Correction: ERp29 as a regulator of Insulin biosynthesis.

[This corrects the article DOI: 10.1371/journal.pone.0233502.].

ERp29 as a regulator of Insulin biosynthesis.

The environment within the Endoplasmic Reticulum (ER) influences Insulin biogenesis. In particular, ER stress may contribute to the development of Type 2 Diabetes (T2D) and Cystic Fibrosis Related Diabetes (CFRD), where evidence of impaired Insulin processing, including elevated secreted Proinsulin/Insulin ratios, are observed. Our group has established the role of a novel ER chaperone ERp29 (ER protein of 29 kDa) in the biogenesis of the Epithelial Sodium Channel, ENaC. The biogenesis of Insulin and ENaC share may key features, including their potential association with COP II machinery, their cleavage into a more active form in the Golgi or later compartments, and their ability to bypass such cleavage and remain in a less active form. Given these similarities we hypothesized that ERp29 is a critical factor in promoting the efficient conversion of Proinsulin to Insulin. Here, we confirmed that Proinsulin associates with the COP II vesicle cargo recognition component, Sec24D. When Sec24D expression was decreased, we observed a corresponding decrease in whole cell Proinsulin levels. In addition, we found that Sec24D associates with ERp29 in co-precipitation experiments and that ERp29 associates with Proinsulin in co-precipitation experiments. When ERp29 was overexpressed, a corresponding increase in whole cell Proinsulin levels was observed, while depletion of ERp29 decreased whole cell Proinsulin levels. Together, these data suggest a potential role for ERp29 in regulating Insulin biosynthesis, perhaps in promoting the exit of Proinsulin from the ER via Sec24D/COPII vesicles.

The KDEL receptor has a role in the biogenesis and trafficking of the epithelial sodium channel (ENaC).

Endoplasmic reticulum protein of 29 kDa (ERp29) is a thioredoxin-homologous endoplasmic reticulum (ER) protein that regulates the biogenesis of cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC). ERp29 may promote ENaC cleavage and increased open probability by directing ENaC to the Golgi via coat complex II (COP II) during biogenesis. We hypothesized that ERp29's C-terminal KEEL ER retention motif, a KDEL variant that is associated with less robust ER retention, strongly influences its regulation of ENaC biogenesis. As predicted by our previous work, depletion of Sec24D, the cargo recognition component of COP II that we previously demonstrated to interact with ENaC, decreases ENaC functional expression without altering ß-ENaC expression at the apical surface. We then tested the influence of KDEL ERp29, which should be more readily retrieved from the proximal Golgi by the KDEL receptor (KDEL-R), and a KEEL-deleted mutant (ΔKEEL ERp29), which should not interact with the KDEL-R. ENaC functional expression was decreased by ΔKEEL ERp29 overexpression, whereas KDEL ERp29 overexpression did not significantly alter ENaC functional expression. Again, ß-ENaC expression at the apical surface was unaltered by either of these manipulations. Finally, we tested whether the KDEL-R itself has a role in ENaC forward trafficking and found that KDEL-R depletion decreases ENaC functional expression, again without altering ß-ENaC expression at the apical surface. These results support the hypothesis that the KDEL-R plays a role in the biogenesis of ENaC and in its exit from the ER through its association with COP II. The cleavage of the extracellular loops of the epithelial sodium channel (ENaC) α and γ subunits increases the channel's open probability and function. During ENaC biogenesis, such cleavage is regulated by the novel 29-kDa chaperone of the ER, ERp29. Our data here are consistent with the hypothesis that ERp29 must interact with the KDEL receptor to exert its regulation of ENaC biogenesis. The classically described role of the KDEL receptor is to retrieve ER-retained species from the proximal Golgi and return them to the ER via coat complex I machinery. In contrast, our data suggest a novel and important role for the KDEL receptor in the biogenesis and forward trafficking of ENaC.

ERp29 regulates epithelial sodium channel functional expression by promoting channel cleavage.

