Lost after translation: insights from pulmonary surfactant ...the role of alveolar epithelial...

Lost after translation: insights from pulmonary surfactant for understandingthe role of alveolar epithelial dysfunction and cellular quality control infibrotic lung disease

Surafel Mulugeta,1 Shin-Ichi Nureki,2 and Michael F. Beers1

1Pulmonary, Allergy, and Critical Care Division; Department of Medicine, University of Pennsylvania Perelman School ofMedicine, Philadelphia, Pennsylvania; and 2Department of Respiratory Medicine and Infectious Diseases, Oita University,Yufu, Oita, Japan

Submitted 5 May 2015; accepted in final form 10 July 2015

Mulugeta S, Nureki S, Beers MF. Lost after translation: insightsfrom pulmonary surfactant for understanding the role of alveolarepithelial dysfunction and cellular quality control in fibrotic lungdisease. Am J Physiol Lung Cell Mol Physiol 309: L507–L525, 2015.First published July 17, 2015; doi:10.1152/ajplung.00139.2015.—Dating back nearly 35 years ago to the Witschi hypothesis, epithelialcell dysfunction and abnormal wound healing have reemerged ascentral concepts in the pathophysiology of idiopathic pulmonaryfibrosis (IPF) in adults and in interstitial lung disease in children.Alveolar type 2 (AT2) cells represent a metabolically active compart-ment in the distal air spaces responsible for pulmonary surfactantbiosynthesis and function as a progenitor population required formaintenance of alveolar integrity. Rare mutations in surfactant systemcomponents have provided new clues to understanding broader ques-tions regarding the role of AT2 cell dysfunction in the pathophysiol-ogy of fibrotic lung diseases. Drawing on data generated from avariety of model systems expressing disease-related surfactant com-ponent mutations [surfactant proteins A and C (SP-A and SP-C); thelipid transporter ABCA3], this review will examine the concept ofepithelial dysfunction in fibrotic lung disease, provide an update onAT2 cell and surfactant biology, summarize cellular responses tomutant surfactant components [including endoplasmic reticulum (ER)stress, mitochondrial dysfunction, and intrinsic apoptosis], and exam-ine quality control pathways (unfolded protein response, the ubiquitin-proteasome system, macroautophagy) that can be utilized to restoreAT2 homeostasis. This integrated response and its derangement willbe placed in the context of cell stress and quality control signaturesfound in patients with familial or sporadic IPF as well as non-surfactant-related AT2 cell dysfunction syndromes associated with afibrotic lung phenotype. Finally, the need for targeted therapeuticstrategies for pulmonary fibrosis that address epithelial ER stress, itsdownstream signaling, and cell quality control are discussed.

alveolar type 2 cells; cell quality control; epithelial ER stress; fibroticlung disease; surfactant proteins

BY THE VERY NATURE OF ITS juxtaposition with the outside world,the epithelium of the distal lung, and specifically the alveolartype 2 (AT2) cell, in the course of performing the biosynthetic,metabolic, and repair/regenerative functions critical to mainte-nance of alveolar homeostasis, must also protect itself from

significant stress imparted by its constant exposure to mechan-ical, metabolic, and environmental factors. To address thisconsiderable threat to protein synthesis, macromolecular turn-over, and maintenance of organelle mass and integrity, eukary-otic cells and higher organisms have evolved a complex net-work of cellular quality control pathways designed to improve,modify, compartmentalize, and/or remove abnormal molecularand organellar substrates that accumulate during the course of“daily life.” Disruption of this network inevitably results in theaccumulation of misfolded proteins/aggregates, complex mac-romolecules, and/or dysfunctional organelles with elaborationof multiple signaling cascades that can drive disease initiation/progression through the triggering of inflammation, cell death,and organ dysfunction. Although cellular quality control andits derangement have been shown to play an important role indisease pathogenesis in other organ systems such as brain,liver, and pancreas, its contribution to pulmonary disease isonly emerging.

Pulmonary fibrosis represents a heterogeneous group ofpostnatal interstitial lung disorders characterized by destructionof pulmonary architecture caused by an abnormal wound repairresponse that ultimately leads to scar formation, organ mal-function, disruption of gas exchange, and respiratory failure. Inadults, idiopathic pulmonary fibrosis (IPF), the most commonsubtype of the larger family of idiopathic interstitial pneumo-nias, is a diffuse parenchymal lung disease of unknown etiol-ogy that affects over 5 million people worldwide and typicallyresults in a need for lung transplantation or in death within 2–5years of diagnosis (81). In the United States, the annualincidence of IPF appears to be rising and is estimated at5–16/100,000 individuals with a prevalence of 13–20/100,000,is more common in men, and increases significantly with age.IPF is characterized histologically by a pattern of usual inter-stitial pneumonia (UIP), in which the peripheral subpleuralparenchyma shows evidence of fibroblastic foci, traction bron-chiectasis, and microscopic honeycombing lined by hyperplas-tic AT2 cells interspersed with areas of normal or nearlynormal lung tissue. Familial forms of pulmonary fibrosis (FPF)can also present in children and are part of the larger spectrumof childhood interstitial lung disease (chILD) (38; 88). Theincompletely defined pathogenesis of IPF (and chILD) hasbeen a major obstacle in developing effective therapies capableof stabilizing or improving lung function for these disorders.

Although research into the details of pathways responsiblefor epithelial cell dysfunction in IPF is still in its infancy, thepulmonary surfactant system has provided an important plat-form and experimental substrates to study the role aberrant

Articles published in this series are intended to provide a thoughtful andlucid linkage of science to the patient. Go here for more information on thisseries and to browse the content (http://www.the-aps.org/mm/Publications/Journals/PIM).

Address for reprint requests and other correspondence: S. Mulugeta, Pul-monary, Allergy, and Critical Care Division, Univ. of Pennsylvania, PerelmanSchool of Medicine, Smilow Center for Translational Research Suite 11-111,3400 Civic Center Blvd., Philadelphia, PA 19104-5159 (e-mail: [email protected]).

Am J Physiol Lung Cell Mol Physiol 309: L507–L525, 2015.First published July 17, 2015; doi:10.1152/ajplung.00139.2015. Physiology In Medicine

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quality control, cell stress, and cytotoxicity in these events. Inthis review, we describe developments in the understanding ofthe molecular mechanisms underlying childhood and adultinterstitial lung diseases (ILDs) from the viewpoint of disease-related mutations in components of the surfactant system[surfactant protein C (SP-C); surfactant protein A (SP-A), ATPbinding cassette class A3 (ABCA3) lipid transporter] and thecellular responses to their expression that could modulatedisease pathogenesis. Data on the elicited quality controlevents and signaling pathways leveraged from a variety of invitro and in vivo models expressing these mutant isoforms willbe examined and placed in the context of translational datafrom patients showing similar molecular signatures in bothfamilial and sporadic IPF and in preclinical and patient datafrom other non-surfactant-related AT2 cell dysfunction syn-dromes associated with a fibrotic lung phenotype.

Alveolar Epithelial Injury and Fibrotic Lung Remodeling:the Witschi Hypothesis Reborn

The health of the distal lung relies on the proper function ofAT2 cells, which play a critical role in the production andmaintenance of pulmonary surfactant, contribute to overallbarrier function, perform xenobiotic metabolism, and partici-pate in lung repair serving as a progenitor population followinglung injury to replace damaged AT1 and AT2 cells (10, 16).The cuboidal-shaped AT2 cells comprise only 3–5% of thealveolar surface area while the remainder is occupied byjuxtaposed AT1 cells that serve to facilitate gas exchange. BothAT1 and AT2 cells have important roles in ion transport in thedistal lung (101). Nevertheless, AT2 cells constitute 60% ofalveolar epithelial cells and 10–15% of all lung cells (36),rendering them a critical component in alveolar homeostasis.

Over 35 years ago, Haschek and Witschi published a theoryon the pathogenesis of lung fibrosis that challenged the thencontemporaneous view of lung fibrosis as an “inflammatorydisease” (56, 165). Although chronic inflammation as a key toits pathogenesis had persisted in some circles, it is not aprominent feature in biopsies of IPF patients (144). Coupledwith the low efficacy or even harmful effects of immunomodu-latory therapies shown in many clinical trials involving thesepatients (62a, 67, 132, 133), a paradigm shift developed awayfrom the so-called “alveolitis” theory of the 1970s and 1980s toagain suggest a pivotal role for alveolar epithelial cells (AEC)and specifically AT2 cell dysfunction in the development of afibrotic lung phenotype. Although not primary drivers of thefibrotic lesion, the exact roles of inflammation and the localimmune milieu in IPF pathogenesis remain to be defined.

