Hsp70 promotes epithelial sodium channel functional expression by increasing its association with coat complex II and its exit from endoplasmic reticulum

The epithelial sodium channel (ENaC) plays an important role in the homeostasis of blood pressure and of the airway surface liquid, and inappropriate regulation of ENaC results in refractory hypertension (in Liddle syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsp70, is not completely understood. Building on the previous suggestion by our group that Hsp70 promotes ENaC functional and surface expression in Xenopus oocytes, we investigated the mechanism by which Hsp70 acts upon ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsp70, treatment with 1 or 2 μg/ml doxycycline increased total Hsp70 expression ~2-fold and ENaC functional expression ~1.4-fold. This increase in ENaC functional expression corresponded to an increase in ENaC expression at the apical surface of the cells and was not present when an ATPase-deficient Hsp70 was similarly overexpressed. The increase in functional expression was not due to a change in the rate at which ENaC was retrieved from the apical membrane. Instead, Hsp70 overexpression increased the association of ENaC with the Sec24D cargo recognition component of coat complex II, which carries protein cargo from the endoplasmic reticulum to the Golgi. These data support the hypothesis that Hsp70 promotes ENaC biogenesis and trafficking to the apical surface of epithelial cells.     

Classically activated macrophages use stable microtubules for matrix metalloproteinase-9 (MMP-9) secretion

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.     

Regulation of Tight Junction Resistance in T84 Monolayers by Elevation in Intracellular Ca2+: A Protein Kinase C Effect

Elevation in intracellular Ca²⁺ acting via protein kinase C (PKC) is shown to regulate tight junction resistance in T₈₄ cells, a human colon cancer line and a model Cl⁻ secretory epithelial cell. The Ca²⁺ ionophore A23187, which was used to increase the intracellular Ca²⁺ concentration, caused a decrease in tight junction resistance in a concentration- and time-dependent manner. Dual Na⁺/mannitol serosal-to-mucosal flux analysis performed across the T₈₄ monolayers treated with 2 μm A23187 revealed that A23187 increased both fluxes and that in the presence of ionophore there was a linear relationship between the Na⁺ and mannitol fluxes with a slope of 56.4, indicating that the decrease in transepithelial resistance was due to a decrease in tight junction resistance. Whereas there was no effect of 0.1 μm A23187, 1 or 2 μm produced a 55% decrease in baseline resistance in 1 hr and 10 μm decreased resistance more than 80%. The A23187-induced decrease in tight junction resistance was partially reversible by washing 3 times with a Ringer's-HCO₃ solution containing 1% BSA. The A23187 effect on resistance was dependent on intracellular Ca²⁺; loading the T₈₄ cells with the intracellular Ca²⁺ chelator BAPTA significantly reduced the decrease in tight junction resistance caused by A23187. This intracellular Ca²⁺ effect was mediated by protein kinase C and not calmodulin. While the protein kinase C antagonist H-7 totally prevented the action of A23187 on tight junction resistance, the Ca²⁺/calmodulin inhibitor W13 did not have any effect. Sphingosine, another inhibitor of PKC, partially reduced the A23187-induced decline in tight junction resistance. The PKC agonist PMA mimicked the A23187 effect on resistance, although the effect was delayed up to 1 hr after exposure. In addition, however, PMA also caused an earlier increase in resistance, indicating it had an additional effect in addition to mimicking the effect of elevating Ca²⁺. The effects of a phospholipase inhibitor (mepacrine) and of inhibitors of arachidonic acid metabolism (indomethacin for the cyclooxygenase pathway, NDGA for the lipoxygenase pathway, and SKF 525A for the epoxygenase pathway) on the A23187 action were also examined. None of these agents altered the A23187-induced decrease in resistance. Monolayers exposed to 2 μm A23187 for 1 hr were stained with fluorescein conjugated phalloidin, revealing that neighboring cells did not part one from another and that A23187 did not have a detectable effect on distribution of F-actin in the perijunctional actomyosin ring. The results indicate that elevation in intracellular Ca²⁺ decreases tight junction resistance in the T₈₄ monolayer, acting through protein kinase C by a mechanism which does not involve visible changes in the perijunctional actomyosin ring. Received: 14 July 1995/Revised: 25 September 1995     

