AJP - Lung Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 293: L720-L729, 2007. First published June 22, 2007; doi:10.1152/ajplung.00025.2007
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/L720    most recent
00025.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mulugeta, S.
Right arrow Articles by Beers, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mulugeta, S.
Right arrow Articles by Beers, M. F.

Misfolded BRICHOS SP-C mutant proteins induce apoptosis via caspase-4- and cytochrome c-related mechanisms

Surafel Mulugeta, Jean Ann Maguire, Jennifer L. Newitt, Scott J. Russo, Adam Kotorashvili, and Michael F. Beers

Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Submitted 18 January 2007 ; accepted in final form 19 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Several mutations within the BRICHOS domain of surfactant protein C (SP-C) have been linked to interstitial lung disease. Recent studies have suggested that these mutations cause misfolding of the proprotein (proSP-C), which initiates the unfolded protein response to resolve improper folding or promote protein degradation. We have reported that in vitro expression of one of these proteins, the exon 4 deletion mutant (hSP-C{Delta}exon4), causes endoplasmic reticulum (ER) stress, inhibits proteasome function, and activates caspase-3-mediated apoptosis. To further elucidate mechanisms and common pathways for cellular dysfunction, various assays were performed by transiently expressing two SP-C BRICHOS domain mutant (BRISPC) proteins (hSP-C{Delta}exon4, hSP-CL188Q) and control proteins in lung epithelium-derived A549 and kidney epithelium-derived (HEK-293) GFPu-1 cell lines. Compared with controls, cells expressing either BRICHOS mutant protein consistently exhibited increased formation of insoluble aggregates, enhanced promotion of inositol-requiring enzyme 1-dependent splicing of X-box binding protein-1 (XBP-1), significant inhibition of proteasome activity, enhanced induction of mitochondrial cytochrome c release, and increased activations of caspase-4 and caspase-3, leading to apoptosis. These results suggest common cellular responses, including initiation of cell-death signaling pathways, to these lung disease-associated BRISPC proteins.

surfactant protein C; misfolding proteins; protein aggregation; endoplasmic reticulum stress; proteasome inhibition

SURFACTANT PROTEIN C (SP-C) is a unique component of pulmonary surfactant, a heterogeneous complex of lipids and proteins that lowers lung surface tension as well as contributes to the pulmonary host defense system. The presence of SP-C in surfactant enhances adsorption and spreading of surfactant phospholipids, thereby promoting surface film formation. Mature SP-C is a hydrophobic peptide with an Mr of 3,700 produced from a 21-kDa bitopic precursor transmembrane protein (proSP-C) that is synthesized and proteolytically processed exclusively in alveolar type II (ATII) cells (1). During subcellular trafficking, proSP-C undergoes homomeric association mediated by the mature domain (51). SP-C is released into the alveolar space via regulated secretion together with phospholipids and other proteins contained in lamellar bodies (specialized secretory organelles of ATII cells).

Multiple reports have described an association between mutations in the gene encoding SP-C (SFTPC) and chronic interstitial lung diseases (ILDs) in both infants and adults (3, 37, 48, 49). Several of these mutations result in amino acid substitutions clustered within the proprotein's COOH-terminal domain, known as BRICHOS, a region of ~100 residues with structural homology to a number of proteins associated with degenerative and proliferative diseases in other organs (46). A deletion mutation (hSP-C{Delta}exon4) has also been described in the intronic region of SFTPC immediately upstream of exon 4 that results in alternative splicing of the proSP-C mRNA and exclusion of exon 4 (38). This event produces a defective precursor protein in which the COOH-terminal domain of proSP-C is foreshortened by 37 amino acids and removes the putative disulfide bond-forming cysteine residue that is believed to be responsible for folding and consequent processing of proSP-C (21). Consistent with the characteristics of other misfolded proteins, unprocessed mutant peptide accumulation in ubiquitinated perinuclear inclusion bodies with subsequent aggresome formation is observed in various lung epithelial cell lines transiently transfected with hSP-C{Delta}exon4 (33, 50). Ubiquitin/proteasome activity is also inhibited in cells expressing hSP-C{Delta}exon4 (33). Furthermore, expression of the mutant protein is retained in the endoplasmic reticulum (ER) and can elicit the unfolded protein response with concomitant production of multiple ER stress species and induction of apoptotic cell death (4, 5, 33). In addition, the presence of proSP-C in ATII cells and the absence of detectable mature SP-C in the alveolar space of patients with heterozygous expression of the hSP-C{Delta}exon4 mutation suggest a dominant negative effect.

A SFTPC mutation that substitutes a conserved leucine residue for glutamine within the BRICHOS domain (hSP-CL188Q) has also been identified in association with familial ILD with pathological features comparable to patients with the hSP-C{Delta}exon4 isoform (49) (Fig. 1A). Immunohistochemical and electron micrograph evaluations of patients’ lung tissue show atypical ATII cells with abnormal lamellar bodies and aberrant subcellular localization of the precursor protein. Transient expression of the mutant proprotein in mouse lung epithelial cell lines has also been shown to induce cell toxicity (49). The structural configuration (1, 18, 21, 51, 53) of proSP-C, coupled with the location of this missense mutation adjacent to a conserved cysteine residue at 189, suggests that the hSP-CL188Q mutation may affect proSP-C folding. This could be achieved via hindrance of disulfide bond formation by the adjacent cysteine 189 residue and/or destabilizing the conformation of the propeptide (17). This is graphically illustrated in Fig. 1A. Moreover, similarities in the clinical manifestations of hSP-C{Delta}exon4 and hSP-CL188Q mutations, together with experimental data available thus far, support the notion that these two mutations could share common pathways for cellular injury and death. We therefore hypothesized that expression of hSP-CL188Q causes apoptotic cell death via multiple cellular responses. We report presently that the two SP-C BRICHOS domain mutant (BRISPC) proteins not only elicit similar cellular responses including induction of ER stress, disruption of the ubiquitin proteasome pathway, deposition of toxic cellular aggregates, and cell death, but also induce previously undescribed (with BRICHOS domain mutations) death-promoting pathways for apoptosis involving caspase-4 and mitochondrial signaling.


Figure 1
View larger version (69K):
[in this window]
[in a new window]