In this “epithelial injury/abnormal wound repair” model,rechampioned in 2001 (144), IPF development and progressionwere proposed to be initiated and sustained by microfoci ofrepeated cycles of AEC injury occur within a larger dysfunc-tional repair process reminiscent of aberrant wound healingdescribed in other organs such as skin, kidney, and liver (18,184). More recently, the “postmodern” link between intrinsicepithelial cell dysfunction and IPF/ILD postulated by Witschihas been firmly established based on multiple lines of experi-mental evidence and clinical correlations:

1) Monogenetic diseases such as Hermansky-Pudlak syn-drome (HPS) are characterized by dysfunctional AT2 cells andILD (114). Mutations in nine different HPS genes disrupt

intracellular cargo protein trafficking leading to dysfunctionallysosome-related organelles (LROs) in various organs. Lamel-lar bodies (LBs), the LROs of AT2 cells, show marked increasein size due to overaccumulation of phospholipids. Two HPSgenetic subgroups, HPS1 and HPS4, manifest a common lungphenotype characterized by the premature development of aUIP-like fibrotic pattern in the fourth decade of life (114).Some pathological features can be recapitulated in mousemodels of HPS, where AT2 cells were shown to be importanteffector cells elaborating proinflammatory cytokines such asmonocyte chemoattractant protein-1 (MCP-1) (5, 191, 192).

2) Depletion of AT2 cells induces spontaneous fibrosis inmice. In a transgenic mouse model using the SP-C genepromoter to express a diphtheria toxin (DT) receptor exclu-sively in AT2 cells, administration of DT in these mice inducedAT2 cell death and increased lung collagen deposition.

3) AEC apoptosis is paramount in mouse models of lungfibrosis generated by single or repetitive exposures to bleomy-cin. Inhibition of apoptosis in these models by use of caspaseinhibitors, Fas/FasL pathway blockade, or genetic deletion ofproapoptotic Bcl2 proteins attenuates fibrosis (164).

4) It has been noted that aberrantly activated lung epithelialcells produce virtually all the mediators responsible for fibro-blast migration, proliferation, and activation with the subse-quent exaggerated extracellular matrix accumulation and de-struction of the lung parenchyma (144, 187).

5) Some of the most compelling evidence for the role of AT2cells in these processes is derived from studies linking patientswith UIP or chILD histopathology with mutations in AT2cell-restricted genes involved in the production of pulmonarysurfactant. To date, more than 60 SP-C (SFTPC), and 150ABCA3 mutations, as well as two mutations in the surfactantprotein A2 gene (SFTPA2) have been identified in affectedchildren and adults (25, 116, 175, 178). As will be detailedbelow, signatures of protein aggregation, endoplasmic reticu-lum (ER) stress, proinflammatory/profibrotic cytokine elabora-tion, altered macroautophagy, and apoptosis defined by modelsystems expressing SFTPC, SFTPA, and ABCA3 mutations arealso present in the lungs and AT2 epithelia of sporadic andfamilial IPF patients (3, 23, 83, 91).

Thus substantial evidence exists that a vulnerable and/ordysfunctional AT2 epithelium is a pivotal player in aberrantinjury/repair responses occurring in IPF and other forms offibrotic lung remodeling.

AT2 CELLS AND THE BIOSYNTHETIC CHALLENGE OFSURFACTANT

The alveolar gas exchange surface is coated with a thin filmof surface active agent (� surfactant), representing a complexheterogeneous mixture of primarily lipids (90% by weight) andsome protein that serves to promote alveolar stability byreducing surface tension at the air-liquid interface along theepithelial lining layer (129). In addition to lipids [mainlyphosphatidylcholine (PC) with one (lyso-PC) or two (DPPC)palmitic acid side chains], biochemical analysis of surfactanthas identified four unique proteins designated surfactant pro-teins: SP-A, SP-B, SP-C, and SP-D (131). A large volume ofliterature has demonstrated that the surface tension-reducingfunction of surfactant stems from the interaction of phospho-lipids and the two low-molecular-weight hydrophobic proteins,

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SP-B and SP-C (reviewed in Ref. 180). The relatively hydro-philic and more abundant oligomeric proteins, SP-A and SP-D,are members of the collectin family of C-type lectins that sharedistinct collagen-like and globular, carbohydrate-binding do-mains. Although SP-A and SP-D do not have a direct functionin the surface tension activity of surfactant, they play anessential role in innate lung host defense (reviewed in Ref.183). Numerous studies have shown that, under pathologicalconditions, SP-A and SP-D can each undergo a variety ofposttranslational modifications (such as oxidation, nitration,S-nitrosylation) that can lead to loss of function (50, 53,102, 196).

AT2 cells synthesize, secrete, and recycle all components ofsurfactant (Fig. 1). Compared with neuroendocrine and many

secretory epithelia (exocrine and endocrine) in other tissues,both functionally and morphologically, the AT2 cell is some-what unique. The diversity of cellular cargo that must bemanaged (phospholipids, hydrophobic proteins, multimeric hy-drophilic proteins, cytokines) creates a significant burden oncell quality control and metabolic demands. Ultrastructurally,AT2 cells lack a dense secretory granule; but instead, trans-mission electron microscopy (EM) reveals the presence ofcytoplasmic, osmiophilic, lamellated organelles (i.e., LBs)long recognized as the storage organelle from which surfactantis released into the alveolar lumen (9). Biochemically, LBsresemble other LROs in that they are acidic, express lysosomalmarkers (CD63; LAMP-1), but also contain surfactant lipids,SP-B, and SP-C as well as a fraction of intracellular SP-A

Fig. 1. Schematic representation of structure, biosynthetic processing, and life cycle of 3 surfactant system components associated with interstitial lung diseasein humans by the alveolar type 2 cell. Biosynthesis: along with phospholipids, surfactant protein (SP)-B, and SP-D, AT2 cells synthesize SP-C, SP-A, andABCA3. SP-C: the human SFTPC gene is transcribed and translated to a 197-amino acid (21-kDa) proprotein (proSP-C21). ProSP-C21 is translocated to theendoplasmic reticulum (ER), is sorted in the Golgi, and enters the regulated secretory pathway where it is processed by 4 endoproteolytic cleavages of NH2 andCOOH propeptide domains as it transits through small or sorting vesicles (SV), multivesicular bodies (MVB), and composite bodies (CB) to lamellar bodies (LB),the site of the final cleavage. Inset, left: topological representation of the bitopic proSP-C showing its type II orientation. The 4 domains of proSP-C includeNH2-terminal targeting domain harboring lysine 6 ubiquitination site (yellow oval) and Nedd4-2 binding “PY” targeting motif (black rectangle); mature domain(depicted in blue) containing juxtamembrane palmitoylation sites (pink bars) and valine-rich �-helical transmembrane hydrophobic region; linker domain, sitefor the common I73T mutant variant (yellow pentagon); BRICHOS domain with the exon4 deletion region (depicted in gray), and L188Q mutation (yellowtriangle). Conserved cysteine residues (C121 and C189) form the primary disulfide-bond (gray bar). ABCA3: initially routed to post-Golgi sorting vesicles,ABCA3 is trafficked via the MVB/CB network to LBs and plasma membrane. ABCA3 undergoes a posttranslational proteolytic cleavage within the proximalNH2-terminal region at distal post-Golgi compartments. Inset, bottom right: predicted topological structure of the ABCA3 transporter. ABCA3 comprises 12putative membrane-spanning helices where the NH2- and COOH-terminal domains and the 2 ABC binding cassettes (ABC1 and ABC2) face the cytosol. TheNH2 targeting signal for Golgi exit (yellow region) and the 2 glycosylation sites within the first luminal loop (orange) are shown. ECD, extracellular domain.SP-A: the human SFTPA gene gives rise to multiple mRNA transcripts that assemble into an 18-mer native structure made up from 6 SP-A trimers. The assembledform of SP-A is secreted by both regulated and constitutive pathways. Posttranslational modifications of SP-A include signal peptide cleavage prior to enteringthe ER, hydroxylation of proline residues, and N-linked glycosylation. Inset, top right: structural representation of a native SP-A. The primary structure of SP-Aprotein consists of 4 domains: a short amino-terminal domain, a collagenous domain, a neck domain, and a carbohydrate recognition domain (CRD). Six of theSP-A trimers composed of 2 SP-A1 molecules (depicted in green) and 1 SP-A2 molecule (depicted in black) give rise to the mature SP-A 18-mer. A prolineresidue in the collagenous domain of SP-A primary structure provides a flexible kink that causes the trimers to bend outward in different directions, giving thenative SP-A 18-mer a “flower bouquet” appearance. Secretion: surfactant phospholipid, processed SP-C, SP-B, and SP-A contained in the LB are released byregulated exocytosis; ABCA3 remains in the LB membrane and is recycled. A portion of SP-A and SP-D is secreted constitutively. Reuptake/degradation/recycling: SP-A, SP-C, together with other surfactant proteins and lipids, are taken up by AT2 cells via endocytosis and are either targeted to the lysosomes fordegradation or recycled back to LBs via endosomes and MVBs.