Opposing roles of IL-10 in acute bacterial infection

Interleukin-10 (IL-10) is recognized as an anti-inflammatory cytokine that downmodulates inflammatory immune responses at multiple levels. In innate cells, production of this cytokine is usually triggered after pathogen recognition receptor (PRR) engagement by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patters (DAMPs), as well as by other soluble factors. Importantly, IL-10 is frequently secreted during acute bacterial infections and has been described to play a key role in infection resolution, although its effects can significantly vary depending on the infecting bacterium. While the production of IL-10 might favor host survival in some cases, it may also result harmful for the host in other circumstances, as it can prevent appropriate bacterial clearance. In this review we discuss the role of IL-10 in bacterial clearance and propose that this cytokine is required to recover from infection caused by extracellular or highly pro-inflammatory bacteria. Altogether, we propose that IL-10 drives excessive suppression of the immune response upon infection with intracellular bacteria or in non-inflammatory bacterial infections, which ultimately favors bacterial persistence and dissemination within the host. Thus, the nature of the bacterium causing infection is an important factor that needs to be taken into account when considering new immunotherapies that consist on the modulation of inflammation, such as IL-10. Indeed, induction of this cytokine may significantly improve the host’s immune response to certain bacteria when antibiotics are not completely effective.     

Transport via the transcytotic pathway makes prostasin available as a substrate for matriptase

The matriptase-prostasin proteolytic cascade is essential for epidermal tight junction formation and terminal epidermal differentiation. This proteolytic pathway may also be operative in a variety of other epithelia, as both matriptase and prostasin are involved in tight junction formation in epithelial monolayers. However, in polarized epithelial cells matriptase is mainly located on the basolateral plasma membrane whereas prostasin is mainly located on the apical plasma membrane. To determine how matriptase and prostasin interact, we mapped the subcellular itinerary of matriptase and prostasin in polarized colonic epithelial cells. We show that zymogen matriptase is activated on the basolateral plasma membrane where it is able to cleave relevant substrates. After activation, matriptase forms a complex with the cognate matriptase inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1 and is efficiently endocytosed. The majority of prostasin is located on the apical plasma membrane albeit a minor fraction of prostasin is present on the basolateral plasma membrane. Basolateral prostasin is endocytosed and transcytosed to the apical plasma membrane where a long retention time causes an accumulation of prostasin. Furthermore, we show that prostasin on the basolateral membrane is activated before it is transcytosed. This study shows that matriptase and prostasin co-localize for a brief period of time at the basolateral plasma membrane after which prostasin is transported to the apical membrane as an active protease. This study suggests a possible explanation for how matriptase or other basolateral serine proteases activate prostasin on its way to its apical destination.     

C terminus of the P2X7 receptor: treasure hunting

P2X receptor (P2XR) is a family of the ATP-gated ion channel family and can permeabilize the plasma membrane to small cations such as potassium, sodium, and calcium, resulting in cellular depolarization. There are seven P2XR that have been described and cloned, with 45% identity in amino acid sequence. Each P2X receptors has two transmembrane domains that are separated by an extracellular loop and an intracellular N and C terminus. Unlike the other P2X receptors, the P2X7R has a larger C terminus with an extra 200 amino acid residues compared with the other receptors. The C terminus of the P2X7R has been implicated in regulating receptor function including signaling pathway activation, cellular localization, protein–protein interactions, and post-translational modification (PTM). In the present review, we discuss the role of the P2X7R C terminus in regards to receptor function, describe the specific domains and motifs found therein and compare the C terminus sequence with others proteins to discover predicted domains or sites of PTM.     