  		Fig. 1. Enhanced green fluorescent protein (EGFP)-tagged surfactant protein C (SP-C) BRICHOS domain mutant (BRISPC) isoforms form insoluble cytosolic aggregates. A: schematic illustration of previously described proSP-C structure (8, 19, 21, 32, 41) as it exists in the endoplasmic reticulum (ER) membrane as a bitopic transmembrane protein with a type II (Ncytosol/Clumen) orientation. The BRICHOS domain (hatched oval circle), the {Delta}exon4 deletion mutation (hatched domain), cysteine residues mediating propeptide folding, and the single substitution mutation of glutamine (Q) for leucine (L) at amino acid 188 of the propeptide are depicted. B and C: fluorescent microscopy of A549 cells 48 h following plasmid introduction of EGFP·hSP-CWT (B) and EGFP·hSP-CL188Q (C) immunostained with Texas red-conjugated CD63 and EEA1 antibodies. Insets from merged images are magnified to illustrate colocalization or noncolocalization (bottom). D: EGFP·hSP-CL188Q-transfected A549 cells were also immunostained with calnexin (left) and ubiquitin (center and right) antibodies. Colocalization of GFP with calnexin and ubiquitin in mutant protein-expressing cells is shown (bottom). E: cell count of expressed EGFP·hSP-C proteins colocalized with markers 48 h following transfection. At least 150 cells per marker from 3 separate experiments were counted. *P < 0.05; **P < 0.001 compared with wild-type hSP-C. F: anti-GFP immunoblot of A549 whole cell lysates partitioned into detergent-soluble (top) and -insoluble fractions (bottom) 72 h following transfection with vehicle only containing no plasmid DNA (ND), vector (EGFPC1), and various isoforms of EGFP-tagged proSP-C (WT, {Delta}exon4, and L188Q). Immunoblotting with beta-actin antibody was used to normalize equal loading (bottom of each fraction). All fluorescent and immunoblot images represent data from at least 3 separate experiments. {Phi}, Lower bands denote partial processing of EGFP·hSP-CWT and EGFP·hSP-CL188Q.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
SP-C cDNA expression. Human proSP-CWT (amino acids 1–197) in pEGFPC1 and pcDNA3 vectors has been previously described, as has proSP-C{Delta}exon4 (50, 51). To create EGFP·hSP-CL188Q, proSP-CWT in pcDNA3 vector was used as a template in a single PCR reaction with two primers, dTCCGGACTCAGATCTATGGATGTGGGCAGCAAAGAGGTCCTG (forward) and dGGAGGCGTCGGATCCCTAGATGTAGTAGAGCGGCACCTCGCCACACTGGGTGCTCACGGCCATGCCCAG (reverse). These primers respectively contain BglII and BamHI restriction enzyme recognition sites for insertion into the pEGFPC1 vector. Human proSP-CWT, proSP-C{Delta}exon4, and proSP-CL188Q in DsRed vector were created using the enhanced green fluorescent protein (EGFP)-tagged wild-type and mutant proSP-C plasmids as templates and a single PCR reaction with two primers. The primers used were dGGACTCAGATCTCGAGAAATGGATGTGGGCAGCAAAGAG (forward) and dTGTGGTATGGGTACCTATGATCAGTTATCTAGATCC (reverse) containing XhoI and KpnI restriction enzyme recognition sites, respectively, for insertion into the DsRed vector. Sequence fidelity of each PCR product was verified by automated DNA sequencing (in both directions) performed at the Core Facility in the Department of Genetics at the University of Pennsylvania. Primers were designed to amplify ~600 bases.

Cell lines. Human lung epithelium (A549)- and kidney epithelium (HEK-293)-derived GFPu-1 cell lines were obtained from American Type Culture Collection (Manassas, VA).

Immunocytochemistry. Immunocytochemistry and fluorescence imaging was performed according to the previously described protocol (33). Following permeabilization, cells on coverslips were immunolabeled with primary antibodies for 1 h at room temperature at the following dilutions: anti-CD63 (Immunotech, Marseilles, France), 1:1,000; anti-calnexin (Stressgen, Victoria, Canada), 1:200; anti-EEA1 (Affinity BioReagents, Golden, CO), 1:100; and anti-ubiquitin (FK2; Affiniti, Devon, UK), 1:100. Texas red-conjugated secondary goat anti-mouse IgG monoclonal or secondary goat anti-rabbit IgG polyclonal antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for visualization at 1:200 dilution.

In situ apoptotic cell death assays. Two separate assays were used for determining apoptosis: 1) activated caspase-3 labeling in permeabilized cells (see Immunocytochemistry) and 2) annexin V binding. The assay for annexin V binding was performed according to the manufacturer's recommendation (Roche Diagnostics, Penzberg, Germany) with minor modifications. A549 cells grown to 70% confluence in 35-mm glass-bottom dishes [with 0.16- to 0.19-mm cover glasses (MatTek, Ashland, MA) for live microscopic observation] were transiently transfected with EGFP/proSP-C constructs (4 µg/dish) using FuGene 6. For positive control, cells in separate 35-mm glass-bottom dishes were treated with TNF-{alpha} (BD Biosciences, Bedford, MA) overnight. Forty-eight hours following the introduction of plasmid DNA, cells were washed twice (5 min each) with phosphate-buffered saline (PBS: 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.2), incubated with incubation buffer (10 mM HEPES/NaOH, pH 7.2, 140 mM NaCl, 5 mM CaCl2) for 10 min, and incubated with annexin V-Alexa647 labeling solution [incubation buffer plus 10 µl of annexin V-Alexa647 (Roche Diagnostics)] for 15 min at room temperature. To control for necrosis, cells were monitored for propidium iodide nuclear labeling by adding 10 µg/ml propidium iodide concomitantly with the annexin V. Cells were visualized with a confocal fluorescence microscope (Olympus, Center Valley, PA).

Immunoblotting. Immunoblotting was performed according to the previously described procedure (33) using successive incubations with either primary polyclonal antisera [anti-GFP (Molecular Probes, Eugene, OR), 1:5,000; anti-FLAG (Rockland Immunochemicals, Gilbertsville, PA), 1:1,000; anti-activated caspase-3 (Sigma, St. Louis, MO), 1:200; anti-cytochrome c (BD Bioscience, San Jose, CA), 1:100; anti-beta-actin (Sigma), 1:2,000] or primary monoclonal antibodies [MTCO2 (mitochondrial marker; AbCam, Cambridge, MA), 1:200; anti-caspase-4 (Stressgen, Victoria, Canada), 1:500] followed by either goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibody, respectively (1:10,000). Bands were visualized by enhanced chemiluminescence using a commercially available kit (ECL Western blotting detection reagents; Amersham, Arlington Heights, IL). Chemiluminescent images were produced either by exposure to film or by direct acquisition using the Kodak 440 imaging system (New Haven, CT).

In vitro proteasome inhibition assay and induction of ER stress and apoptosis. A proteasome inhibition assay has been described previously (33). To induce ER stress and/or apoptosis, cells were treated overnight, at the indicated concentrations, with either tunicamycin Streptomyces sp. or thapsigargin (both from Sigma) for ER stress, TNF-{alpha} (BD Biosciences) for caspase-3 activation and apoptosis, or etoposide (Sigma) for ER stress-independent induction of apoptosis.

Mitochondrial cytochrome c release. Determination of mitochondrial cytochrome c release to the cytosol was performed according to a previously described procedure with minor modification (40). Cell pellets collected by scraping dishes and centrifuging at 300 g were washed twice with 500 µl of PBS. For protein enrichment, cells from two separate 30-mm culture dishes were used per fraction. Pellets were resuspended in 150 µl of ice-cold permeabilizing buffer (75 mM KCl, 1 mM NaH2HPO4, 250 mM sucrose, 230 µg/ml digitonin) containing protease inhibitors and incubated on ice for 15 min for plasma membrane permeabilization and release of soluble cytosolic components. To monitor the level of cell permeability, a trypan blue dye exclusion assay was used on sample aliquots. Cells were centrifuged at 5,000 g, 4°C for 5 min. Supernatant was transferred into a new tube and centrifuged again. The supernatant of this last centrifugation contained the cytosolic fraction, which was mixed with an equal volume of 2x RIPA buffer (1x RIPA buffer is 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 0.2 mM sodium orthovanadate, and 50 mM sodium fluoride, in PBS) containing protease inhibitors. For the organelle fraction including the mitochondria, the pellet from the first centrifugation following permeabilization was washed twice with 500 µl of PBS (6,000 g, 4°C) for 5 min and resuspended in 40 µl of 1x RIPA buffer.