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(121). LBs release these contents into the alveolus via regu-lated exocytosis regulated by a variety of signaling pathways(reviewed in Ref. 134). In contrast much of the synthesizedSP-A and all SP-D is secreted via a non-LB, constitutivepathway. In addition to biosynthesis, AT2 cells participate inthe uptake, catabolism, and reutilization of surfactant. Proteinand lipid components are endocytosed via clathrin-dependentand -independent mechanisms, routed through the central vac-uolar system [early endosomes (EE), late endosomes/multive-sicular bodies (LE/MVB)] and then sorted to lysosomes fordegradation or to LB for resecretion.

SP-C: a Unique Secretory Product

Of the four surfactant proteins, only SP-C is synthesizedexclusively by the AT2 cell. The 2.4-kb gene encoding humanSFTPC is located on chromosome 8p and is organized into sixexons (I through V coding, VI untranslated) and five introns,which produce a 0.9-kb mRNA encoding either a 191- or197-amino acid 21-kDa proprotein (proSP-C21). The proteinform isolated and sequenced from lung lavage fractions (SP-C3.7) is a lipid-avid peptide composed of 33–35 highly con-served amino acids containing a high content of Val, Ile, andLeu (�60–65% of the primary sequence), which in aqueoussolution self-aggregates, adopting �-sheet conformation andforming amyloid fibrils. Thus SP-C represents a structurallyand functionally challenging substrate for the AT2 cell in whichthe proSP-C21 propeptide is trafficked through the regulatedsecretory pathway as an integral type II bitopic transmembrane(TM) protein (Ncytosol/Clumen), undergoing four endoproteolyticcleavages of its flanking NH2 and COOH propeptides to yield themature, biophysically active 3.7-kDa form found in secretedsurfactant (Fig. 1B) (reviewed in Ref. 13). These posttranslationalevents as well as the behavior of disease-causing proSP-C mutantsare influenced (directly and indirectly) by four functional domainsand structural motifs contained within proSP-C21 (Fig. 1B, inset):

1) NH2 targeting domain (PY motif). A series of mutagenesisstudies identified and characterized a tryptophan-tryptophan(WW) domain-interacting PPxY (PY) motif within the NH2

propeptide that directs post-ER/Golgi proSP-C trafficking todistal compartments (34, 66, 84). The PPDY motif specificallyinteracts with two members of an E3 ubiquitin ligase family,Nedd4 and Nedd4-2 (Nedd4-L), with Nedd4-2 exerting thedominant effect on proSP-C transport via catalysis of monou-biquitination (a well-known signal for lysosomal targeting ofproteins) of proSP-C21 at lysine residue 6 of the cytosolic NH2

propeptide (34).2) Mature SP-C (TM) domain. In phospholipid-rich environ-

ments the mature SP-C domain forms a very stable and rigid�-helix and functions as a noncleavable signal anchor peptidefor proSP-C21, facilitating its translocation to the ER (33, 75).Additionally, a conserved pair of juxtamembrane basic aminoacids, lysine (K) and arginine (R) (at positions 34 and 35 ofhuman proSP-C), play an essential role in the determination ofthe propeptide’s type II TM topology (75, 109).

3) The BRICHOS domain. The distal proSP-C21 COOH-terminus (residues 94–197), known as the BRICHOS domain,shares a high degree of structural homology with domainsidentified in over 300 proteins (59). The term BRICHOS wasderived from the original disease-associated family membersincluding BRI2, linked to familial British and Danish Demen-

tia; Chondromodulin-1, related to chondrosarcoma; and SP-C(139). The structural similarity of these three is punctuated bythe fact that each exist as integral type II TM proteins com-prised of a cytosolic region, a TM segment, a linker domain,and BRICHOS domain containing a pair of conserved cysteineresidues The human SFTPC encodes four cysteines in theBRICHOS domain. Cysteines 121 and 189 are positionallyconserved across species and form the primary folding func-tion. Johannson’s group has also modeled a second disulfidebridge between cysteine 120 and 148 (see Refs. 18, 181).

For proSP-C, mutation of either one of these cysteine resi-dues results initially in proximal (ER) retention and eventualformation of aggregates of unprocessed polyubiquitinated pro-protein in vitro (69). Functionally, in addition to promotingproSP-C folding, the BRICHOS domain appears to have anintramolecular chaperone-like activity safeguarding the meta-stable, �-sheet-prone, mature SP-C domain from amyloid fibrilformation (51, 65, 154, 182). Amyloid deposits composed ofmature SP-C have also been observed in lung tissue samplesfrom some ILD patients with mutations in the BRICHOSdomain (181).

4) Linker domain. As the name signifies, the proximalCOOH-terminal propeptide (residues 59–93) is linearly posi-tioned between the mature SP-C (TM) and BRICHOS domainsand through formation of �-hairpin structure thereby facilitatesdocking of the BRICHOS domain to the TM segment. Impor-tantly, mutations in the linker domain do not induce cytosolicaggregation of proSP-C but instead result in its mistraffickingand accumulation in plasma membrane, early endosomes, andLE/MVB (15, 58) (discussed in detail later).

Surfactant Protein A: a Multimeric Glycoprotein AT2Product

SP-A, the most abundant surfactant protein, is a luminal,multifunctional sialoglycoprotein of Mr 28–36,000 that con-tains a COOH-terminal C-type lectin motif, a triple helicalcollagen domain, and carbohydrate recognition domain (CRD)(183) (Fig. 1, inset). In humans, there are two genes (SFTPA1and SFTPA2), 4.5 kb each, located on chromosome 10. Theamino acid differences that distinguish SP-A1 from SP-A2are in general conservative and located mainly in the colla-gen-like domain with only one shown to affect proteinstructure. Like SP-D, SP-A is synthesized by airway cells,including nonciliated (club/Clara) cells. Although it hasbeen localized to extrapulmonary sites, including salivaryglands, lacrimal glands, and the female urogenital tract, theexact physiological relevance of its expression in theseorgans remains to be defined (reviewed in Ref. 78).

The biosynthesis of SP-A differs markedly from SP-C (andSP-B). In contrast to proteolytic processing of a larger propro-tein, posttranslational processing of the SP-A primary transla-tion product consists of NH2 signal peptide cleavage, N-linkedglycosylation, and proline hydroxylation followed by trimericassociation of monomers via coiled-coil bundling of �-helicesin the neck thought to be composed of two SP-A1 and oneSP-A2 monomer(s). The completed oligomeric structure ofSP-A represents a higher order 18-mer assembled from sixtrimeric subunits linked by interchain disulfide bonding(Fig. 1, inset).




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Metabolic labeling studies have demonstrated that the intra-cellular trafficking and secretion of nascent SP-A may involveboth a LB-dependent (42) and a LB-independent (constitutive)pathway (122). Since alveolar SP-A can also be taken up viaendocytosis and incorporated into LBs it has been difficult toaccurately gauge the contribution of each pathway. Beyond thepresence of a signal peptide and N-linked glycosylation, struc-tural motifs and signals regulating trafficking of SP-A are notdefined.

ABCA3: a Transporter Critical to AT2 Cell Homeostasis

A key component in the formation of LBs is the ABCA3glycoprotein, a member of the ABC superfamily TM trans-porter proteins that use ATP energy to drive various substrates,ranging from small ions (e.g., ABCC7 (CFTR) � Cl�) to largemolecules (e.g., ABCA1 � cholesterol) across the plasma andintracellular membranes. ABCA3 belongs to a subclass (class A)of ABC transporters that are involved in a variety of lipid trans-location events in multiple cell types, including macrophages,epithelial cells, and neurons, with each localized in distinct sub-cellular compartments (71).

Human ABCA3 has been mapped to chromosome 16p13.3and encodes a 1,704-amino acid protein (35). AlthoughABCA3 mRNA is detected in many tissues, the ABCA3message is highly expressed in AT2 cells and ABCA3 proteinlocalized in the LB-limiting membrane (110, 186). ABCA3 hasbeen shown in vitro to transport phosphatidylcholine, phos-phatidylglycerol, sphingomyelin, and cholesterol into lyso-somes of model cell line systems (8, 30, 31, 41, 110). Func-tionally homozygous null mutations of ABCA3 described inneonates with respiratory failure are associated with the pres-ence of abnormal electron dense bodies on transmission EM,further demonstrating its importance as one of the criticalregulators of LB biogenesis and lung surfactant metabolism (8,25, 31).

The structure of ABCA3 is schematically illustrated in Fig.1. To date studies have identified two functional domains in themolecule: 1) xLxxKN targeting motif: nearly all ABCA sub-family of transporters share significant homology in theirNH2-terminal domain, including a highly conserved motif(xLxxKN) (71) that we have shown to be essential for directingthese proteins to post-Golgi compartments (12). Since eachABCA transporter has a unique subcellular destination andfunction, subsequent sorting of ABCA3 to LB requires addi-tional unidentified signal(s). 2) N-linked glycosylation: site-directed mutagenesis of two asparagine residues within the firstNH2-terminal luminal loop of the transporter impairs proteinstability and disrupts its anterograde trafficking, resulting inproteasomal degradation and cell stress (14).