The epithelial Na(+) channel (ENaC) plays a key role in the regulation of blood pressure and airway surface liquid volume. ERp29 is a 29-kDa thioredoxin-homologous endoplasmic reticulum (ER) protein that has only a single cysteine instead of the usual thioredoxin CXXC motif. Our group previously demonstrated that ERp29 promotes biogenesis of the cystic fibrosis transmembrane conductance regulator (CFTR). On the basis of similarities of CFTR and ENaC trafficking, we hypothesized that ERp29 would also regulate ENaC biogenesis and functional expression. In epithelial cells, overexpression of wild-type (wt) ERp29 increased ENaC functional expression [amiloride-sensitive short-circuit current (Isc)] in Ussing chamber experiments, as well as the abundance of the cleaved form of γ-ENaC in whole cell lysates. In contrast, siRNA-mediated depletion of ERp29 or overexpression of a mutant ERp29 lacking its single cysteine (C157S ERp29) decreased ENaC functional expression. Cells in which wt ERp29 was overexpressed had a smaller fractional increase in amiloride-sensitive Isc when trypsin was applied to the apical surface to activate uncleaved ENaC, while cells in which C157S ERp29 was overexpressed or ERp29 was depleted had a significantly greater fractional increase in amiloride-sensitive Isc in response to trypsin. Interestingly, these observations were not associated with altered expression of ß-ENaC at the apical surface. Instead, ERp29 appeared to promote the interaction of ß-ENaC with the Sec24D cargo recognition component of the coat complex II ER exit machinery. Together, these data support the hypothesis that ERp29 directs ENaC toward the Golgi, where it undergoes cleavage during its biogenesis and trafficking to the apical membrane.

Hsc70 negatively regulates epithelial sodium channel trafficking at multiple sites in epithelial cells.

The epithelial sodium channel (ENaC) plays an important role in homeostasis of blood pressure and of the airway surface liquid, and excess function of ENaC results in refractory hypertension (in Liddle's syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsc70, is not completely understood. Our previously published data suggest that Hsc70 negatively affects ENaC activity and surface expression in Xenopus oocytes; here we investigate the mechanism by which Hsc70 acts on ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αßγ-ENaC and with tetracycline-inducible overexpression of Hsc70, treatment with 5 µg/ml doxycycline increased total Hsc70 expression 20%. This increase in Hsc70 expression led to a decrease in ENaC activity and surface expression that corresponded to an increased rate of functional ENaC retrieval from the cell surface. In addition, Hsc70 overexpression decreased the association of newly synthesized ENaC subunits. These data support the hypothesis that Hsc70 inhibits ENaC functional expression at the apical surface of epithelia by regulating ENaC biogenesis and ENaC trafficking at the cell surface.

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 αßγ-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.

4-Phenylbutyrate stimulates Hsp70 expression through the Elp2 component of elongator and STAT-3 in cystic fibrosis epithelial cells.

Sodium 4-phenylbutyrate (4PBA) corrects trafficking of ΔF508-CFTR in Cystic Fibrosis (CF) epithelia, which is hypothesized to, at least in part, result from increased expression of Hsp70 (stress-induced 70 kDa heat shock protein). To identify other 4PBA-regulated proteins that may promote correction of ΔF508 trafficking, we performed differential display RT-PCR on mRNA from IB3-1 CF bronchiolar epithelial cells treated for 0-24 h with 1 mM 4PBA. In this screen, a STAT-3 (signal transducer and activator of transcription-3)-interacting protein, StIP-1 that regulates STAT-3 activation had transiently increased expression. StIP-1 is identical to Elongator protein 2 (Elp2), a component of the Elongator complex that regulates RNA polymerase II. Previous studies have suggested that Elongator regulates Hsp70 mRNA transcription, and that the Hsp70 promoter contains functional STAT-3-binding sites. We therefore tested the hypothesis that 4PBA increases Hsp70 expression by an Elongator- and STAT-3-dependent mechanism. 4PBA treatment of IB3-1 CF bronchiolar epithelial cells caused transiently increased expression of Hsp70 protein, as well as Elp2 protein and mRNA. Elp2 depletion by transfection of small interfering RNAs, reduced both Elp2 and Hsp70 protein expression. 4PBA also caused transient activation of STAT-3, and increased abundance of nuclear proteins that bind to the STAT-3-responsive element of the Hsp70 promoter. Luciferase reporter assays demonstrated that both Elp2 overexpression and 4PBA increase Hsp70 promoter activity, while Elp2 depletion blocked the ability of 4PBA to stimulate Hsp70 promoter activity. Together, these data suggest that Elp2 and STAT-3 mediate, at least in part, the stimulation of Hsp70 expression by 4PBA.