Triton X-soluble and -insoluble fractionation assay. Fractionation assay to separate Triton X-soluble from Triton X-insoluble proteins was performed as previously described (31) with some modification. A549 cells in 30-mm culture dishes were transiently transfected with EGFP-tagged proSP-C constructs (10 µg/dish) using the calcium phosphate method. At 72 h posttransfection, cells were washed twice with 500 µl of ice-cold PBS and lysed in ice-cold lysis buffer [50 mM Tris·HCl (pH 7.2), 150 mM NaCl, 2 mM EDTA, 1% (vol/vol) Triton X-100, and .05% (vol/vol) sodium deoxycholate] containing protease inhibitors. For protein enrichment, cells from two separate 30-mm culture dishes were used per fraction. Lysed cells were sedimented at 16,000 g for 30 min at 4°C. The supernatants were transferred into new tubes and centrifuged again at 16,000 g for 30 min at 4°C. The supernatants from the second centrifugation were saved as the Triton X-soluble fraction. Pellets from the initial fractionation were washed twice with lysis buffer and solubilized with SDS buffer (lysis buffer containing 1% SDS in place of Triton X-100) and saved as the Triton X-insoluble fraction. SDS-PAGE sample buffer was added to both the supernatant and pellet fractions for subsequent electrophoresis.

Statistical analyses. Experimental data were analyzed by one-way analysis of variance with the Tukey-Kramer post hoc test using GraphPad InStat software, version 3.0 for Windows (GraphPad Software, San Diego, CA). All values are means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The SP-CL188Q mutant is trafficked to ubiquitinated cytosolic inclusion bodies and perinuclear aggregate. We have reported previously that the hSP-C{Delta}exon4 BRISPC propeptide localizes to both the ER and in ubiquitinated perinuclear aggregates when transiently expressed in various epithelial cell lines including A549, MLE15, and HEK-293 cells (33). Moreover, we have shown that non-BRICHOS domain mutations of SP-C, including SP-CE66K and SP-CI73T, are targeted to early endosome compartments (3, 48). To determine the trafficking pattern of hSP-CL188Q, we transfected A549 cells with EGFP-tagged wild-type (EGFP·hSP-CWT) and mutant (EGFP·hSP-CL188Q) isoforms of SP-C. At 48 h posttransfection, EGFP·hSP-CWT was localized to CD63 (a marker for lamellar bodies and lysosomes)-positive and EEA1 (an early endosome marker)-negative vesicles (Fig. 1B), the expected target vesicle for the wild-type isoform of SP-C. In contrast, EGFP·hSP-CL188Q was rarely observed in CD63-positive vesicles (Fig. 1C, left, arrows) and was not detected in EEA1-containing vesicles (Fig. 1C, right). Instead, three distinct patterns of trafficking are apparent predominantly localizing EGFP·hSP-CL188Q to the calnexin-positive ER compartment (Fig. 1D, left) and ubiquitin-positive inclusions with punctate cytosolic distribution and perinuclear-confined localization (Fig. 1D, center and right). Quantitative analyses of these hSP-C isoforms revealed that EGFP·hSP-CWT had minimal colocalization with either ubiquitin or calnexin (Fig. 1E). By contrast, both EGFP·hSP-C{Delta}exon4 and EGFP·hSP-CL188Q showed a significant amount of colocalization with calnexin (>20%) and ubiquitin (>40%) (Fig. 1E).

BRISPCs form insoluble cytosolic aggregates. To date, direct biochemical evidence of solubility of BRISPC proteins has not been described. To determine the susceptibility of these proteins to form insoluble aggregates, lysates from A549 cells transfected with various isoforms of EGFP-tagged SP-C proteins or vector alone (EGFPC1) were partitioned into detergent-soluble and -insoluble fractions (Fig. 1F). Immunoblots of SDS-PAGE-separated proteins from cell lysates harvested 72 h following transfection showed that EGFP·hSP-CWT and EGFPC1 were almost exclusively partitioned in soluble fractions. In contrast, a substantial portion of either EGFP·hSP-C{Delta}exon4 or EGFP·hSP-CL188Q proteins segregated with Triton X-insoluble fractions (Fig. 1F), indicating a tendency for these BRISPC proteins to form insoluble aggregates.

Expression of hSP-CL188Q inhibits ubiquitin/proteasome function. Accumulation of protein aggregates suggests an ubiquitin/proteasome system (UPS) that is overwhelmed by chronic production of misfolded proteins and is unable to sustain their degradation (20, 26). To address whether expression of hSP-CL188Q inhibits the UPS, we used GFPu-1 cells. GFPu-1 is a human kidney epithelium (HEK-293)-derived, stably transfected cell line expressing a short degron (CL1) tagged to GFP (10). GFP stability in these cells can be used as a marker to determine relationships between protein expression and UPS dysfunction (2). Under normal conditions, the GFP/degron expression is short-lived, since the efficient UPS of HEK cells rapidly degrades the product soon after translation, before any detectable green fluorescence is observed (Fig. 2A, bottom left). However, conditions that impair the UPS, thereby inhibiting proteasome degradation, will promote the accumulation of the GFP-tagged peptide. Control treatment with lactacystin, a proteasome inhibitor, resulted in GFP expression across the entire cell culture population (Fig. 2A, bottom center). Cells transiently transfected with DsRed-tagged hSP-CWT (Fig. 2A, top left) showed no GFP fluorescence (Fig. 2A, top center), indicating that the UPS is not affected by the presence of hSP-CWT. In contrast, cells expressing DsRed·hSP-CL188Q (Fig. 2A, middle left) frequently exhibited GFP fluorescence (Fig. 2A, middle center). Immunoblot of GFPu-1 whole cell lysates using anti-GFP showed a dose-dependent increase in GFP protein levels in samples where cells were treated with lactacystin (Fig. 2B). In addition, significant increases in GFP protein levels were observed in GFPu-1 whole cell lysates transfected with either hSP-C{Delta}exon4 or hSP-CL188Q compared with those transfected with vector or hSP-CWT (Fig. 2B). Together, these results demonstrate the capacity of BRISPC protein expression to inhibit the UPS.


Figure 2
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 2. BRISPC expression inhibits proteasome function. A: characteristic fluorescent microscopy images of GFPu-1 cells 48 h following plasmid introduction of either DsRed·hSP-CL188Q (middle left) or DsRed·hSP-CWT (top left) showing the presence (center) or absence (top center) of GFP expression, respectively. Control GFPu-1 cell populations before (bottom left) and after (bottom center) treatment with lactacystin show GFP expression only after treatment. A phase-contrast image of lactacystin-treated cells is shown at bottom right. B: representative anti-GFP immunoblot bands (top) and histogram of quantified immunoblot bands (bottom) of GFPu-1 whole cell lysate samples transfected with either vector (DsRed) or DsRed-tagged isoforms of proSP-C. Expression of GFP in control cells treated with lactacystin is shown in right 2 lanes. beta-Actin was used to normalize for equal loading. Data were obtained from 3 separate experiments, each consisting of at least 2 culture dishes per construct. *P < 0.05; **P < 0.001. Lac, lactacystin.