To summarize, AT2 cells represent a metabolically activecellular component of the distal lung responsible for the bio-synthesis, assembly, and complex intracellular trafficking of adiverse and challenging set of cargo, resident proteins, andorganelles that often mix in multiple subcellular compartmentsof the secretory, endocytic, and degradative (lysosomal) path-ways.

CELLULAR QUALITY CONTROL

In 2001, Nogee and colleagues (117) first reported a muta-tion in the COOH-terminal encoding region of SFTPC that

resulted in deletion of exon 4 and its 37 amino acids (SP-C�exon4) in both an infant and mother with ILD. Shortly after,a second report described a different SFTPC mutation resultingin substitution of glutamine for leucine at position 188 of theSP-C propeptide (SP-CL188Q) in a large kindred of whom thebiopsies of 11 adults showed IPF/UIP patterns and nonspecificinterstitial pneumonia (NSIP) patterns were present in threechildren. Using a variety of model systems, we and otherssubsequently demonstrated that expression of SP-C�exon4 orSP-CL188Q in vitro resulted in intracellular SP-C protein ag-gregation and cell death by apoptosis. Importantly, all affectedpatients with SFTPC-related ILD reported to date have beenheterozygous for the mutant allele, suggesting a toxic gain offunction mechanism.

To preserve homeostasis in the face of a variety of exoge-nous and endogenous insults that challenge the integrity oftheir molecular and organelle components, eukaryotic cellshave developed a quality control network integrating variouscombinations of the following processes: 1) sensing mecha-nisms designed to detect the presence of unwanted proteins,nucleic acids, macromolecules, and organelles; 2) effectorpathways composed of chaperone/cochaperone/enzyme sys-tems designed to improve macromolecular integrity (e.g., pro-tein folding), facilitate normal trafficking, or repair damagedorganelles and DNA-based structures (e.g., telomeres); and 3)degradative components such as the ubiquitin-proteasome sys-tem (UPS) and autophagosome-lysosomal-endosomal path-ways (principally macroautophagy will be considered here)acting synergistically to restore protein/organelle integrity andremove defective or unwanted substrates. In addition, signalingpathways emanating from many of these component piecesserve to modulate the overall composition of the quality controlnetwork.

Quality Control Lessons for the Lung from Other Organs

At the NHLBI workshop “Malformed Protein Structure andProteostasis in Lung Diseases,” the role that disorders ofprotein folding and degradation play in the pathobiology oflung disorders and their associated complications was high-lighted (7). By extension, recent discoveries have also impli-cated organelle dysfunction and turnover, in particular inmitochondria or in the endosomal-lysosomal system, in thepathogenesis of some age-dependent (e.g., IPF), age-acceler-ated (e.g., HPS-induced fibrosis), and environmentally modi-fied (e.g., tobacco) lung disorders, pointing to a critical role forthe need to edit and remove damaged organelles to maintainlung health. A major conclusion from this workshop was thatresearch into the mechanisms and pathways underlying protein(and organelle) quality control in the lung and the coupling ofalterations in quality control to lung disease has been largelyunderdeveloped.

By contrast, much of the progress made in our understandingthe role of quality control pathways in disease has been derivedfrom studies of a seemingly diverse set of chronic, degenera-tive disorders observed in a variety of organ systems includingthe central nervous system (CNS), liver, and pancreas. Hun-tington’s and Alzheimer’s diseases represent progressive neu-rodegenerative disorders associated with expression or alteredprocessing of a mutant TM protein (analogous to proSP-C) thatundergoes a fatal conformational rearrangement that unmasks




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an intrinsic ability to self-associate (aggregate), induce aber-rant cell signaling/cytotoxicity, and/or be deposited in nonna-tive subcellular or tissue compartments. Similarly, liver diseasefrom expression of the mutant Z allele of alpha-1-antitrypsin(Z-A1AT) is associated with ER retention and cytosolic aggre-gation of Z-A1AT in hepatocytes leading to their eventualdeath. The sheer magnitude of biosynthetic capacity for insulinand other hormones in islet cells of the endocrine pancreasrequires an equally robust quality control system, which ap-pears to fail in models of Type 2 diabetes. Furthermore,disorders such as Parkinson’s disease have supplied the recog-nition that protein “conformational diseases” are often accom-panied by mitochondrial dysfunction and, like all organelles,must be “recycled,” requiring an additional layer of cell qualitycontrol (130). Finally, lipid storage disorders such as Ni-emann-Pick C (NPC) disease that are marked by severeneurodegeneration and lung disease have been shown toacquire a defective cell quality control phenotype (princi-pally impaired macroautophagy) (140).

Together, from basic and translational studies of these dis-eases using these abnormal protein isoforms as substrates, thecellular responses to misfolded proteins and dysfunctionalorganelles have been dissected out in the context of cellularquality control.

Protein Quality Control (Proteostasis)

Mutant protein accumulation within the ER and cytosol iscountered by a complex mechanism aimed at restoring proteinhomeostasis or “proteostasis” (6). Proteostasis refers to thecontrol of concentration, formation, binding interactions, qua-ternary structure, and location of individual proteins making upthe proteome by readapting the innate biology of the cell, oftenthrough transcriptional and translational changes. The proteo-stasis network comprises processes controlling protein synthe-sis, folding, trafficking, aggregation, and degradation and isregulated by the integration of inputs from numerous cellsensing and signaling pathways. Failure of proteostasis to copewith misfolding-prone proteins, aging, or metabolic/environ-mental stress can trigger or exacerbate conformational disease.

The major components of a prototypical quality controlresponse to aberrant conformers include the following:

The unfolded protein response. As an initial response, highlyspecific signaling pathways, evolved to ensure that ER protein-folding capacity is not overwhelmed, are collectively termedthe unfolded protein response (UPR) (reviewed in detail in Ref.73). The UPR pathways are governed by three ER TM proteinsensors: PKR-like endoplasmic reticulum kinase (PERK), ac-tivating transcription factor 6 (ATF6), and inositol-requiringenzyme 1 (IRE-1) (illustrated in Fig. 2). Their activation byabnormally folded proteins initiate one or more of three signalingcascades with two objectives: 1) generation, translocation, andbinding of select transcription factors (XBP-1, ATF-6p50, ATF4)to nuclear ER-stress-responsive elements to expand ER refoldingcapacity by upregulating a variety of chaperones (e.g., Bip,GRP94, calnexin, protein disulfide isomerase); and 2) throughphosphorylated eif2� to promote translation attenuation at thelevel of the ribosome. Importantly, molecular signatures generatedby these events can be used to detect UPR activation in cells andin tissues: IRE1 activation is heralded by appearance of splicedXBP1 mRNA or XBP1 protein; PERK activation can be con-

firmed by detection of phosphor-eIF2�, CHOP/GADD153, andATF4; ATF6 cleavage fragments confirm engagement of thispathway. The relative contributions of each UPR pathway to theproteostatic response are protein substrate and cell specific.

ER-associated protein degradation. If the UPR is unsuccess-ful at restoring normal protein conformation, misfolded orunassembled protein targets are removed from the ER byretrotranslocation and ultimately destroyed by the ubiquitin-proteasome system (UPS) depicted in Fig. 2 (107, 170). Theproteasome is the main protein degradation machinery of thecell and degrades �90% of all cellular proteins. Covalentattachment of one or more K48-linked polyubiquitin chain(s)to a lysine residue(s) represents the primary targeting signal fordirection to 26S proteasomes and then degraded into smallpeptides. In addition to a role in protein quality control, proteindegradation by the proteasome is essential for diverse cellularfunctions such as apoptosis, transcription, cell signaling, andimmune responses (reviewed in Ref. 119, 141). The protea-some itself is composed of a cylinder-shaped catalytic coreparticle, the 20S proteasome, which can be capped at either endby several different 19S regulator complexes when ATP/Mg2�

is present (48). The 20S proteasome alone participates but isinefficient at protein degradation therefore activation mediatedby these 19S regulatory particles is crucial and can contributeto differential proteasome responses to physiological or path-ological conditions (reviewed in Ref. 106).

The 26S proteasome used for ER-associated protein degra-dation (ERAD) is subject to failure. In a variety of systems,overexpression of misfolded protein isomers and conformerscan actually inhibit global UPS function, leading to the cyto-solic accumulation of both the primary mutant as well asbystander proteins (17). In addition, environmental factorssuch as cigarette smoke can directly impair proteasome activityin lung cells (without affecting proteasome expression), result-ing in accumulation of polyubiquitinated proteins (168).