ERp29 regulates DeltaF508 and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) trafficking to the plasma membrane in cystic fibrosis (CF) and non-CF epithelial cells.

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o- WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells.

Regulation of endogenous ENaC functional expression by CFTR and ΔF508-CFTR in airway epithelial cells.

The functional expression of the epithelial sodium channel (ENaC) appears elevated in cystic fibrosis (CF) airway epithelia, but the mechanism by which this occurs is not clear. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) alters the trafficking of endogenously expressed human ENaC in the CFBE41o⁻ model of CF bronchial epithelia. Functional expression of ENaC, as defined by amiloride-inhibited short-circuit current (I(sc)) in Ussing chambers, was absent under control conditions but present in CFBE41o⁻ parental and ΔF508-CFTR-overexpressing cells after treatment with 1 µM dexamethasone (Dex) for 24 h. The effect of Dex was mimicked by incubation with the glucocorticoid hydrocortisone but not with the mineralocorticoid aldosterone. Application of trypsin to the apical surface to activate uncleaved, "near-silent" ENaC caused an additional increase in amiloride-sensitive I(sc) in the Dex-treated cells and was without effect in the control cells, suggesting that Dex increased ENaC cell surface expression. In contrast, Dex treatment did not stimulate amiloride-sensitive I(sc) in CFBE41o⁻ cells that stably express wild-type (wt) CFTR. CFBE41o⁻ wt cells also had reduced expression of α- and γ-ENaC compared with parental and ΔF508-CFTR-overexpressing cells. Furthermore, application of trypsin to the apical surface of Dex-treated CFBE41o⁻ wt cells did not stimulate amiloride-sensitive I(sc), suggesting that ENaC remained absent from the surface of these cells even after Dex treatment. We also tested the effect of trafficking-corrected ΔF508-CFTR on ENaC functional expression. Incubation with 1 mM 4-phenylbutyrate synergistically increased Dex-induced ENaC functional expression in ΔF508-CFTR-overexpressing cells. These data support the hypothesis that wt CFTR can regulate the whole cell, functional, and surface expression of endogenous ENaC in airway epithelial cells and that absence of this regulation may foster ENaC hyperactivity in CF airway epithelia.

ERp29 restricts Connexin43 oligomerization in the endoplasmic reticulum.

Connexin43 (Cx43) is a gap junction protein that forms multimeric channels that enable intercellular communication through the direct transfer of signals and metabolites. Although most multimeric protein complexes form in the endoplasmic reticulum (ER), Cx43 seems to exit from the ER as monomers and subsequently oligomerizes in the Golgi complex. This suggests that one or more protein chaperones inhibit premature Cx43 oligomerization in the ER. Here, we provide evidence that an ER-localized, 29-kDa thioredoxin-family protein (ERp29) regulates Cx43 trafficking and function. Interfering with ERp29 function destabilized monomeric Cx43 oligomerization in the ER, caused increased Cx43 accumulation in the Golgi apparatus, reduced transport of Cx43 to the plasma membrane, and inhibited gap junctional communication. ERp29 also formed a specific complex with monomeric Cx43. Together, this supports a new role for ERp29 as a chaperone that helps stabilize monomeric Cx43 to enable oligomerization to occur in the Golgi apparatus.

Abnormal regulatory interactions of I148T-CFTR and the epithelial Na+ channel in Xenopus oocytes.

