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.

Molecular Chaperones as Targets to Circumvent the CFTR Defect in Cystic Fibrosis.

Cystic Fibrosis (CF) is the most common autosomal recessive lethal disorder among Caucasian populations. CF results from mutations and resulting dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CFTR is a cyclic AMP-dependent chloride channel that is localized to the apical membrane in epithelial cells where it plays a key role in salt and water homeostasis. An intricate network of molecular chaperone proteins regulates CFTR's proper maturation and trafficking to the apical membrane. Understanding and manipulation of this network may lead to therapeutics for CF in cases where mutant CFTR has aberrant trafficking.

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.

A molecular signature of normal breast epithelial and stromal cells from Li-Fraumeni syndrome mutation carriers.

Specific changes in gene expression during cancer initiation should enable discovery of biomarkers for risk assessment, early detection and targets for chemoprevention. It has been previously demonstrated that altered mRNA and proteome signatures of morphologically normal cells bearing a single inherited "hit" in a tumor suppressor gene parallel many changes observed in the corresponding sporadic cancer. Here, we report on the global gene expression profile of morphologically normal, cultured primary breast epithelial and stromal cells from Li-Fraumeni syndrome (LFS) TP53 mutation carriers. Our analyses identified multiple changes in gene expression in both morphologically normal breast epithelial and stromal cells associated with TP53 haploinsufficiency, as well as interlocking pathways. Notably, a dysregulated p53 signaling pathway was readily detectable. Pharmacological intervention with the p53 rescue compounds CP-31398 and PRIMA-1 provided further evidence in support of the central role of p53 in affecting these changes in LFS cells and treatment for this cancer. Because loss of signaling mediated by TP53 is associated with the development and survival of many human tumors, identification of gene expression profiles in morphologically normal cells that carry "one-hit" p53 mutations may reveal novel biomarkers, enabling the discovery of potential targets for chemoprevention of sporadic tumors as well.

Timeless Maintains Genomic Stability and Suppresses Sister Chromatid Exchange during Unperturbed DNA Replication.

Genome integrity is maintained during DNA replication by coordination of various replisome-regulated processes. Although it is known that Timeless (Tim) is a replisome component that participates in replication checkpoint responses to genotoxic stress, its importance for genome maintenance during normal DNA synthesis has not been reported. Here we demonstrate that Tim reduction leads to genomic instability during unperturbed DNA replication, culminating in increased chromatid breaks and translocations (triradials, quadriradials, and fusions). Tim deficiency led to increased H2AX phosphorylation and Rad51 and Rad52 foci formation selectively during DNA synthesis and caused a 3-4-fold increase in sister chromatid exchange. The sister chromatid exchange events stimulated by Tim reduction were largely mediated via a Brca2/Rad51-dependent mechanism and were additively increased by deletion of the Blm helicase. Therefore, Tim deficiency leads to an increased reliance on homologous recombination for proper continuation of DNA synthesis. Together, these results indicate a pivotal role for Tim in maintaining genome stability throughout normal DNA replication.

ATR and H2AX cooperate in maintaining genome stability under replication stress.

Chromosomal abnormalities are frequently caused by problems encountered during DNA replication. Although the ATR-Chk1 pathway has previously been implicated in preventing the collapse of stalled replication forks into double-strand breaks (DSB), the importance of the response to fork collapse in ATR-deficient cells has not been well characterized. Herein, we demonstrate that, upon stalled replication, ATR deficiency leads to the phosphorylation of H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of homologous recombination and fork restart. Because H2AX has been shown to play a facilitative role in homologous recombination, we hypothesized that H2AX participates in Rad51-mediated suppression of DSBs generated in the absence of ATR. Consistent with this model, increased Rad51 focal accumulation in ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR and H2AX lead to synergistic increases in chromatid breaks and translocations. Importantly, the ATM and DNA-PK phosphorylation site on H2AX (Ser(139)) is required for genome stabilization in the absence of ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal role in suppressing DSBs during DNA synthesis in instances of ATR pathway failure. These results imply that ATR-dependent fork stabilization and H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress double-strand breaks as a layered response network when replication stalls.