Autophagy. The process of macroautophagy (hereafter re-ferred to as autophagy) was originally described as a cellularresponse to fasting starvation. However, an important emerg-ing concept is that autophagy also functions as a second majordegradation pathway for the targeted removal of long-lived,aggregation-prone proteins (e.g., huntingtin, �-synuclein, andneuroserpins) (82, 89). An extensive discussion of the au-tophagic process is beyond the scope of this review; the readeris referred to several excellent recent reviews (32, 94, 113). Inbrief, as illustrated in Fig. 2, autophagy is a dynamic processcontrolled by at least 30 known autophagy (Atg) genes whoseassembly and turnover enclose cytosolic materials and organ-elles by generation of isolation membranes (phagophores) thatmature to form double membrane-bound vesicles (“autopha-gosomes”) that are readily identified by EM. Subsequently, theautophagosome containing captured cargo fuses with the lyso-some for degradation of the contents. The entire process ishighly regulated from inputs from a variety of sensing andsignaling mechanisms including mammalian target of rapamy-cin (mTOR1), Class III phosphoinositide 3-kinases (PI3K), andseveral mTOR-independent pathways. The selectivity of au-tophagy for misfolded protein is dependent on the recognitionof ubiquitinated aggregates by ubiquitin-binding receptorssuch as p62/sequestosome-1 (p62/SQSTM1) (125).

Various reports have confirmed the existence of a linkbetween ERAD/UPS and autophagy in the clearance of mis-




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folded proteins (28, 124), since perturbations in the fluxthrough either pathway can affect the activity of the othersystem (85). Evidence also exists for cross talk between theUPR and autophagy mediated by several pathways includingan eIF2�/ATF4 axis and JNK1 (105, 171).

Aggresomes, JUNQ, and IPOD. Current consensus holdsthat direct cellular toxicity from aberrant mutant protein con-formers is associated with nonnative soluble protein oligomersand that the formation of large aggregates is instead cytopro-tective. To limit toxicity from misfolded proteins accumulating

in the cytosol or nucleus that escape degradation by either theUPS and/or autophagy, mammalian cells contain an additionalcellular quality control-based defense that involves the recog-nition, segregation, and active compartmentalization of ubiq-uitylated protein oligomers/microaggregates in a microtubule-dependent manner to pericentriolar, membrane-free, vimentin-enwrapped perinuclear structures termed aggresomes (68).Work by several groups has shown that, in addition to proteinaggregates, Atgs and lysosomes can also be directed to ag-gresomes in a process dependent on the microtubule deactylase

Fig. 2. Quality control machinery utilized by cells in response to the expression of misfolded or unassembled proteins and dysfunctional organelles. Unfoldedprotein response (UPR): 1 or more of the 3 ER proximal sensors for misfolded proteins, IRE1, PERK, and ATF6, are activated by misfolded SP-C to initiatethe downstream signaling of the UPR. These sensors are typically maintained in an inactive state as they are bound to the molecular chaperone, Bip/GRP78. Thepresence of increasing load of misfolded SP-C proteins within the ER requires the availability of Bip/GRP78 to bind to the misfolding proteins and consequentlyBip/GRP78 dissociates from the sensors sites to meet this requirement and to allow 3 downstream signaling pathways to activate the following. 1) ATF6: whenBip is released from ATF6p90, this transmembrane sensor is first translocated to Golgi and subjected to cleavage by S1 and S2 protease generating a solublecytosolic form (ATF6p50) that traffics to the nucleus where it binds to ER-stress-responsive elements (ERSE) to upregulate chaperone production. 2) IRE1, inthe absence of Bip binding, undergoes phosphodimerization and acquires specific endonuclease activity for cytosolic splicing of XBP1 mRNA. The translatedXBP1, a transcription factor, also binds to ERSEs in the nucleus. 3) PERK activation results in both phosphorylation of cytosolic eIF2� leading to a generalizedtranslation repression plus selective upregulation of transcription factors such as CHOP/GADD153 and ATF4 to promote a specific subset of gene expressioninvolved in expanding proteostatic capacity. ER-associated protein degradation (ERAD) involves a multistep process consisting of recognition of misfolded cargoby ER-resident proteins [e.g., EDEM, protein disulfide isomerase (PDI), Bip/Grp78] followed by their retrotranslocation out of the ER through translocons andsubsequent polyubiquitination mediated by cytosolic E1/E2/E3 ubiquitin ligases. Targeting of ubiquitin-decorated coformers to the 26S proteasome results ineventual degradation. Autophagy: cytosolic macroaggregates are targeted for degradation by autophagy. Activation of a cascade of upstream events (not depicted)mediated by a complex composed of Beclin 1, class III PI3K, and other components initiates isolation membrane assembly and the eventual formation of aphagophore. The Atg5-Atg12-Atg16L complex and atg8 (LC3-I) phosphatidylethanolamine (PE) conjugate recruit and through elongation envelop cytosolicubiquitinated cargos including protein aggregates and mitochondria, leading to the formation of autophagosome. Subsequently, the autophagosome fuses withlysosomes, resulting in an acidified and functional autophagolysosome (“autolysosome”), which also contains lysosome-derived acid hydrolases, to promotedegradation of internalized cytosolic content. Aggresome formation: as a last coping mechanism, failure of UPR/ubiquitin-proteasome system (UPS)/autophagynetwork to control mutant protein levels results in transport of aggregate forms of misfolded protein in a microtubule-dependent manner to the microtubuleorganizing center (MTOC) near the nucleus, resulting in the formation of aggresomes.




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HDAC6, suggesting that aggresomes may function as a deliv-ery and concentration system to improve the efficiency ofautophagic degradation (63, 74).

Recently, our understanding of cellular aggregate depositionsites has been further expanded by the discovery of two otherdistinct types of deposition sites that can concurrently existwithin a single cell (70). Like aggresomes, one site is locatedin close proximity to the nucleus and functions as a qualitycontrol center that attracts chaperones and proteasomes toeither refold or degrade misfolded aggregated proteins. Thistype of deposition site, which contains ubiquitylated misfoldedproteins, was termed “JUxta Nuclear Quality control compart-ment” (JUNQ) and is a dynamic structure that rapidly ex-changes proteins with the cytosol. The other aggregate depositionsite, located near cytosolic vacuoles, was termed “insoluble pro-tein deposit” (IPOD) and sequesters immobile substrates that haveno ubiquitin signal. The interrelationships of aggresomes, JUNQ,and IPOD to each other and to the UPS and macroautophagy inmammalian cells remain to be established.

Consequences of Failed Protein Quality Control: ER Stress,Aggregation, and Apoptosis

If the integrated proteostatic program (combined efforts ofUPR, ERAD, and autophagy) fails to control mutant proteinlevels, a series of deleterious consequences for the cell canensue. Illustrated in Fig. 3, these include the following events:

1) ER stress. In addition to its normal adaptive signalingdescribed previously, prolonged or severe overload of theER with client proteins can activate “alarm signals” thatoverlap with some of the same signaling transduction cas-cades associated with innate immunity (185, 194). Termed“ER stress,” UPR sensors (particularly IRE1) can thenactivate downstream MAP kinases such as JNK to promotethe elaboration of proinflammatory cytokines and growthfactors from affected cells (115, 166). The proof of principlethat the balance of UPR/ER stress can determine cytopro-tection/cytotoxicity has been demonstrated repeatedlywhereby overexpression or knockdown of Grp78/Bip can

Fig. 3. Pathways to AT2 cell injury/dysfunction defined by using mutant proSP-C isoforms as prototype substrates. The expression of proSP-C mutants canproduce 1 of 2 profiles of aberrant cellular responses. Misfolding BRICHOS proSP-C (Group A1): ER stress marks the state in which the UPR can no longermaintain ER homeostasis because of overwhelming expression of misfolded SP-C, leading to a cascade of cellular disruption and injury: 1) Dysfunction of ERADdue to an overloaded and defective UPS. This event can be further exacerbated by second hits such as cigarette smoke and viruses. 2) ER stress-dependentrecruitment of TRAF2 by IRE1 to activate JNK that promotes upregulation and release of cytokines. 3) Induction of apoptotic pathways by ER stress-inducedactivation of either the PERK/EIF2�/ATF4 network to trigger CHOP activation, IRE1/TRAF2 activation of caspase 4/12, and/or cytochrome c release fromdysfunctional mitochondria. All 3 intersect downstream at the activation of caspase 3. 4) A failure of aggresomal compartmentalization or autophagy alsocontributes to the eventual accumulation of toxic macroaggregates. Mistrafficked Linker (non-BRICHOS) proSP-C (Group B): mutations in the COOH-terminusoutside BRICHOS domain of proSP-C reported to date appear to be initially mistargeted to the plasma membrane via a constitutive pathway. Because of properprocessing failure, proSP-C in the plasma membrane can be reinternalized and trafficked through early endosomes to late endosomes (LE)/MVB. The enhancedpresence of aberrantly trafficked and misprocessed proSP-C in these compartments leads to a functional disruption of normal endosome/amphisome/lysosometurnover within the autophagic machinery that ultimately results in a distal block in autophagy. Such a block leads to the accumulation of abnormally largeautophagic vacuoles containing undegraded, organellar, and proteinaceous debris populated with autophagy markers including LC3 and P62, as well as parkin,a protein critical for autophagy-dependent removal of dysfunctional mitochondria (i.e., mitophagy). *Potential sites for organelle disruption by the expressionof mistrafficked proSP-C.