 
Aggregate prone BRISPCs induce apoptotic cell death via activation of caspase-3. It has been shown in other aggregate prone proteins that mutant protein expression results in apoptosis via activation of caspase-3 (27, 33). To determine whether expression of hSP-CL188Q elicits a similar response, we assayed A549 cells transfected with EGFP·hSP-CL188Q for apoptosis and caspase-3 activation by fluorescence labeling. Plasma membranes of live cells containing mutant aggregates were frequently labeled with annexin V (Fig. 3A, bottom), an agent that binds translocated (inner to outer leaflet) plasma membrane phosphatidylserine during apoptosis. The absence of nuclear labeling by propidium iodide in these cells (Fig. 3A, bottom) substantiates apoptotic and not necrotic cell death. Caspase-3 labeling (using an antibody that recognizes the activated form of caspase-3) was also more frequently observed in cells containing EGFP·hSP-CL188Q (Fig. 3, A, top, and B) than in those transfected with EGFP·hSP-CWT (Fig. 3B).


Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 3. BRISPC proteins activate dose-dependent caspase-3-mediated apoptosis. A: EGFP·hSP-CL188Q-transfected A549 cells (top and bottom left) were immunostained (48 h following introduction of plasmid) with Texas red-conjugated anti-activated caspase-3 (top center) or were stained with propidium iodide (bottom, second from left) and Alexa647-tagged annexin V (bottom, third from left) simultaneously. Activated caspase-3 in cells expressing the mutant protein is shown at top right (merged image). The presence of plasma membrane phosphatidylserine binding to annexin V and the absence of nuclear propidium iodide labeling in EGFP·hSP-CL188Q-expressing cells is shown at bottom right (merged image). Images represent results from at least 3 separate experiments. Bars, 5 µm. B: cell count of expressed EGFP·hSP-C proteins colocalized with caspase-3 48 h following transfection. At least 150 cells per marker from 3 separate experiments were counted. *P < 0.01; **P < 0.005 compared with wild-type hSP-C. C: representative immunoblot bands of activated caspase-3 (top) from A549 cell lysates (transfected with various concentrations of plasmids in 30-mm culture dishes) and histogram (bottom) of quantified bands are from at least 3 separate experiments. Overnight treatment of cells with TNF-{alpha} was used for positive control (right 2 bars). beta-Actin was used to normalize for equal loading. *P < 0.05 compared with 10 µg/dish of hSP-CWT-containing plasmid. **P < 0.001 compared with 10 µg/dish of hSP-CL188Q-containing plasmid.

 
Caspase-3 activation by BRISPC proteins was confirmed using whole cell lysate immunoblot analysis (Fig. 3C), where cell lysates from A549 cells treated with positive control TNF-{alpha}, a known inducer of apoptosis in A549 cells, showed caspase-3 activation in a dose-dependent manner. Moreover, 48 h following transfection, significant dose-dependent activation of caspase-3 was observed in both hSP-C{Delta}exon4- and hSP-CL188Q-expressing cells compared with cells expressing either hSP-CWT or vector (Fig. 3C).

BRISPC expression induces inositol-requiring enzyme 1-dependent XBP-1 mRNA splicing. Accumulation of unfolded proteins in the ER cells activates an intracellular signaling pathway termed the unfolded protein response (UPR). One of the major proximal sensors of the UPR is inositol-requiring enzyme 1 (IRE1), an ER transmembrane protein kinase/endoribonuclease that initiates the UPR signal by regulating synthesis of transcription factors such as the X-box binding protein-1 (XBP-1) through XBP-1 mRNA splicing. XBP-1 in turn regulates ER-resident chaperone genes such as BiP/GRP78, HEDJ, EDEM, and HDJ-2/HSP40 (28).

To determine ER involvement in response to BRISPC protein expression, we utilized the ERAI plasmid construct, which has been used as an indicator for ER stress (14). The ERAI construct contains a partial sequence of XBP-1, including the 26-nucleotide (nt) ER stress-specific intron, fused to the gene encoding venus (a variant of green fluorescent protein) with a FLAG tag at the NH2 terminus. Under normal conditions, a stop codon that is added between the XBP-1 and venus sequences eliminates the venus florescence expression. However, during ER stress, the 26-nt intron is spliced out by IRE1, resulting in the frame shift of the fusion mRNA and the expression of the fluorescent protein.

IRE1 participation was assessed using a strategy in which various isoforms of DsRed-tagged proSP-C plasmid constructs were cotransfected with the ERAI construct in A549 cells. This particular ERAI plasmid lacks the DNA-binding domain (DBD) of XBP-1 (XBP-1{Delta}DBD), which promotes nuclear translocation. Consequently, expression of the fusion proteins remains cytosolic. Compared with DsRed·hSP-CWT, cells transfected with DsRed·hSP-CL188Q consistently expressed the XBP-1·venus fusion protein (Fig. 4A). Similar results were obtained with DsRed·hSP-C{Delta}exon4 (data not shown). Immunoblot analyses of FLAG-tagged XBP-1 expression in cell lysates cotransfected with ERA1 and various isoforms of SP-C confirmed that spliced XBP-1 expression was significantly higher when either BRISPC mutant (hSP-C{Delta}exon4 or hSP-CL188Q) was expressed compared with hSP-CWT or vector alone (Fig. 4B). Moreover, control experiments showed that the ER stress-inducing reagent thapsigargin promoted the expression of spliced XBP-1 in a dose-dependent manner, whereas etoposide, a non-ER stress inducer of apoptosis, had no effect (Fig. 4B). These results suggest that misfolded BRISPC proteins induce ER stress, leading to IRE1-dependent activation of XBP-1.


Figure 4
View larger version (50K):
[in this window]
[in a new window]

  		Fig. 4. BRISPC expression activates inositol-requiring enzyme 1 (IRE1)-dependent unfolded protein response. A: DsRed-tagged hSP-CWT (top) and hSP-CL188Q (bottom) were cotransfected in A549 cells with an ERA1 plasmid (XBP1{Delta}DBD) expressing both FLAG and venus proteins, but only during IRE1-spliced X-box binding protein-1 (XBP-1) expression. Florescence microscopy 48 h posttransfection showing ERA1 expression only in DsRed·hSP-CL188Q (bottom, second from left)- and not in DsRed·hSP-CWT (top, second from left)-transfected cells. Merged and phase-contrast images are shown. B: representative anti-FLAG immunoblot bands (top) and relative intensity of beta-actin immunoblot bands (bottom) of A549 cell lysate samples obtained 48 h posttransfection with ERA1, DsRed, and DsRed-tagged isoforms of proSP-C. Protein bands from cells transfected with ERA1 and treated overnight with either thapsigargin for positive control or with etoposide for negative control are also shown. Data were obtained from 3 separate experiments, each consisting of at least 2 culture dishes per construct. *P < 0.05; **P < 0.005. Thapsi, thapsigargin; Etop, etoposide.

