Alterations of protein folding or Ca(2+) levels within the endoplasmic reticulum (ER) result in the unfolded-protein response (UPR), a process considered as an endogenous inducer of inflammation. Thereby, understanding how genetic factors modify UPR is particularly relevant in chronic inflammatory diseases such as asthma. Here we identified that ORMDL3, the only genetic risk factor recently associated to asthma in a genome wide study, alters ER-mediated Ca(2+) homeostasis and facilitates the UPR. Heterologous expression of human ER-resident transmembrane ORMDL3 protein increased resting cytosolic Ca(2+) levels and reduced ER-mediated Ca(2+) signaling, an effect reverted by co-expression with the sarco-endoplasmic reticulum Ca(2+) pump (SERCA). Increased ORMDL3 expression also promoted stronger activation of UPR transducing molecules and target genes while siRNA-mediated knock-down of endogenous ORMDL3 potentiated ER Ca(2+) release and attenuated the UPR. In conclusion, our findings are consistent with a model in which ORMDL3 binds and inhibits SERCA resulting in a reduced ER Ca(2+) concentration and increased UPR. Thus, we provide a first insight into the molecular mechanism explaining the association of ORMDL3 with proinflammatory diseases.

Mitochondria-associated membranes (MAMs) are central microdomains that fine-tune bioenergetics by the local transfer of calcium from the endoplasmic reticulum to the mitochondrial matrix. Here, we report an unexpected function of the endoplasmic reticulum stress transducer IRE1α as a structural determinant of MAMs that controls mitochondrial calcium uptake. IRE1α deficiency resulted in marked alterations in mitochondrial physiology and energy metabolism under resting conditions. IRE1α determined the distribution of inositol-1,4,5-trisphosphate receptors at MAMs by operating as a scaffold. Using mutagenesis analysis, we separated the housekeeping activity of IRE1α at MAMs from its canonical role in the unfolded protein response. These observations were validated in vivo in the liver of IRE1α conditional knockout mice, revealing broad implications for cellular metabolism. Our results support an alternative function of IRE1α in orchestrating the communication between the endoplasmic reticulum and mitochondria to sustain bioenergetics. Cellular organelles are no longer conceived as unconnected structures with isolated functions, but as dynamic and integrated compartments. The best-characterized membrane contact sites bridge the endoplasmic reticulum (ER) and mitochondria 1. The ER-the largest organelle in eukaryotic cells-controls protein folding, lipid synthesis and calcium storage. The folding capacity of the ER is constantly challenged by physiological demands and disease states. To sustain proteostasis, cells engage the unfolded protein response (UPR) 2 , a Carreras-Sureda et al.

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP 2 ), although the structural rearrangements occurring on PIP 2 binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP 2 binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence 121 KRWRK 125 , which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP 2 from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP 2 facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5′-6'-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP 2 -binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP 2 mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4-PIP 2 interacting site and conditions that deplete PIP 2 or restrict access of TRPV4 to PIP 2 -in the case of PACSIN3-change tail conformation and negatively affect channel activation by hypotonicity and heat.structure | regulation | thermosensitivity T he transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel that responds to osmotic (1-4), mechanical (5-7), and temperature stimulation (8), thereby contributing to many different physiological functions, including cellular (4, 9) and systemic volume homeostasis (10), vasodilation (11, 12), nociception (13), epithelial hydroelectrolyte transport (14), bladder voiding (15), ciliary beat frequency regulation (7,16,17), chondroprotection (18), and skeletal regulation (19). The osmotic (20) and mechanical (7, 16) sensitivity of TRPV4 depends on the activation of phospholipase A 2 and subsequent production of the arachidonic acid metabolite 5′-6′-epoxyeicosatrienoic acid (EET), whereas the mechanism leading to temperature-mediated activation (observed only in intact cells) is not known at present (21). EETindependent TRPV4 activation by membrane stretch in excised patches from oocytes also has been reported (22), in apparent contradiction to early reports claiming lack of activation by membrane stretch (1). Several studies have characterized TRPV4 domains implicated in channel regulation by calmodulin (23, 24), prot...

The widely distributed TRPV4 cationic channel participates in the transduction of both physical (osmotic, mechanical, and heat) and chemical (endogenous, plant-derived, and synthetic ligands) stimuli. In this chapter we will review TRPV4 expression, biophysics, structure, regulation, and interacting partners as well as physiological and pathological insights obtained in TRPV4 animal models and human genetic studies.

Macrophages exert potent effector functions against invading microorganisms but constitute, paradoxically, a preferential niche for many bacterial strains to replicate. Using a model of infection by Salmonella Typhimurium, we have identified a molecular mechanism regulated by the nuclear receptor LXR that limits infection of host macrophages through transcriptional activation of the multifunctional enzyme CD38. LXR agonists reduced the intracellular levels of NAD in a CD38-dependent manner, counteracting pathogen-induced changes in macrophage morphology and the distribution of the F-actin cytoskeleton and reducing the capability of non-opsonized Salmonella to infect macrophages. Remarkably, pharmacological treatment with an LXR agonist ameliorated clinical signs associated with Salmonella infection in vivo, and these effects were dependent on CD38 expression in bone-marrow-derived cells. Altogether, this work reveals an unappreciated role for CD38 in bacterial-host cell interaction that can be pharmacologically exploited by activation of the LXR pathway.