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modulate both ER stress markers and susceptibility to celldeath in vitro and in vivo (72, 179).

2) Intrinsic apoptosis. In mammalian cells, ER stress canalso induce “intrinsic apoptosis” via one or more pathways (80,185). One involves the cleavage and activation of the ER-membrane-localized caspase-4/12 (60). In another, a complexgenerated by IRE1 and TNF-receptor-associated factor 2(TRAF2) recruited to the ER membrane signals downstreameffectors such as JNK (62). In some systems, PERK via anATF4-mediated pathway and/or IRE1 via XBP-1 can upregu-late C/EBP-homologous protein (CHOP), a transcriptional re-pressor of expression of prosurvival Bcl2 proteins. Addition-ally, an ER-mitochondria death-signaling cascade mediatedthrough a cytochrome c/apoptotic proteasome activating factor 1(APAF1)/caspase 9 apoptosome mechanism has been described ina variety of model systems (52). Further downstream integrativedeath signaling can involve activated caspase cascades that varyamong cell types, by pathway and by inciting substrate. Asexamples, numerous reports have linked ER stress-mediated ap-optosis of pancreatic beta cells to a caspase 4/12-mediated acti-vation of caspase 3 (reviewed in Ref. 123). Similarly, amyloid-beta (A�) also induces apoptosis via activation of caspases 4/12and then caspase 3 in neuroblastoma cell lines (61); however,neurons expressing misfolded PolyQ72 huntingtin undergocaspase 8-mediated apoptotic events (138).

3) Protein aggregation. Best exemplified by chronic neuro-degenerative disease, the intracellular or extracellular accumu-lation of an abnormal protein isoform (such as A� amyloidprecursor protein, �-synuclein, or polyglutamine huntingtin-1)with inherent instability can lead to time-dependent neuronalcell toxicity and the histopathological appearance of inclusionbodies, amyloid fibrils, and protein macroaggregates in thecytosol or within the nucleus (29, 76, 135, 137). As will bediscussed in detail subsequently, given the propensity of SP-C3.7 to form �-amyloid structures (51), it is not surprising thatexpression of some SFTPC mutations results in generation ofSDS-insoluble proSP-C macroaggregates in vitro (111, 112) aswell as intracellular amyloid formation in vitro and in vivo(182).

4) Mitochondrial dysfunction. ER stress can induce mitochon-drial dysfunction through both physical and functional communi-cations between mitochondria and the ER network, which canfurther exacerbate apoptotic events (reviewed in Ref. 169).

Quality Control for Dysfunctional Organelles: Mitophagy

In addition to the utilization of macroautophagy for selectiveremoval of polyubiquitinated protein aggregates (aggrephagy),autophagy can target and remove specific subcellular compo-nents (selective autophagy) including invading pathogens (xe-nophagy), lipids (lipophagy), and dysfunctional cellular organ-elles such as mitochondria or ER. Degradation of dysfunctionalmitochondria, termed mitophagy, is a remarkably specificprocess. When a single mitochondrion is uncoupled via pho-toirradiation, this and only this mitochondrion will be degraded(79). Additionally, global disruption of mitochondrial functionin cells treated by using an uncoupler such as carbonyl cyanide3-chlorophenylhydrazone results in selective mitochondrialdegradation, leaving other organelles intact.

The process of mitophagy requires the coordination of cy-tosolic factors and signals assembled at the outer mitochondrial

membrane (reviewed in Refs. 189, 190). A key step in theinitiation of the process involves PTEN-induced putative ki-nase 1 (PINK1), stabilized in the outer mitochondrial mem-brane in response to lowered TM potential (�), which thenrecruits the E3 ligase parkin, resulting in K-63-linked polyu-biquitin chains of a variety of mitochondrial protein substratessuch as mitofusins (Mfn1; mfn2). The subsequent recognitionand sequestration of the ubiquitin decorated mitochondrionutilizes much of the same autophagy machinery commissionedfor misfolded protein removal. For example, the ubiquitin-binding adaptor p62/SQM1, which recruits ubiquitinated pro-tein cargo into autophagosomes by binding to LC3, alsoaccumulates on mitochondria following their parkin-mediatedubiquitination and facilitates their engulfment by the phago-phore/autophagosome.

Aging, Cellular Quality Control, and Parenchymal LungDisease

The effects of aging on cellular quality control capacity havebeen well documented, particularly in the CNS where pertur-bations to proteostasis and mitochondrial function are hall-marks of age-related neurodegenerative disorders (108). Anage-dependent decrease in proteasome activity has also beenobserved in other tissues including skin, muscle, heart, liver,kidney, and eye, each with a variety of consequences (reviewedin Ref. 95). Aging has been shown to markedly downregulateautophagic activity (136). Given their key role in processingcellular constituents, either to the lysosome for degradation orto other cellular compartments for immune activation, au-tophagy and the UPS represent important cytoprotective strat-egies against aging, especially for postmitotic cells. As proof ofconcept, expansion of either proteasome activity or autophagyin the CNS extends lifespan and protects organisms fromsymptoms of proteotoxic stress (96).

Aging is a known risk factor for IPF (81). Many of thehallmarks of aging (e.g., genomic instability, telomere attrition,epigenetic alterations, and cellular senescence) have been pro-posed as essential mechanisms for the development of IPF;however, these disturbances are not restricted to IPF (reviewedin Ref. 145). Telomerase mutations have been described in IPFfamilies but also in chronic obstructive pulmonary disease (4,150). A recent report has described enhanced sensitivity of thealveolar stem cell function of AT2 cells from mice with shorttelomere syndromes subjected to exogenous “second hits”providing a potential mechanism for their role in aberrantinjury/repair processes in IPF pathogenesis (2).

In addition to these pathways, several lines of evidence linkage-dependent alterations in cell quality control, AT2 cellfunction, and susceptibility to fibrosis. Aged mice are suscep-tible to fibrosis from herpesvirus infection with increasedexpression of ER stress markers (161). In a preliminary report,lung tissue from aged mice displays decreased proteasomalactivity and alterations in 26S proteasome assembly (27).Recently, Bueno et al. (23) have found that AT2 cells from thelungs of both IPF patients and aged wild-type mice exhibitmarked accumulation of dysmorphic and dysfunctional mito-chondria. This was associated with both an upregulation of ERstress markers and a downregulation of PINK1. Moreover,young PINK1-deficient mice had premature development ofsimilarly dysmorphic, dysfunctional mitochondria in AT2 cells




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and were vulnerable to apoptosis and development of lungfibrosis in a viral challenge model (23). Interestingly, in atransgenic mouse model of atg5 deficiency, a 90% decrease inautophagy was well tolerated in young adult mice but resultedin alveolar septal thickening and altered lung mechanics inaged animals, consistent with accumulation of damage overtime (54). Collectively, these data suggest that aging hasmarked effects on both the proteostasis network and autophagyin the lung epithelia and that IPF, like many neurodegenerativeconditions, may reflect an acquired and potentially aged-re-lated defect in one or more arms of cellular quality control.

SURFACTANT MUTATIONS, EPITHELIAL DYSFUNCTION, ANDIPF PATHOGENESIS

The three components of the surfactant system associatedwith ILD opportunistically represent distinct substrates (a

bitopic integral membrane protein, a soluble luminal pro-tein, and a polytopic membrane transporter) with which toassess cellular responses and aberrant signaling present inthe distal lung epithelia in IPF. As the first of these to bedescribed, SFTPC represents a starting point to understandAT2 cell dysfunction in both sporadic IPF and FPF. Sincethe initial descriptions of the clinical proSP-C mutants,SP-C�exon4 and SP-CL188Q, nearly 15 years ago, over 60additional SFTPC mutations have been reported in theliterature in association with parenchymal lung disease(summarized in Table 1). These can be grouped on the basisof their functional behavior and domain location within theproSP-C sequence. The potential targets and various pro-cesses that have been revealed by their expression in cells,preclinical animal models, and patients are schematicallyillustrated in Fig. 3.