 
Caspase-4 is activated by the expression of BRISPCs. Prolonged ER stress can induce apoptosis via caspase-4 activation (13). Caspase-4 is a member of the caspase-1 subfamily that includes the mouse homolog caspase-12. Localized to the ER membrane, caspase-4 is cleaved during ER stress, leading to the activation of caspase-3 and apoptosis. Caspase-4 involvement during apoptotic induction by BRISPCs was evaluated in transiently transfected A549 cells. Control studies showed that an apoptosis-inducing cytokine (TNF-{alpha}) and known ER stressors tunicamycin and thapsigargin induced procaspase-4 cleavage in a dose-dependent manner (Fig. 5A). In contrast, etoposide, a non-ER stress inducer of apoptosis, at a dose imparting a comparable extent of cell death to the ER stressors, failed to cleave the procaspase (Fig. 5A). These results are consistent with previous reports showing equivalent procaspase-4 cleavage in human neuroblastoma (HK-N-SH) and carcinoma (HeLa) cells (13), implying ER stress-specific intrinsic caspase-4 activation in A549 cells. Similarly, procaspase-4 cleavage was significantly induced 48 h posttransfection in hSP-C{Delta}exon4- and hSP-CL188Q- compared with either hSP-CWT- or vector-expressing cells (Fig. 5B), indicating a potential role for caspase-4 in the apoptotic pathway caused by the expression of these misfolded proteins.


Figure 5
View larger version (49K):
[in this window]
[in a new window]

  		Fig. 5. ER stress-dependent activation of caspase-4 by BRISPC expression. A: control experiments showing representative immunoblot bands (top) and histogram of quantified immunoblot bands (bottom) of dose-dependent cleavage of procaspase-4 in A549 cells by overnight treatment with TNF-{alpha}, tunicamycin, and thapsigargin. Etoposide was unable to cleave caspase-4 (right 2 lanes). Tmn, tunicamycin. B: characteristic immunoblot bands (top) and histogram of quantified bands (bottom) of A549 cells transfected with EGFPC1 (vector) or various isoforms of hSP-C showing procaspase-4 cleavages. Positions of procaspase, cleaved (activated) caspase-4, and beta-actin as a control for equal loading are indicated. {Phi}, Likely by-product of cleaved caspase-4 by an unknown process consistent with previously reported data (13). Data were obtained from at least 3 separate experiments, each consisting of at least 2 culture dishes per construct. *P < 0.05.

 
Cytochrome c is released into the cytosol in BRISPC-expressing cells. Cytochrome c is a soluble protein localized to the intermembrane space and peripherally attached to the surface of the inner mitochondrial membrane. In response to a variety of apoptosis-inducing agents, cytochrome c can be released from mitochondria into the cytosol (25) and participates in the signaling cascade that induces caspase-3 activation and apoptosis (30). To evaluate cytochrome c involvement during BRISPC expression, A549 cells either treated with control reagent overnight or transfected with various isoforms of SP-C for 48 h were harvested and partitioned into cytosolic and organelle fractions. The purity of the fractionated samples was confirmed by immunoblotting using a mitochondrial resident protein-specific antibody (Fig. 6, B and E). Although cytochrome c remained predominantly partitioned within the mitochondria-containing organelle fractions (Fig. 6, A and D, bottom), its dose-dependent enrichment in the cytosol fraction was apparent in control experiments when A549 cells were treated with etoposide (Fig. 6, A, top, and C). Treatment of cells with the ER stressor tunicamycin did not elicit cytochrome c release (data not shown). Moreover, the cytosolic fraction of cells expressing either hSP-C{Delta}exon4 or hSP-CL188Q contained a significantly higher quantity of cytochrome c than those expressing either hSP-CWT or vector (Fig. 6D, top, and F). Together, these results suggest induction of ER stress-independent mitochondrial cytochrome c release by the expression of the BRISPC proteins.


Figure 6
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 6. Cytochrome c is released from the mitochondria to the cytosol in BRISPC protein-expressing cells. A549 cells were either treated overnight with a control reagent, etoposide, or allowed to express either vector or various hSP-C isoforms for 48 h. Cells were harvested and partitioned into cytosolic and organelle fractions. Cytochrome c in the designated fractions was identified using an anti-cytochrome c antibody (A and D), and the relative intensity of the bands was quantified (C and F). beta-Actin was used to normalize for equal loading. The purity of the fractionated samples was confirmed by immunoblot, using a resident control mitochondrial protein-specific antibody (MTCO2) (B and E). Data were obtained from at least 3 separate experiments, each consisting of at least 2 culture dishes per construct. *P < 0.05. Cyto. c., cytochrome c.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have previously demonstrated that one BRISPC protein (hSP-C{Delta}exon4) induces the UPR and apoptosis in a manner similar to that described for misfolded proteins in other systems (20, 22, 26, 27). The present work extends these observations by linking UPR to apoptosis, identifying an additional apoptotic pathway mediated by mitochondrial dysfunction, and characterizing cell death signaling mechanisms common to the expression of two well-known BRISPC proteins that may extend to other BRISPC isoforms.

Inhibition of proteasome function by both BRISPC isoforms was demonstrated using GFPu-1 cells (Fig. 3), suggesting a direct cause-and-effect relationship between BRISPC mutants and disruption of the UPS as proposed for other misfolded mutant proteins associated with conformational diseases. Other aggregate-prone misfolded mutant proteins linked to conformational diseases, such as the polyglutamine repeat expansion of the huntingtin protein associated with Huntington disease, the cystic fibrosis transmembrane conductance regulator mutant (CFTR{Delta}F508) linked to cystic fibrosis, and the {alpha}-synuclein protein associated with Parkinson's disease, have all been shown to form cytosolic accumulation of their respective misfolded proteins that involves inhibition of the UPS (2, 7, 36, 42). Using GFPu-1 cells, studies have demonstrated that transient expression of the expanded huntingtin proteins and CFTR{Delta}F508 caused almost total inhibition of the UPS (2). The subsequent formation of cytosolic aggregates of these misfolded proteins implies a further decline of UPS function due to a positive feedback mechanism. Mutant {alpha}-synuclein expression has been shown to significantly decrease proteasome activity in GFPu-1 and other cells as well as to cause selective toxicity to catecholaminergic neurons when expressed in primary midbrain cultures (7, 42). Moreover, the polyglutamine repeat-associated spinocerebrocellular atrophy protein (SCA3) of Machado-Joseph disease shows several similarities with the BRISPC proteins in terms of cellular response to their expression. In addition to inhibition of proteasome function, expression of SCA3 results in ER stress, formation of cytosolic aggregates, and apoptosis (36). Together, these findings further support common cellular responses to protein expression of BRISPC and other proteins associated with conformational diseases.

The in vitro inhibition of proteasome function by BRISPC proteins is cell model independent. Both A549 cells (33) and HEK-293 cells (5), transiently and constitutively expressing hSP-C{Delta}exon4, respectively, have altered cellular activity. In HEK-293 cells that constitutively express low levels of hSP-C{Delta}exon4, the toxic effect of the mutant propeptide is ameliorated through a cellular adaptive response involving the NF-{kappa}B pathway (5). However, when infected with virus, the proteasome function of these stably transfected cells was further inhibited, leading to the accumulation of the propeptide and increased susceptibility to virus-induced cell death. This suggests that environmental factors such as viral infection play an important role in the development and progress of lung diseases associated with BRISPC mutations.