The mechanisms underlying regulatory interactions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na(+) channel (ENaC) in Xenopus oocytes are controversial. CFTR's first nucleotide binding domain (NBD-1) may be important in these interactions, because mutations within NBD-1 impair these functional interactions. We hypothesized that an abnormal CFTR containing a non-NBD-1 mutation and able to transport chloride would retain regulatory interactions with murine ENaC (mENaC). We tested this hypothesis for I148T-CFTR, where the mutation is located in CFTR's first intracellular loop. I148T-CFTR has been associated with a severe CF phenotype, perhaps because of defects in its regulation of bicarbonate transport, but it transports chloride similarly to wild-type CFTR in model systems (Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. Nature 410: 94-97, 2001). cRNAs encoding alphabetagamma-mENaC and I148T-CFTR were injected separately or together into Xenopus oocytes. mENaC and CFTR functional expression were assessed by two-electrode voltage clamp. mENaC whole oocyte expression was determined by immunoblotting, and surface expression was quantitated by surface biotinylation. Injection of I148T-CFTR cRNA alone yielded high levels of CFTR functional expression. In coinjected oocytes, mENaC functional and surface expression was not altered by activation of I148T-CFTR with forskolin/ IBMX. Furthermore, the CFTR potentiator genistein both enhanced functional expression of I148T-CFTR and restored regulation of mENaC surface expression by activated I148T-CFTR. These data suggest that the ability to transport chloride is not a critical determinant of regulation of mENaC by activated CFTR in Xenopus oocytes and provide further evidence that I148T-CFTR is dysfunctional despite maintaining the ability to transport chloride.

Regulatory interactions of N1303K-CFTR and ENaC in Xenopus oocytes: evidence that chloride transport is not necessary for inhibition of ENaC.

Regulatory interactions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na(+) channel (ENaC) are readily apparent in Xenopus oocytes. However, the mechanism underlying these interactions remains controversial. CFTR's first nucleotide binding fold (NBD-1) may be important in these interactions, as dysfunctional CFTRs containing mutations within NBD-1, such as DeltaF508 and G551D, lack such functional interactions with murine ENaC (mENaC). We hypothesized that a dysfunctional CFTR containing a non-NBD-1 mutation would retain regulatory interactions with mENaC and tested this hypothesis for N1303K-CFTR, where the mutation is located in CFTR's second nucleotide binding fold (NBD-2). cRNA for alphabetagamma-mENaC and N1303K-CFTR was injected separately or together into Xenopus oocytes. ENaC and CFTR functional expression was assessed by two-electrode voltage clamp. Injection of N1303K (class II trafficking mutation) yielded low levels of CFTR function on activation with forskolin and 3-isobutyl-1-methylxanthine (IBMX). In coinjected oocytes, N1303K did not alter mENaC functional expression or surface expression before activation of N1303K. This is similar to our prior observations with DeltaF508. However, unlike our observations with DeltaF508, activation of N1303K acutely decreased mENaC functional and surface expression, and N1303K currents were enhanced by coinjection of mENaC. Furthermore, genistein only mildly enhanced the functional expression of N1303K-CFTR and did not improve regulation of ENaC by N1303K-CFTR. These data suggest that a structurally and functionally intact CFTR NBD-1 in activated CFTR can regulate mENaC surface expression independent of Cl(-) transport in Xenopus oocytes.

Differential effects of Hsc70 and Hsp70 on the intracellular trafficking and functional expression of epithelial sodium channels.

The members of the cytoplasmic 70-kDa heat shock protein family are involved in appropriate folding and trafficking of newly synthesized proteins in the cell. Hsc70, which is expressed constitutively, and Hsp70, the expression of which is stress- and heat shock-induced, are often considered to have similar cellular functions in this regard, but there are suggestions that the intracellular functions of these homologous but not identical proteins may differ. We tested the hypothesis that Hsc70 and Hsp70 would have differential effects on the expression of the epithelial sodium channel (ENaC). In Xenopus oocytes, overexpression of human Hsc70 decreased the functional (defined as amiloride-sensitive whole-oocyte current) and surface expression of murine ENaC (mENaC) in a concentration-dependent fashion. In contrast, coinjection of a moderate amount of Hsp70 cRNA (10 ng) increased the functional and surface expression of mENaC, whereas a higher amount of coinjected Hsp70 cRNA (30 ng) decreased mENaC functional and surface expression. The increase in mENaC functional expression with coinjection of 10 ng of Hsp70 cRNA was antagonized by the additional coinjection of Hsc70 cRNA in a concentration-dependent fashion. These data are consistent with Hsc70 and Hsp70 having differential and antagonistic effects with regard to the intracellular trafficking of mENaC in oocytes, which may have an impact on our understanding and potential treatment of diseases of aberrant ion channel trafficking.

Differential modulation of a polymorphism in the COOH terminus of the alpha-subunit of the human epithelial sodium channel by protein kinase Cdelta.