Table 1. Summary of reported phenotypic features for surfactant component mutations

Mutation (Domain) Clinical Diagnosis Lung Phenotype (in vivo)Subcellular Localization

Trafficking Cellular Responses (in vitro) References

SFTPA2

F198S (CRD)G231V (CRD)

Familial pulmonaryfibrosis

Total BAL [SP-A] Normal ER retentionIntracellular aggregationNot secreted

(�) ER stress, cleared by ERAD (�)TGF�1 elaboration

99, 100, 175

SFTPC

Group A1�Exon4 (BRICHOS)L188Q (BRICHOS)G100S (BRICHOS)

NSIP (Children)IPF/UIP (Adult)

Absence of mature SP-C (humans)Arrested lung development (mice)ER stress (humans; mice)1Sensitivity to bleomycin (mice)Epithelial cytotoxicity

ER retention¡ aggresomesIntracellular aggregatesERAD requires Erdj 4/5MG132 blocks degradation4-PBA improves aggregates

(�) ER stress(�) Apoptosis(�) Incomplete or absent proSP-C processing(�) IL-8/TGF�1 expression(�) Polyubiquitinated isoforms

21, 39, 97,98, 100,111, 112,116, 117,120, 153,159, 160,173, 193

Group A2L110R (BRICHOS)P115L (BRICHOS)A116D (BRICHOS)

Unspecified ILDUnspecified ILDUnspecified chILD

Phenotype not reported EEA-1 (�); Syntaxin2(�) Intracellular aggregation

2 PC secretion(�) Aberrant processing, 2 cell viability1 HSP response(�) Congo red aggregates

160, 193

Group B1E66K (Linker)I73T (Linker)

NSIP/PAP (Child)IPF/UIP (Adult)

1 Phospholipid; 1SP-A, PAS positivestaining

Biopsy: PM and EE localizationMisprocessed SP-C (BAL)Misprocessed SP-B (BAL)

Plasmamembrane¡EE¡LE/MVB

(�) Aberrantly processed protein(�) Late autophagy block2 Mitophagy1 Mysfunctional mitochondria

1, 19, 24,26, 49, 116,118, 128,152

Group B2�91–93 (Non-BRICHOS) NSIP/PAP 2 BAL SP-B

1 BAL SP-A 2 Surfactant surface tension(�) Intracellular aggregates(�) Congo red staining

Plasma membrane‡EEA1 (�) compartments‡

Not reported 55, 181

Group CP30L (NH2-terminal) Unspecified ILD Phenotype not reported (�) ER retention 1 Bip expression

(�) Polyubiquitinated isoforms13, 116, 160

ABCA3

Group I (Trafficking Defective)L101P (1st luminal loop)R280C (1st cytosolic loop)L982P (3rd luminal loop)G1221S (11th TM domain)L1553P (COOH-terminal)Q1591P (COOH-terminal)

Surfactantdeficiency*

RDS*chILD†

Phenotype not reportedPhenotype not reportedPhenotype not reported

(�) ER retentionNon-LRO cytosolic vesicles

(�) ER stress 30, 31, 103,147, 172,177

Group II (Functionally Defective)R43L (1st luminal loop)D253H (1st luminal loop)E292V (1st cytosolic loop)N568D (ABC1)E690K (ABC1)T1114M (8thTM domain)T1173R (1st luminal loop)L1580P (COOH-terminal)

Surfactantdeficiency*RDS* chILD(CPI)†

Reduced SP-B and SP-C (�) ER retentionLysosomes or LROs (normal)

Impaired lipid transportImpaired ATP hydrolysisImpaired ATP bindingAbnormal LBs1 IL8 secretion

20, 25, 103,104, 147,148, 177

*Seen with homozygous or compound heterozygous ABCA3 expression; †found with heterozugous ABCA3 expression. ‡S. Mulugeta, unpublishedobservations. BAL, bronchoalveolar lavage; chILD, interstitial lung disease of childhood; CPI, chronic pneumonitis of infancy; CRD, carbohydrate recognitiondomain; EE, early endosomes; EEA1, early endosomal antigen-1; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; HSP, heat shockresponse; ILD, interstitial lung disease; IPF, idiopathic pulmonary fibrosis; LBs, ; LE/MVB, ; LRO, lysosome-related organelle; NSIP, nonspecific interstitialpneumonitis; PAP, pulmonary alveolar proteinosis; PAS, periodic acid Schiff; 4-PBA, 4-phenylbutyric acid; PC, phosphatidyl choline; PM, plasma membrane;RDS, respiratory distress syndrome of newborn; SP, surfactant protein; TM, ; UIP, usual interstitial pneumonitis.




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Clues from SFTPC BRICHOS Mutations: Aggresomes, ERStress, Cytokines, EMT, and Apoptosis

The Group A1 mutations prone to misfolding tend to clusteraround the conserved cysteine residues (cys121/cys189) in theSP-C BRICHOS domain. When the prototypical SP-C�exon4

and SP-CL188Q BRICHOS mutants are expressed in a variety ofin vitro model systems, they are initially retained in the ER, failto undergo appropriate proteolytic processing, do not end up inLB, and have a high propensity to form intracellular aggre-gates. Alanine mutagenesis of either or both cysteines gener-ates laboratory-mutant isoforms that indicate these aggregatesare actually aggresomes whose formation can be blocked withmicrotubule inhibitors, exacerbated by proteasome inhibitors,and rescued by the chemical chaperone 4-phenylbutyric acid(4-PBA) (69, 153). Aggresomes are also observed with clinicalBRICHOS mutants (173). Additional mutations in the moreproximal BRICHOS domain termed Group A2 (A116D;L110R) are less well characterized but are also excluded fromLRO/LB, partially localize in LAMP3/EEA1 (LE/MVB) com-partments, induce a UPR, and form Congo red aggregates invitro (160, 193). The sole Group C mutant reported to date(P30L) is also ER retained and polyubiquitinated (presumablyfor ERAD) (13, 160).

Using SP-C�exon4 and SP-CL188Q mutants as substrates,misfolded BRICHOS conformers are also capable of activatingone of more of the three UPR sensors, IRE1/XBP1, ATF6, andPERK/eIF2�, in both cell lines and primary rodent AT2 cells(97, 98, 111, 112). The ultimate failure of the UPR to correctmutant protein load and alleviate ER stress appears to induce aseries of downstream events that then contribute to AT2 celldysfunction and promote aberrant injury/repair responses.

Sounding the alarm: inflammation. Persistent UPR activa-tion by these mutant isoforms appears to induce an ER-mediated “alarm” response. Transient expression of SP-C BRI-CHOS domain mutants promoted IL-8 release from culturedepithelial cells. The release of IL-8 was mechanistically linkedto the activation of JNK signaling and was associated withactivation of nuclear factor-kappa light-chain-enhancer of ac-tivated B cells (NF-B) (97). Stably transfected cell linesexpressing SP-C�exon4 appear to adapt to chronic ER stress viaan NF-B-dependent pathway and suggest this may be aprosurvival response, but one capable both of elaboratingadditional inflammatory mediators and of being exploited bysecond hits such as viral infections (22).

Apoptosis. Given the role of AT2 cells as progenitors (16),their depletion poses a threat to the overall capacity of the lungfor repair/regeneration. ER stress induced by mutant proSP-Ccan induce intrinsic apoptotic death via induction of numerouspathways including caspase 4 activation, mitochondrial cyto-chrome c release, c-Jun NH2-terminal kinase (JNK) signaling,and caspase 3 activation (97, 98, 111, 112). Interestingly,apoptosis induced by pharmacological proteasome inhibitionor overexpression of an SP-C BRICHOS mutant can be sig-nificantly inhibited by angiotensin receptor blockade (sarala-sin) or the angiotensin II counterregulatory peptide Ang1-7,showing that ER stress-induced intrinsic apoptosis in humanAECs can involve autocrine pathways such as the ANGII/ANG1-7 system (163).

Changes in cell phenotype. Although somewhat controver-sial as to their contribution to total fibrotic burden, a variety of

studies have suggested that epithelial cells in the lungs andother organs can undergo phenotypic changes with the adop-tion of mesenchymal phenotypes. Termed epithelial-mesen-chymal transition (EMT), this phenomenon probably repre-sents an adaptive response to cellular stress. In the alveolarepithelium and in cell lines, EMT driven by ER stress inducedeither pharmacologically or by using proSP-C mutants hasbeen shown. In primary rodent AT2 cells, Zhong et al. (195)have shown that treatment with tunicamycin or thapsigargininduces �-smooth muscle actin expression and decreased E-cadherin that was accompanied by morphological changesconsistent with a shift to a mesenchymal phenotype. Overex-pression of SP-C�exon4 in this model largely recapitulated theseeffects. Tanjore et al. (157) reported similar findings followinginduction of ER stress by overexpression of the SP-CL188Q

mutant and identified SMAD2/3 signaling as a mediator ofthese events. Importantly, both studies demonstrated a role forSRC family kinases and suggest that therapies based on miti-gating ER stress may have pluripotent benefits.

Beyond SP-C: Aberrant Cellular Responses to OtherSurfactant Component Substrates

The cellular responses observed above are not idiosyncraticwith proSP-C BRICHOS mutants, given that studies utilizingmutant SFTPA2 and ABCA3 substrates have both corroboratedthe pathways discussed above and extended the findings withseveral new observations.

