Donna L. Cioffi

Contribution of endogenously expressed Trp1 to a Ca2+-selective, store-operated Ca2+ entry pathway

Previous work from our laboratory has demonstrated that infection with influenza A/Bangkok/1/79 (H3N2), a relatively mild strain of the virus, caused much more severe pneumonitis in selenium (Se)‐deficient mice than in Se‐adequate mice. Here we report that the increased virulence observed in the Se‐deficient mice is due to mutations in the influenza virus genome, resulting in a more virulent genotype. Most of the mutations occurred in the gene for the M1 matrix protein, an internal protein that is thought to be relatively stable. A total of 29 nucleotide changes were observed in this gene, and all 29 changes were identical in three separate isolates taken from three different Se‐deficient mice. In contrast, only one to three mutations were seen in the genes for the hemagglutinin or neuraminidase proteins, surface antigens that are known to be highly variable. Once the mutations have occurred, even hosts with normal nutritional status are susceptible to the newly virulent strain. This work, in conjunction with our earlier work with coxsackievirus, shows that specific nutritional deficiencies can have a profound impact on the genome of RNA viruses. Poor nutritional status in the host may contribute to the emergence of new viral strains.

Dominant regulation of interendothelial cell gap formation by calcium-inhibited type 6 adenylyl cyclase

Acute transitions in cytosolic calcium ([Ca2+]i) through store-operated calcium entry channels catalyze interendothelial cell gap formation that increases permeability. However, the rise in [Ca2+]i only disrupts barrier function in the absence of a rise in cAMP. Discovery that type 6 adenylyl cyclase (AC6; EC 4.6.6.1) is inhibited by calcium entry through store-operated calcium entry pathways provided a plausible explanation for how inflammatory [Ca2+]i mediators may decrease cAMP necessary for endothelial cell gap formation. [Ca2+]i mediators only modestly decrease global cAMP concentrations and thus, to date, the physiological role of AC6 is unresolved. Present studies used an adenoviral construct that expresses the calcium-stimulated AC8 to convert normal calcium inhibition into stimulation of cAMP, within physiologically relevant concentration ranges. Thrombin stimulated a dose-dependent [Ca2+]i rise in both pulmonary artery (PAECs) and microvascular (PMVEC) endothelial cells, and promoted intercellular gap formation in both cell types. In PAECs, gap formation was progressive over 2 h, whereas in PMVECs, gap formation was rapid (within 10 min) and gaps resealed within 2 h. Expression of AC8 resulted in a modest calcium stimulation of cAMP, which virtually abolished thrombin-induced gap formation in PMVECs. Findings provide the first direct evidence that calcium inhibition of AC6 is essential for endothelial gap formation.

Activation of the endothelial store-operated ISOC Ca2+ channel requires interaction of protein 4.1 with TRPC4

Store-operated calcium (SOC) entry represents the principal Ca2+ entry pathway into nonexcitable cells. Despite intensive investigation, mechanisms underlying activation of SOC entry have remained elusive. The endothelial ISOC channel is a Ca2+-selective SOC entry channel to which the transient receptor potential (TRP) proteins TRPC1 and TRPC4 contribute subunits. Activation of ISOC is specifically regulated by the spectrin–actin membrane skeleton; however, the nature of coupling between the ISOC channel and membrane skeleton is unknown. Here we demonstrate that protein 4.1 is an essential component of the ISOC channel gating mechanism. Protein 4.1 interacts with TRPC4 and the membrane skeleton. Deletion of the protein 4.1 binding domain on TRPC4 or peptide competition to the protein 4.1 binding domain prevents ISOC activation. These findings reveal that interaction of protein 4.1 with TRPC4 is required for activation of the endothelial ISOC channel.

On the endothelial cell ISOC

Ca2+ store depletion activates both Ca2+ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca2+ selective current, I(SOC). Indeed, I(SOC) is a relatively small inward Ca2+ current that exhibits an approximate +40mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I(SOC), although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I(SOC) requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca2+ store depletion and channel activation. Thus, at present findings indicate a TRPC4-protein 4.1 physical linkage regulates I(SOC) activation following Ca2+ store depletion.

Regulation of Endothelial Cell Barrier Function by Store-Operated Calcium Entry

Transient receptor potential (TRP) channels fulfill important and diverse signaling functions, and are generally conserved among species. The canonical subfamily of TRP proteins, TRPC channels, possesses 7 isoforms that combine in various ways to form heteromultimers. In endothelium, TRPC1 and TRPC4 form subunits of a channel that selectively conducts calcium. This channel is activated by calcium depletion in the endoplasmic reticulum, and thus TRPC1/TRPC4 forms the molecular basis of a store operated calcium entry pathway. TRPC4 interacts with protein 4.1, which tethers the channel to the membrane skeleton and represents a gating mechanism required for calcium permeation. In response to inflammatory agonists such as thrombin and bradykinin, the generation of inositol 1,4,5‐trisphosphate transiently depletes endoplasmic reticulum calcium and activates the TRPC1/TRPC4 channel. Calcium permeation through this channel triggers cytoskeletal reorganization that is necessary to disrupt the endothelial cell barrier and increase permeability. Thus, inhibition of the TRPC1/TRPC4 channel provides a putative anti‐inflammatory strategy.