Phosphatidylserine is involved in the ferrichrome-induced plasma membrane trafficking of Arn1 in Saccharomyces cerevisiae

Arn1 is an integral membrane protein that mediates the uptake of ferrichrome, an important nutritional source of iron, in Saccharomyces cerevisiae. In the absence of ferrichrome, Arn1p is sorted directly from the trans-Golgi network to the vacuolar lumen for degradation. In the presence of low levels of ferrichrome, the siderophore binds to a receptor domain on Arn1, triggering the redistribution of Arn1 to the plasma membrane. When extracellular ferrichrome levels are high, Arn1 cycles between the plasma membrane and intracellular vesicles. To further understand the mechanisms of trafficking of Arn1p, we screened 4580 viable yeast deletion mutants for mislocalization of Arn1-GFP using synthetic genetic array technology. We identified over 100 genes required for trans-Golgi network-to-vacuole trafficking of Arn1-GFP and only two genes, SER1 and SER2, required for the ferrichrome-induced plasma membrane trafficking of Arn1-GFP. SER1 and SER2 encode two enzymes of the major serine biosynthetic pathway, and the Arn1 trafficking defect in the ser1Δ strain was corrected with supplemental serine or glycine. Plasma membrane trafficking of Hxt3, a structurally related glucose transporter, was unaffected by SER1 deletion. Serine is required for the synthesis of multiple cellular components, including purines, sphingolipids, and phospholipids, but of these only phosphatidylserine corrected the Arn1 trafficking defects of the ser1Δ strain. Strains with defects in phospholipid synthesis also exhibited alterations in Arn1p trafficking, indicating that the intracellular trafficking of some transporters is dependent on the phospholipid composition of the cellular membranes.     

A Tyrosine-based Motif Localizes a Drosophila Vesicular Transporter to Synaptic Vesicles in Vivo

Vesicular neurotransmitter transporters must localize to synaptic vesicles (SVs) to allow regulated neurotransmitter release at the synapse. However, the signals required to localize vesicular proteins to SVs in vivo remain unclear. To address this question we have tested the effects of mutating proposed trafficking domains in Drosophila orthologs of the vesicular monoamine and glutamate transporters, DVMAT-A and DVGLUT. We show that a tyrosine-based motif (YXXY) is important both for DVMAT-A internalization from the cell surface in vitro, and localization to SVs in vivo. In contrast, DVGLUT deletion mutants that lack a putative C-terminal trafficking domain show more modest defects in both internalization in vitro and trafficking to SVs in vivo. Our data show for the first time that mutation of a specific trafficking motif can disrupt localization to SVs in vivo and suggest possible differences in the sorting of VMATs versus VGLUTs to SVs at the synapse.     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

Tissue specification and intracellular distribution of actin isoforms inVicia faba L.

Summary Two dimensional gel electrophoresis of total cell protein extracts from not expanded, and primary leaves, petioles, and roots ofVicia faba resulted in four actin isoforms at 43 kDa with pI values from 5.9 to 6.05. In contrast to root extracts, in all leaf extracts an additional immunoreactive polypeptide with a molecular mass of 51 kDa and pI 5.75 was detected. This polypeptide was present in high amounts in protein extracts of purified chloroplasts, whereas no actin isoform at 43 kDa could be demonstrated. Compared to the tissue extracts, two actin isoforms at 43 kDa with pI values of 5.9 and 6.0 were enriched, when purified plasma membranes and the membranous fraction of vacuoles were analysed. In contrast, the soluble protein fraction of the plasma membrane preparation contained only two isoactins with pI values of 5.95 and 6.05 and a molecular mass of 43 kDa. These results indicate, that the four actin isoforms at 43 kDa detected in all examined tissues ofV. faba fulfill different functions at specific intracellular compartments, for example, the anchorage of actin microfilaments to membranes.Abbreviations BSA bovine serum albumin - BCIP 5-bromo-4-chloro-3-indolyl phosphate - DDM n-decyl -D-maltopyranoside - EDTA ethylenediamine-tetraacetic acid - HG n-hexyl -D-glucopyranoside - IEF isoelectrical focusing - MES morpholinoethanesulfonic acid - 2-ME 2 mercaptoethanol - NBT nitro blue tetrazolium - pCMB p-chloromercuribenzoic acid - PVP polyvinylpyrrolidone - Tris tris (hydroxymethyl) aminomethane