The A663T polymorphism of the alpha-subunit of the human epithelial sodium channel (hENaC) increases the functional and surface expression of alphabetagamma-hENaC in Xenopus laevis oocytes. The context of this residue in the COOH terminus of alpha-hENaC is important for this effect, as a homologous change in murine ENaC (mENaC), A692T, does not alter functional and surface expression of mENaC. Query of a phosphoprotein database suggested that the alpha-T663 residue might be phosphorylated by PKCdelta. General inhibition of PKC with calphostin C decreased the functional and surface expression of alphaT663-hENaC and not alphaA663-hENaC, and was without effect on alphaA692-mENaC, alphaT692-mENaC, and a chimeric m(1-678)/h(650-669)alphaT663, mbetagamma-ENaC. These data suggest that residues outside of the alpha-hENaC COOH terminus are important for modulation of alphaT663-hENaC trafficking by PKC. In contrast, expression of PKCdelta decreased the functional and surface expression of alphaT663-hENaC and the functional expression of m(1-678)/h(650-669)alphaT663, mbetagamma-ENaC, and was without effect on alphaA663-hENaC, alphaA692-mENaC, or alphaT692-mENaC. PKCdelta did not phosphorylate the COOH terminus of either alphaT663-hENaC or alphaA663-hENaC in vitro, suggesting that it acts indirectly to regulate hENaC trafficking. alphaT663-hENaC was retrieved from the oocyte membrane more slowly than alphaA663-hENaC, and calphostin C increased the rate of alphaT663-hENaC removal from the oocyte membrane to a rate similar to that of alphaA663-hENaC. In contrast, PKCdelta did not alter the rate of removal of alphaT663-hENaC from the oocyte membrane, suggesting that PKCdelta altered rates of alphaT663-hENaC biosynthesis and/or delivery to the plasma membrane. These data are consistent with PKC isoform-specific effects on the intracellular trafficking of alphaT663- vs. alphaA663-hENaC.

Genistein improves regulatory interactions between G551D-cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) in addition to its well defined Cl(-) channel properties regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and non-epithelial cells, whereas the presence of ENaC increases CFTR functional expression. This interregulation is reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl(-) channels are increased when CFTR is co-expressed with alphabetagamma-mouse ENaC (mENaC) and conversely when the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, different functional regulatory interactions were observed between G551D-CFTR and alphabetagamma-mENaC. The co-expression of G551D-CFTR and alphabetagamma-mENaC resulted in a 5-fold increase in G551D-CFTR Cl(-) current compared with oocytes expressing G551D-CFTR alone. Oocytes co-injected with both G551D-CFTR and ENaC expressed an amiloride-sensitive whole cell current that was similar to that observed before and after G551D-CFTR activation with forskolin/isobutylmethylxanthine. Treatment with genistein both enhanced the functional expression of G551D-CFTR and improved regulatory interactions between G551D-CFTR and ENaC. These data suggest that genistein may be useful in patients with cystic fibrosis and the G551D-CFTR mutation.

Genistein restores functional interactions between Delta F508-CFTR and ENaC in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its Cl(-) channel properties, has regulatory interactions with other epithelial ion channels including the epithelial Na(+) channel (ENaC). Both the open probability and surface expression of wild type CFTR Cl(-) channels are increased significantly when CFTR is co-expressed in Xenopus oocytes with alphabetagamma-ENaC, and conversely, the activity of ENaC is inhibited following wild type CFTR activation. Using the Xenopus oocyte expression system, a lack of functional regulatory interactions between DeltaF508-CFTR and ENaC was observed following activation of DeltaF508-CFTR by forskolin and isobutylmethylxanthine (IBMX). Whole cell currents in oocytes expressing ENaC alone decreased in response to genistein but increased in response to a combination of forskolin and IBMX followed by genistein. In contrast, ENaC currents in oocytes co-expressing ENaC and DeltaF508-CFTR remained stable following stimulation with forskolin/IBMX/genistein. Furthermore, co-expression of DeltaF508-CFTR with ENaC enhanced the forskolin/IBMX/genistein-mediated activation of DeltaF508-CFTR. Our data suggest that genistein restores regulatory interactions between DeltaF508-CFTR and ENaC and that combinations of protein repair agents, such as 4-phenylbutyrate and genistein, may be necessary to restore DeltaF508-CFTR function in vivo.