The ER has developed a tightly regulated quality control mechanism such that even the slightest alteration in a protein's makeup that disturbs folding efficacy can cause the recognition of the nascent protein as misfolded, leading to subsequent accumulation and/or retrotranslocation of the protein to the cytosol (22, 45). In the cytosol, misfolded proteins are primarily destined for proteasome-mediated ER-associated degradation (ERAD). However, persistent production of aberrant proteins may shift the balance between synthesis by the ER to degradation of mutant proteins by the proteasome, resulting in accumulation of the misfolded proteins in the form of cytosolic aggregates. We have found that the BRISPC isoforms examined show both ER retention and formation of cytosolic aggregates (33) with an appreciable accumulation of insoluble forms (Fig. 1D). Moreover, we have previously shown time and expression level dependency of hSP-C{Delta}exon4 in the formation of aggregates (33). Similar results can be seen in hSP-CL188Q-expressing cells (Mulugeta S, unpublished observation). However, unlike hSP-C{Delta}exon4, which appeared to be entirely targeted to ERAD or to aggresomes depending on the level of expression (4, 33), a relatively small population of the hSP-CL188Q propeptides were properly targeted in CD63-positive vesicles (Fig. 1C, arrows) and were processed similarly to the wild-type isoform (Fig. 1F). Although the conformational changes of the hSP-C{Delta}exon4 deletion mutation may be severe, missense mutation of hSP-CL188Q may present a more moderate change, sometimes resulting in relatively preserved propeptide folding efficacy. Therefore, it is likely that some hSP-CL188Q mutant proteins are folded or refolded properly, thereby escaping the degradation pathway and resulting in propeptide processing similar to that of the wild-type isoform. This phenomenon has been well characterized for the CFTR protein, where 70–80% of the newly synthesized wild-type CFTR proteins are normally destined to ERAD, whereas the remaining 20–30% are properly transported to the cell surface (15, 52).

ER stress appears to be a consequence of an insufficient ER adaptive response due to persistent production of misfolded proteins (22, 57). The accumulation of these proteins leads to the initiation of apoptosis, which in BRISPC-expressing cells as well as in various other systems appears to be mediated by caspase-4 (13, 34, 56). Localized in the ER membrane, cleavage of this cysteine protease has been shown when cells are specifically treated with ER stress-inducing agents but not with ER stress-independent apoptotic stimuli. Caspase-12, the mouse homolog to human caspase-4, appears to be the major pathway that induces apoptosis in response to ER stress in mice (22, 56). Caspase-12-deficient mice are resistant to ER stress-induced apoptosis, but cells from these mice can be led to apoptosis by non-ER stress apoptotic agents (35). Caspase-4 involvement has also been implicated in the neurodegenerative disorder of Alzheimer's disease. The extracellular plaques (containing amyloid-beta peptide) found in the brains of Alzheimer's patients cause neuronal cytotoxicity (55) mediated by caspase-4 (13) and caspase-12 (34) in human and murine cells, respectively. However, recent studies have demonstrated that caspase-4 may not be required for ER stress-induced apoptosis in some systems (39). Using murine and human cell lines that lack the murine caspase-12 and the human caspase-4, respectively, Obeng and Boise (39) have shown that caspase-12/-4 deficiency did not protect the cells from ER stress-induced apoptosis triggered by tunicamycin, thapsigargin, or brefeldin A.

The release of the apoptotic factor cytochrome c from mitochondria by the expression of BRISPC proteins (Fig. 6) raises the possible involvement of mitochondria with at least three subcellular events that include ER stress, impairment of the UPS, and protein aggregates. The role of ER stress in mitochondrial cytochrome c release has been previously described in some cell systems. In rodent cells, mitochondrial cytochrome c is released in a caspase-12-independent (12) and caspase-8-dependent (16) manner when cells are treated with ER stress-inducing agents. In contrast, the mutant form of {alpha}-synuclein has been shown to induce ER stress, proteasome dysfunction, and mitochondrial cytochrome c release accompanied by activation of caspases-12 and -3 in differentiated PC12 cells (47), which actually parallels our findings in BRISPC-expressing A549 cells. We have discovered that when A549 cells are treated with the ER stress-inducing agent tunicamycin, cytochrome c is not released (data not shown), implying that BRISPC induction of cytochrome c is an ER stress-independent signaling pathway. We have also demonstrated that BRISPC expression inhibits proteasome function (Fig. 2) and forms detergent-insoluble cytosolic aggregates of the propeptide (Fig. 1). Furthermore, a recent report indicates that although the BRICHOS domain of wild-type proSP-C functions to prevent aggregation of the propeptide, mutation in this domain appears to impede this function (17). Aggregate formation in other systems is also frequently accompanied by mitochondrial cytochrome c release and apoptosis (6, 43, 54). Moreover, intra- and/or extracellular expression of aggregate prone proteins is associated with mitochondrial cytochrome c release in several conformational diseases such as Alzheimer's disease (24, 44), Huntington disease (23), and Parkinson's disease (47). Similar to beta-amyloid aggregates of Alzheimer's disease, SP-C can form beta-sheet amyloid fibrils (9, 11, 29) in solution comparable to those hSP-C amyloid fibrils found in lung lavage obtained from patients with pulmonary alveolar proteinosis (11). Collectively, these studies suggest that the cytochrome c release observed in BRISPC-expressing A549 cells is likely to be a product of UPS impairment and aggregate formation rather than a consequence of ER stress.

We have previously shown that proSP-C self-associates (via the mature domain) during its biosynthesis as it traffics from the ER to lamellar bodies through the Golgi, small vesicles, multivesicular bodies, and composite bodies (51). We have also demonstrated that hSP-C{Delta}exon4 is transiently retained in the ER, is not proteolytically processed, and ultimately acts as a dominant negative to direct SP-CWT to aggregates (50). In vivo, patients heterozygous for SP-C{Delta}exon4 do not express mature SP-C (38). Such a phenomenon not only inhibits the wild-type isoform from being processed and secreted but also may increases the severity of ER stress and the rate of aggregate formation. Thus the heterotypic association of wild-type proSP-C with its mutant isoforms may also contribute to the development of lung diseases linked to BRISPC proteins.

In summary, the data from the present study show that expression of BRISPC isoforms elicits similar cellular reaction, suggesting activation of common cell signaling pathways in response to these misfolded proteins. The study has identified caspase-4 as one of the intermediate signals that activates caspase-3, thereby linking ER stress to apoptosis. In addition, through ER stress-independent and perhaps dysfunctional proteasome- and aggregate-dependent intermediates, cytochrome c is released from the mitochondria into the cytosol (Fig. 6). Moreover, induction of ER stress demonstrated in this study supports data from earlier reports showing that BRISPC expression resulted in the upregulation of ER stress response chaperone genes such as HDJ2/HSP40 and Bip/GRP78 (4, 5, 33). Investigation of the molecular mechanisms such as those described in this report will provide important information to understanding disease progression and may offer insights regarding specific targets for therapeutic intervention.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-19737, HL074064, and P50 HL-56401 (to M. F. Beers) and HL-074064 Minority Supplement Program (to S. Mulugeta), American Lung Association Dalsemer Research Grant DA-188-N (to S. Mulugeta), and the Parker B. Francis Foundation Fellowship Grant (to S. Mulugeta).