T lymphocytes rely on a Ca(2+) signal known as store-operated calcium entry (SOCE) for their activation. This Ca(2+) signal is generated by activation of a T-cell receptor, depletion of endoplasmic reticulum (ER) Ca(2+) stores and activation of Ca(2+) release-activated Ca(2+) currents (I(CRAC)). Here, we report that the ER protein orosomucoid like 3 (ORMDL3), the product of the ORMDL3 gene associated with several autoimmune and/or inflammatory diseases, negatively modulates I(CRAC), SOCE, nuclear factor of activated T cells nuclear translocation and interleukin-2 production. ORMDL3 inhibits the Ca(2+) influx mechanism at the outer mitochondrial membrane, resulting in a Ca(2+)-dependent inhibition of I(CRAC) and reduced SOCE. The effect of ORMDL3 could be mimicked by interventions that decreased mitochondrial Ca(2+) influx and reverted by buffering of cytosolic Ca(2+) or activation of mitochondrial Ca(2+) influx. In conclusion, ORMDL3 modifies key steps in the process of T-lymphocyte activation, providing a functional link between the genetic associations of the ORMDL3 gene with autoimmune and/or inflammatory diseases.

Disorders of water balance are among the most common and morbid of the electrolyte disturbances, and are reflected clinically as abnormalities in the serum sodium concentration. The transient receptor potential vanilloid 4 (TRPV4) channel is postulated to comprise an element of the central tonicity-sensing mechanism in the mammalian hypothalamus, and is activated by hypotonic stress in vitro. A nonsynonymous polymorphism in the TRPV4 gene gives rise to a Pro-to-Ser substitution at residue 19. We show that this polymorphism is significantly associated with serum sodium concentration and with hyponatremia (serum sodium concentration <135 mEq/L) in 2 non-Hispanic Caucasian male populations; in addition, mean serum sodium concentration is lower among subjects with the TRPV4 P19S allele relative to the wild-type allele. Subjects with the minor allele were 2.4؊6.4 times as likely to exhibit hyponatremia as subjects without the minor allele (after inclusion of key covariates). Consistent with these observations, a human TRPV4 channel mutated to incorporate the TRPV4 P19S polymorphism showed diminished response to hypotonic stress (relative to the wild-type channel) and to the osmotransducing lipid epoxyeicosatrienoic acid in heterologous expression studies. These data suggest that this polymorphism affects TRPV4 function in vivo and likely influences systemic water balance on a population-wide basis.association study ͉ osmoregulation ͉ sodium ͉ transient receptor potential ͉ cell volume regulation S ystemic osmolality is among the most tightly regulated of physiological parameters. In humans, aberrant water balance is associated with neurological dysfunction and death. Even subtle changes in systemic osmolality cause reversible defects in coordination and cognition (1, 2). Clinically, water balance is reflected in the serum (or plasma) sodium concentration. Water excess relative to total body sodium content results in hyponatremia, the most prevalent electrolyte abnormality in hospitalized patients (reviewed in refs. 3 and 4).In mammals, systemic water balance is regulated via the renal water-conserving role of the hormone arginine vasopressin. Release of arginine vasopressin from the posterior pituitary into the circulation is governed by the hypothalamic sensor(s) of systemic osmolality. Ample evidence suggests that the transient receptor potential channel, TRPV4, comprises an element of the central sensor of low osmolality. TRPV4 is the mammalian ortholog of a C. elegans osmosensing protein (5, 6). In rodents, the channel is expressed in the osmosensing nuclei of the brain (5), among other sites. In vitro, TRPV4 is activated by hypotonicity (5-7) and by a number of lipid agonists, including phorbol ester derivatives (8); the channel also participates in cell regulatory volume decrease (9, 10). Osmotic and mechanical sensitivity of TRPV4 is ultimately conferred by the arachidonic acid metabolites and epoxyeicosatrienoic acids (EET), following phospholipase A 2 activation (11-13). Other signaling pathways involving in...

Background: Knockdown of orosomucoid-like (ORMDL) proteins releases serine palmitoyltransferase (SPT) activity. Results: Significant changes in SPT activity were detected when all three ORMDLs were overexpressed. Sphingolipids do not modify SPT-ORMDL interaction but rearrange ORMDLs. Macrophages suppress ORMDLs to induce de novo ceramide synthesis. Conclusion: Coordinated ORMDL expression regulation strongly influences SPT activity. Significance: SPT-ORMDL complex presents transcriptional and post-translational regulation.