6,12,13,29C31), whereas it has been recently demonstrated that chr17q12-21 SNPs affect expression most prominently in T-cells (9). Here, we addressed the role of ORMDL3 in SL metabolism and asthma in newly generated reporter mice, full KO mice (from a Bacterial Artificial Chromosome (BAC)-transgene (did not impact on key asthma parameters in various allergen driven asthma models. These data do not support the currently prevailing paradigm that drives asthma by interfering with SPT activity or sphingolipid homeostasis. Methods Mice gene (Fig. 1A). This construct contains a sequence that consists of an En2 splice acceptor site, an internal ribosome entry site, a LacZ sequence, a polyA-tail, a loxP site, and a neomycin coding sequence driven by a human -actin promoter that is flanked by 2 Flp recombinase target (FRT) sites. ORMDL3 knockout (reportermice as a useful tool to study ORMDL3 expressionA)mRNA expression levels in lungs from mice. Expression values are shown relative to means of the wildtype group. Data were pooled from 2 experiments (n=7,6,4; means +/-SEM). C)Western blot showing -galactosidase expression in liver, lung, brown adipose tissue (BAT) and white adipose tissue (WAT) in three individual reportermice. -tubulin was used as a loading control. D)transcript levels in lung, BAT, WAT and liver in wildtype mice. Expression values are shown relative to means of lung samples (means +/- SEM). E)Immunohistochemistry analysis of -galactosidase expression (blue) on lung OCT-inflated cryosections and WAT of reportermice. Periodic-acid Schiff staining was used as counterstaining. A = airway; Bv = blood vessel; Alv = alveoli. F)Scheme representing the acute house dust mite (HDM)-dependent asthma model. G)Western blot showing -galactosidase expression in lung tissue from mock- and HDM-challenged reportermice. Models of allergic asthma The HDM-induced asthma model was performed as described TG-02 (SB1317) TG-02 (SB1317) before (35). In brief, mice were sensitized intratracheally (i.t.) on day 0 with 1 g HDM extract (Greer Laboratories, Lenoir, USA) or saline, followed by 10 g intranasal (i.n.) challenges from day 6 to 10. On day 14, mice were euthanized by an overdose pentobarbital. In the chronic HDM-induced asthma model, mice were instilled i.n. with 10 ug HDM, or saline as a control, three times a week for 5 weeks. Asthma features were determined 3 days after the last challenge. In the (Greer Laboratories) three times a week for 3 weeks. All i.t. and i.n. treatments were given in 80 and 40 ul PBS, respectively, and under light isoflurane anesthesia. Bronchoalveolar lavage (BAL) was performed using 3x1ml of EDTA-containing PBS (0,5 mM). Blood was obtained from the iliac vein in non-coated Eppendorf tubes to.