   ACKNOWLEDGMENTS
 
We thank Dr. Masayuki Miura of the University of Tokyo (Tokyo, Japan) for the generous gift of the ERAI plasmids.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Mulugeta, Pulmonary, Allergy, and Critical Care Division, Univ. of Pennsylvania School of Medicine, Vernon & Shirley Hill Pavilion, Suite H418, 380 South Univ. Ave., Philadelphia, PA 19104 (e-mail: mulugeta{at}mail.med.upenn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Beers MF. Lung Surfactant: Cellular and Molecular Processing. Austin, TX: Landes, 1998.
  2. Bence NF, Sampat RM, Kopito RR. Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292: 1552–1555, 2001.[Abstract/Free Full Text]
  3. Brasch F, Griese M, Tredano M, Johnen G, Ochs M, Rieger C, Mulugeta S, Muller KM, Bahuau M, Beers MF. Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene. Eur Respir J 24: 30–39, 2004.[Abstract/Free Full Text]
  4. Bridges JP, Wert SE, Nogee LM, Weaver TE. Expression of a human surfactant protein C mutation associated with interstitial lung disease disrupts lung development in transgenic mice. J Biol Chem 278: 52739–52746, 2003.[Abstract/Free Full Text]
  5. Bridges JP, Xu Y, Na CL, Wong HR, Weaver TE. Adaptation and increased susceptibility to infection associated with constitutive expression of misfolded SP-C. J Cell Biol 172: 395–407, 2006.[Abstract/Free Full Text]
  6. Cardinale A, Filesi I, Mattei S, Biocca S. Evidence for proteasome dysfunction in cytotoxicity mediated by anti-Ras intracellular antibodies. Eur J Biochem 270: 3389–3397, 2003.[Web of Science][Medline]
  7. Ciechanover A, Schwartz AL. Ubiquitin-mediated degradation of cellular proteins in health and disease. Hepatology 35: 3–6, 2002.[CrossRef][Web of Science][Medline]
  8. Conkright JJ, Bridges JP, Na CL, Voorhout WF, Trapnell B, Glasser SW, Weaver TE. Secretion of surfactant protein C, an integral membrane protein, requires the N-terminal propeptide. J Biol Chem 276: 14658–14664, 2001.[Abstract/Free Full Text]
  9. Dluhy RA, Shanmukh S, Leapard JB, Kruger P, Baatz JE. Deacylated pulmonary surfactant protein SP-C transforms from alpha-helical to amyloid fibril structure via a pH-dependent mechanism: an infrared structural investigation. Biophys J 85: 2417–2429, 2003.[Web of Science][Medline]
  10. Gilon T, Chomsky O, Kulka RG. Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae. EMBO J 17: 2759–2766, 1998.[CrossRef][Web of Science][Medline]
  11. Gustafsson M, Thyberg J, Naslund J, Eliasson E, Johansson J. Amyloid fibril formation by pulmonary surfactant protein C. FEBS Lett 464: 138–142, 1999.[CrossRef][Web of Science][Medline]
  12. Häcki J, Egger L, Monney L, Conus S, Rosse T, Fellay I, Borner C. Apoptotic crosstalk between the endoplasmic reticulum and mitochondria controlled by Bcl-2. Oncogene 19: 2286–2295, 2000.[CrossRef][Web of Science][Medline]
  13. Hitomi J, Katayama T, Eguchi Y, Kudo T, Taniguchi M, Koyama Y, Manabe T, Yamagishi S, Bando Y, Imaizumi K, Tsujimoto Y, Tohyama M. Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death. J Cell Biol 165: 347–356, 2004.[Abstract/Free Full Text]
  14. Iwawaki T, Akai R, Kohno K, Miura M. A transgenic mouse model for monitoring endoplasmic reticulum stress. Nat Med 10: 98–102, 2004.[CrossRef][Web of Science][Medline]
  15. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, Riordan JR. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83: 129–135, 1995.[CrossRef][Web of Science][Medline]
  16. Jimbo A, Fujita E, Kouroku Y, Ohnishi J, Inohara N, Kuida K, Sakamaki K, Yonehara S, Momoi T. ER stress induces caspase-8 activation, stimulating cytochrome c release and caspase-9 activation. Exp Cell Res 283: 156–166, 2003.[CrossRef][Web of Science][Medline]
  17. Johansson H, Nordling K, Weaver TE, Johansson J. The Brichos domain-containing C-terminal part of pro-surfactant protein C binds to an unfolded poly-Val transmembrane segment. J Biol Chem 281: 21032–21039, 2006.[Abstract/Free Full Text]
  18. Johansson J. Structure and properties of surfactant protein C. Biochim Biophys Acta 1408: 161–172, 1998.[Medline]
  19. Johnson AL, Braidotti P, Pietra GG, Russo SJ, Kabore A, Wang WJ, Beers MF. Post-translational processing of surfactant protein-C proprotein: targeting motifs in the NH2-terminal flanking domain are cleaved in late compartments. Am J Respir Cell Mol Biol 24: 253–263, 2001.[Abstract/Free Full Text]
  20. Johnston JA, Ward CL, Kopito RR. Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143: 1883–1898, 1998.[Abstract/Free Full Text]
  21. Kabore AF, Wang WJ, Russo SJ, Beers MF. Biosynthesis of surfactant protein C: characterization of aggresome formation by EGFP chimeras containing propeptide mutants lacking conserved cysteine residues. J Cell Sci 114: 293–302, 2001.[Abstract]
  22. Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin Invest 110: 1389–1398, 2002.[CrossRef][Web of Science][Medline]
  23. Kiechle T, Dedeoglu A, Kubilus J, Kowall NW, Beal MF, Friedlander RM, Hersch SM, Ferrante RJ. Cytochrome c and caspase-9 expression in Huntington's disease. Neuromolecular Med 1: 183–195, 2002.[CrossRef][Web of Science][Medline]
  24. Kim HS, Lee JH, Lee JP, Kim EM, Chang KA, Park CH, Jeong SJ, Wittendorp MC, Seo JH, Choi SH, Suh YH. Amyloid beta peptide induces cytochrome c release from isolated mitochondria. Neuroreport 13: 1989–1993, 2002.[CrossRef][Web of Science][Medline]
  25. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275: 1132–1136, 1997.[Abstract/Free Full Text]
  26. Kopito RR. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10: 524–530, 2000.[CrossRef][Web of Science][Medline]
  27. Kopito RR, Ron D. Conformational disease. Nat Cell Biol 2: E207–E209, 2000.[CrossRef][Web of Science][Medline]
  28. Lee AH, Iwakoshi NN, Glimcher LH. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol 23: 7448–7459, 2003.[Abstract/Free Full Text]
  29. Li J, Hosia W, Hamvas A, Thyberg J, Jornvall H, Weaver TE, Johansson J. The N-terminal propeptide of lung surfactant protein C is necessary for biosynthesis and prevents unfolding of a metastable alpha-helix. J Mol Biol 338: 857–862, 2004.[CrossRef][Web of Science][Medline]
  30. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479–489, 1997.[CrossRef][Web of Science][Medline]
  31. Ma J, Lindquist S. Conversion of PrP to a self-perpetuating PrPSc-like conformation in the cytosol. Science 298: 1785–1788, 2002.[Abstract/Free Full Text]
  32. Mulugeta S, Beers MF. Processing of surfactant protein C requires a type II transmembrane topology directed by juxtamembrane positively charged residues. J Biol Chem 2003.
  33. Mulugeta S, Nguyen V, Russo SJ, Muniswamy M, Beers MF. A surfactant protein C precursor protein BRICHOS domain mutation causes endoplasmic reticulum stress, proteasome dysfunction, and caspase 3 activation. Am J Respir Cell Mol Biol 32: 521–530, 2005.[Abstract/Free Full Text]
  34. Nakagawa T, Yuan J. Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J Cell Biol 150: 887–894, 2000.[Abstract/Free Full Text]
  35. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, Yuan J. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403: 98–103, 2000.[CrossRef][Medline]
  36. Nishitoh H, Matsuzawa A, Tobiume K, Saegusa K, Takeda K, Inoue K, Hori S, Kakizuka A, Ichijo H. ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16: 1345–1355, 2002.[Abstract/Free Full Text]
  37. Nogee LM. Abnormal expression of surfactant protein C and lung disease. Am J Respir Cell Mol Biol 26: 641–644, 2002.[Free Full Text]
  38. Nogee LM, Dunbar AE, III, Wert SE, Askin F, Hamvas A, Whitsett JA. A mutation in the surfactant protein C gene associated with familial interstitial lung disease. N Engl J Med 344: 573–579, 2001.[Free Full Text]
  39. Obeng EA, Boise LH. Caspase-12 and caspase-4 are not required for caspase-dependent endoplasmic reticulum stress-induced apoptosis. J Biol Chem 280: 29578–29587, 2005.[Abstract/Free Full Text]
  40. Paquette JC, Guerin PJ, Gauthier ER. Rapid induction of the intrinsic apoptotic pathway by L-glutamine starvation. J Cell Physiol 202: 912–921, 2005.[CrossRef][Web of Science][Medline]
  41. Pastrana B, Mautone AJ, Mendelsohn R. Fourier transform infrared studies of secondary structure and orientation of pulmonary surfactant SP-C and its effect on the dynamic surface properties of phospholipids. Biochemistry 30: 10058–10064, 1991.[CrossRef][Medline]
  42. Petrucelli L, O'Farrell C, Lockhart PJ, Baptista M, Kehoe K, Vink L, Choi P, Wolozin B, Farrer M, Hardy J, Cookson MR. Parkin protects against the toxicity associated with mutant alpha-synuclein: proteasome dysfunction selectively affects catecholaminergic neurons. Neuron 36: 1007–1019, 2002.[CrossRef][Web of Science][Medline]
  43. Qiu JH, Asai A, Chi S, Saito N, Hamada H, Kirino T. Proteasome inhibitors induce cytochrome c-caspase-3-like protease-mediated apoptosis in cultured cortical neurons. J Neurosci 20: 259–265, 2000.[Abstract/Free Full Text]
  44. Rodrigues CM, Sola S, Brito MA, Brondino CD, Brites D, Moura JJ. Amyloid beta-peptide disrupts mitochondrial membrane lipid and protein structure: protective role of tauroursodeoxycholate. Biochem Biophys Res Commun 281: 468–474, 2001.[CrossRef][Web of Science][Medline]
  45. Rutkowski DT, Kaufman RJ. A trip to the ER: coping with stress. Trends Cell Biol 14: 20–28, 2004.[CrossRef][Web of Science][Medline]
  46. Sanchez-Pulido L, Devos D, Valencia A. BRICHOS: a conserved domain in proteins associated with dementia, respiratory distress and cancer. Trends Biochem Sci 27: 329–332, 2002.[CrossRef][Web of Science][Medline]
  47. Smith WW, Jiang H, Pei Z, Tanaka Y, Morita H, Sawa A, Dawson VL, Dawson TM, Ross CA. Endoplasmic reticulum stress and mitochondrial cell death pathways mediate A53T mutant alpha-synuclein-induced toxicity. Hum Mol Genet 14: 3801–3811, 2005.[Abstract/Free Full Text]
  48. Stevens PA, Pettenazzo A, Brasch F, Mulugeta S, Baritussio A, Ochs M, Morrison L, Russo SJ, Beers MF. Nonspecific interstitial pneumonia, alveolar proteinosis, and abnormal proprotein trafficking resulting from a spontaneous mutation in the surfactant protein C gene. Pediatr Res 57: 89–98, 2005.[CrossRef][Web of Science][Medline]
  49. Thomas AQ, Lane K, Phillips J, III, Prince M, Markin C, Speer M, Schwartz DA, Gaddipati R, Marney A, Johnson J, Roberts R, Haines J, Stahlman M, Loyd JE. Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred. Am J Respir Crit Care Med 165: 1322–1328, 2002.[Abstract/Free Full Text]
  50. Wang WJ, Mulugeta S, Russo SJ, Beers MF. Deletion of exon 4 from human surfactant protein C results in aggresome formation and generation of a dominant negative. J Cell Sci 116: 683–692, 2003.[Abstract/Free Full Text]
  51. Wang WJ, Russo SJ, Mulugeta S, Beers MF. Biosynthesis of Surfactant Protein C (SP-C). Sorting of SP-C proprotein involves homomeric association via a signal anchor domain. J Biol Chem 277: 19929–19937, 2002.[Abstract/Free Full Text]
  52. Ward CL, Omura S, Kopito RR. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83: 121–127, 1995.[CrossRef][Web of Science][Medline]
  53. Weaver TE. Synthesis, processing and secretion of surfactant proteins B and C. Biochim Biophys Acta 1408: 173–179, 1998.[Medline]
  54. Wojcik C. Regulation of apoptosis by the ubiquitin and proteasome pathway. J Cell Mol Med 6: 25–48, 2002.[Web of Science][Medline]
  55. Yankner BA, Dawes LR, Fisher S, Villa-Komaroff L, Oster-Granite ML, Neve RL. Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer's disease. Science 245: 417–420, 1989.[Abstract/Free Full Text]
  56. Yoneda T, Imaizumi K, Oono K, Yui D, Gomi F, Katayama T, Tohyama M. Activation of caspase-12, an endoplasmic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent mechanism in response to the ER stress. J Biol Chem 276: 13935–13940, 2001.[Abstract/Free Full Text]
  57. Zhang K, Kaufman RJ. The unfolded protein response: a stress signaling pathway critical for health and disease. Neurology 66: S102–S109, 2006.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Med. Genet.Home page
L Guillot, R Epaud, G Thouvenin, L Jonard, A Mohsni, R Couderc, F Counil, J de Blic, R A Taam, M Le Bourgeois, et al.
New surfactant protein C gene mutations associated with diffuse lung disease
J. Med. Genet., July 1, 2009; 46(7): 490 - 494.
[Abstract] [Full Text] [PDF]