Essential Role of a Ca2+-Selective, Store-Operated Current (ISOC) in Endothelial Cell Permeability. Determinants of the Vascular Leak Site

Store-operated calcium (SOC) entry is sufficient to disrupt the extra-alveolar, but not the alveolar, endothelial cell barrier. Mechanism(s) underlying such insensitivity to transitions in cytosolic calcium ([Ca2+]i) in microvascular endothelial cells are unknown. Depletion of stored Ca2+ activates a larger SOC entry response in extra-alveolar (pulmonary artery; PAECs) than alveolar (pulmonary microvascular; PMVECs) endothelial cells. In vivo permeation studies revealed that Ca2+ store depletion activates similar nonselective cationic conductances in PAECs and PMVECs, while only PAECs possess the calcium-selective, store-operated Ca2+ entry current, ISOC. Pretreatment with the type 4 phosphodiesterase inhibitor, rolipram, abolished thapsigargin-activated ISOC in PAECs, and revealed ISOC in PMVECs. Rolipram pretreatment shifted the thapsigargin-induced fluid leak site from extra-alveolar to alveolar vessels in the intact pulmonary circulation. Thus, our results indicate ISOC provides a [Ca2+]i source that is needed to disrupt the endothelial cell barrier, and demonstrate that intracellular events controlling ISOC activation coordinate the site-specific vascular response to inflammation.

Orai1 Determines Calcium Selectivity of an Endogenous TRPC Heterotetramer Channel

Rationale: Canonical transient receptor potential 4 (TRPC4) contributes to the molecular composition of a channel encoding for a calcium selective store-operated current, ISOC, whereas Orai1 critically comprises a channel encoding for the highly selective calcium release activated calcium current, ICRAC. However, Orai1 may interact with TRPC proteins and influence their activation and permeation characteristics. Endothelium expresses both TRPC4 and Orai1, and it remains unclear as to whether Orai1 interacts with TRPC4 and contributes to calcium permeation through the TPRC4 channel. Objective: We tested the hypothesis that Orai1 interacts with TRPC4 and contributes to the channels selective calcium permeation important for endothelial barrier function. Methods and Results: A novel method to purify the endogenous TRPC4 channel and probe for functional interactions was developed, using TRPC4 binding to protein 4.1 as bait. Isolated channel complexes were conjugated to anti-TRPC protein antibodies labeled with cy3-cy5 pairs. Förster Resonance Energy Transfer among labeled subunits revealed the endogenous protein alignment. One TRPC1 and at least 2 TRPC4 subunits constituted the endogenous channel (TRPC1/4). Orai1 interacted with TRPC4. Conditional Orai1 knockdown reduced the probability for TRPC1/4 channel activation and converted it from a calcium-selective to a nonselective channel, an effect that was rescued on Orai1 reexpression. Loss of Orai1 improved endothelial cell barrier function. Conclusion: Orai1 interacts with TRPC4 in the endogenous channel complex, where it controls TRPC1/4 activation and channel permeation characteristics, including calcium selectivity, important for control of endothelial cell barrier function.

Organization and function of the FKBP52 and FKBP51 genes.

Best established as components of steroid hormone receptor complexes, it is now clear that the large molecular weight immunophilins, FKBP52 and FKBP51, play important regulatory roles elsewhere in the cell. This review outlines what is known about the organization of the genes, FKBP4 and FKBP5, respectively, encoding these proteins and describes their diverse actions in the nervous system, reproduction, and cancer. The organization of FKBP4 and FKBP5 is very similar among the chordates, and gene expression is influenced by both genetic and epigenetic mechanisms. Recent studies identifying roles of FKBP52 and FKBP51 in the regulation of the microtubule-associated protein tau and microtubule assembly are discussed, as is their interaction with and influence on the transient receptor potential canonical (TRPC) subfamily of ion channel proteins.

Store-Operated Calcium Entry Channels in Pulmonary Endothelium: The Emerging Story of TRPCS and Orai1

Cells of diverse origin utilize shifts in cytosolic calcium concentrations as intracellular signals to elicit physiological responses. In endothelium, inflammatory first messengers increase cytosolic calcium as a signal to disrupt cell-cell borders and produce inter-cellular gaps. Calcium influx across the plasma membrane is required to initiate barrier disruption, although the calcium entry mechanism responsible for this effect remains poorly understood. This chapter highlights recent efforts to define the molecular anatomy of the ion channel responsible for triggering endothelial cell gap formation. Resolving the identity and function of this calcium channel will pave the way for new anti-inflammatory therapeutic targets.

Terminal sialic acids are an important determinant of pulmonary endothelial barrier integrity

The surface of vascular endothelium bears a glycocalyx comprised, in part, of a complex mixture of oligosaccharide chains attached to cell-surface proteins and membrane lipids. Importantly, understanding of the structure and function of the endothelial glycocalyx is poorly understood. Preliminary studies have demonstrated structural differences in the glycocalyx of pulmonary artery endothelial cells compared with pulmonary microvascular endothelial cells. Herein we begin to probe in more detail structural and functional attributes of endothelial cell-surface carbohydrates. In this study we focus on the expression and function of sialic acids in pulmonary endothelium. We observed that, although pulmonary microvascular endothelial cells express similar amounts of total sialic acids as pulmonary artery endothelial cells, the nature of the sialic acid linkages differs between the two cell types such that pulmonary artery endothelial cells express both α(2,3)- and α(2,6)-linked sialic acids on the surface (i.e., surficially), whereas microvascular endothelial cells principally express α(2,3)-linked sialic acids. To determine whether sialic acids play a role in endothelial barrier function, cells were treated with neuraminidases to hydrolyze sialic acid moieties. Disruption of cell-cell and cell-matrix adhesions was observed following neuraminidase treatment, suggesting that terminal sialic acids promote endothelial barrier integrity. When we measured transendothelial resistance, differential responses of pulmonary artery and microvascular endothelial cells to neuraminidase from Clostridium perfringens suggest that the molecular architecture of the sialic acid glycomes differs between these two cell types. Collectively our observations reveal critical structural and functional differences of terminally linked sialic acids on the pulmonary endothelium.