Home page
 ERRHome page
P. W. Noble
Epithelial fibroblast triggering and interactions in pulmonary fibrosis
Eur. Respir. Rev., December 1, 2008; 17(109): 123 - 129.
[Abstract] [Full Text] [PDF]

Home page
 ERRHome page
J. E. Loyd
Gene expression profiling: can we identify the right target genes?
Eur. Respir. Rev., December 1, 2008; 17(109): 163 - 167.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
W. E. Lawson, P. F. Crossno, V. V. Polosukhin, J. Roldan, D.-S. Cheng, K. B. Lane, T. R. Blackwell, C. Xu, C. Markin, L. B. Ware, et al.
Endoplasmic reticulum stress in alveolar epithelial cells is prominent in IPF: association with altered surfactant protein processing and herpesvirus infection
Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1119 - L1126.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
M. Dong, J. P. Bridges, K. Apsley, Y. Xu, and T. E. Weaver
ERdj4 and ERdj5 Are Required for Endoplasmic Reticulum-associated Protein Degradation of Misfolded Surfactant Protein C
Mol. Biol. Cell, June 1, 2008; 19(6): 2620 - 2630.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/L720    most recent
00025.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mulugeta, S.
Right arrow Articles by Beers, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mulugeta, S.
Right arrow Articles by Beers, M. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.