SFTPA (SP-A). In 2009, Wang and colleagues (175) usinggenomewide linkage scanning, reported on two kindredswith pulmonary fibrosis and lung adenocarcinomas in whichrare heterozygous missense mutations were identified inSFTPA2 alleles. Resulting from the substitution of valinefor glycine at codon 231 (G231V) or serine for phenylala-nine at codon 198 (F198S) in the CRD of the SP-A protein,when expressed in lung cell lines or primary murine AT2cells, the resulting mutant SFTPA2 protein products wereproximally retained, failed to be secreted, and activated theUPR (99). Subsequently the same group found elevatedamounts of TGF-�1, a pleotropic profibrotic cytokine linkedto both myofibroblast activation and EMT, in the bronchoal-veolar lavage (BAL) of patients expressing the G231Vmutation. Primary mouse AT2 cells expressing G231V invitro also elaborated significant amounts of TGF-� as wellas two TGF-� binding proteins (LTBP-1 and LTBP-4)(100).

ABCA3. Although not restricted to any particular domain,functional characterization of a subgroup of ABCA3 mutations(including L101P, L982P, L1553P, Q1591P, G1221S) by tran-sient or stable expression results in their total or partial reten-tion in the ER (30, 103). Designated as “Type I” (Table 1), thismutant subgroup can enhance expression of Bip, increaseXBP1 splicing, and induce apoptosis in vitro (177). Recentwork with both the Type I clinical ABCA3 mutants as well aslaboratory-generated glycosylation-deficient constructs sug-gest that the misfolded isoforms are substrates for ERAD (14,177). Taken in total these data are highly supportive of acommon model of ER stress and failed proteostasis frommisfolded surfactant components.




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A Role for Induced AT2 ER Stress and Apoptosis in IPFPathogenesis

Mechanistic relationships between ER stress-induced epi-thelial dysfunction and lung fibrosis identified from the in vitrostudies described above have been further strengthened usingmouse models. Bridges et al. (21) reported that transgenic miceconstitutively expressing SP-C�exon4 exhibit a gene dose-de-pendent arrest of lung morphogenesis, induction of Bip expres-sion, and aberrant proSP-C processing. More recently, condi-tional expression of SP-CL188Q in AT2 cells of adult mice atinduced levels of �20% of the endogenous gene expressionproduced ER stress (90). Although lacking evidence of grossfibrosis at baseline, these mice exhibited a greater sensitivity tolow-dose bleomycin with marked fibrotic changes and en-hanced AT2 cell apoptosis. The direct role of ER stress in thisprocess was further supported by exposure of wild-type mice totunicamycin treatment to independently induce ER stress,which again enhanced the fibrotic response to bleomycin (90).

ER Stress Signatures Are Not Exclusive to Cells, Mice, orEven SFTPC IPF Families

The molecular signatures for ER stress and UPR pathwaysidentified by the in vitro systems and mouse models have beensubsequently detected in the lungs of IPF patients. Lawson andcolleagues (91) demonstrated evidence of UPR activationmarkers (including BiP, EDEM, and XBP1) in FPF patientsexpressing SP-CL188Q mutations as well as in samples bothfrom non-SFTPC FPF patients and in sporadic cases of IPF.Korfei et al. (83) extended these findings by reporting molec-ular signatures of multiple UPR pathways and apoptosis inAT2 cells including ATF6, ATF4, XBP1, CHOP, Bax, andcaspase 3 exclusively in patients with sporadic IPF with path-ological features of UIP. Thus chronic ER stress in the alveolarepithelium likely represents a broad mechanism for the patho-genesis of ILD (156).

AT2 Quality Control Responses

An important concept, especially in the context of qualitycontrol issues discussed above, is that the cellular pathologyand molecular signatures of ER stress induced by BRICHOSproSP-C expression must invariably result from of an imbal-ance in or failure of the cellular quality control network toaccommodate the offending protein conformers and dysfunc-tional organelles. Very little is currently known about theactual quality control repertoire of AT2 cells. Using microarrayanalyses, Dong et al. (39) identified genes specifically inducedin response to expression of SP-C�exon4 and SP-CL188Q andshowed that two BiP cochaperones, ER-localized DnaJ homo-logues ERdj4 and ERdj5, selectively bind to mutant but notwild-type proSP-C and were required for successful retrotrans-location and proteasomal degradation of BRICHOS mutantSP-C substrates. In a preliminary report, we have shown thatalthough primary human AT2 cells exhibit a significant degreeof both proteasomal activity and autophagy, these cells appearto rely primarily on ERAD for control either of misfoldedproSP-C conformers or more generally of a variety of PolyQhuntingin substrates (57). Similarly, Maitra and colleagues (99)have shown that primary mouse AT2 cells in vitro rely almostexclusively on MG132-sensitive proteasomal activity to clear

misfolded SP-A2 mutants. Although not appearing to have aprimary role in basal proteostasis, the function of autophagyeither as a backup to ERAD and/or in the quality control ofAT2 organelles such as mitochondria, LB, or nonprotein sub-strates remains an important area for further investigation.

Beyond ER Stress and Apoptosis: Dominant Negative orLoss-of-Function Effects

Patients heterozygous for the SP-C�exon4 mutation do notexpress mature SP-C (117). Mechanistically, because proSP-Csorting involves homomeric association of proSP-C monomersmediated by the mature SP-C domain, through heterotypicinteractions, SP-C BRICHOS mutants can act as dominantnegatives to direct wild-type proSP-C away from normal rout-ing and into aggregates (174). Because SP-B and SP-C are feltto be functionally redundant with respect to overall surfactantbiophysical activity, the question of whether a lack of matureSP-C in surfactant would contribute to IPF pathogenesis isunanswered. As expected, SP-C null mice are viable at birthand grow normally without a lung phenotype (45) but exhibitenhanced sensitivity to bleomycin fibrosis (93) and respiratorysyncytial virus infection (47). However, when the originalstrain of SP-C null mice are backcrossed onto a differentgenetic background (129/Sv), the absence of SP-C is associ-ated with spontaneous inflammation and lung remodeling (46),suggesting that SP-C deficiency is a potential disease modify-ing factor.

In a related fashion, when expressed in primary rodent AT2cells, the disease-causing human SPA2 mutants are not se-creted (99). Furthermore, in epithelial cell lines simultaneouslytransfected with SP-A2 isoforms and wild-type SP-A1, coim-munoprecipitation resulted in recovery of wild-type SP-A2from the medium but not SP-A2 mutants (G231V or F198S)(175). Although total SP-A levels do not appear to be alteredby heterozygous expression of SP-A2 mutants in vivo, giventhat the SP-A trimer and 18-mer are from combinations ofSP-A1 and SP-A2, the resultant consequences of an SP-A1restricted SP-A on innate lung immunity and/or IPF pathogen-esis remains to be clarified.

In contrast to posttranslationally acquired SP-C or SP-Adeficiency, functionally impaired ABCA3 mutations (desig-nated Type II mutations) have been identified, with homozy-gous expression producing severe loss of function phenotypes(30, 103, 104) (Table 1). Affected patients as well as ABCA3null mice demonstrate complete absence of protein expression,deformed LBs, and die from respiratory distress shortly afterbirth (31, 43, 147, 172), indicating a critical role in productionof pulmonary surfactant. However, heterozygous expression ofABCA3 mutations, with a resultant partial loss-of-functioncaused either by ER retention (Type I), improper lipid-pumpfunction (Type II), or both (Type I/II compound heterozygotes)have each been reported and appear to act as genetic modifiersof lung diseases associated with SFTPC mutations in children(24). In one adult FPF kindred all carrying the same SFTPCmutation, a functional genetic variant in ABCA3 (E292V) alsofound in the previous pediatric study appeared to also affectdisease penetrance, suggesting interplay between the two ge-notypes (37), not surprising given their overlapping biologywithin the AT2 cell.




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SFTPC Trafficking Mutations: a New Phenotype of AcquiredQuality Control Disruption

Beginning in 2004, a series of missense mutations clusteringwithin a 40-amino-acid span encoded on exon 3 of humanSFTPC was reported, heralding an emerging diversity of mo-lecular and clinical phenotypes induced by SFTPC expression(19, 55, 152). The first of these detailed a de novo heterozy-gous missense mutation (g.1286T�C) resulting in substitutionof threonine for isoleucine (I73T) in an infant with NSIP (19).Interestingly, the identical SP-CI73T mutation (both inheritedand sporadic) has reappeared in several other published reportsof parenchymal lung disease in children and adults and is nowfelt to be the most common SFTPC mutation (26, 116).Collectively the Group B mutations (E66K; I73T; �91–93)(55, 152) all reside within the linker domain of proSP-C (Fig.1). Although presentation and course can vary (1, 128), over-lapping features that emerge include interstitial lung remodel-ing with intra-alveolar accumulation of surfactant (lipids,SP-A, SP-B), the presence of aberrantly processed proSP-C,and abnormal AT2 cell cytoplasmic inclusions on EM,suggesting abnormalities in surfactant homeostasis (19, 44,58, 152). Importantly since mature SP-C can be detected inBAL from these patients, the non-BRICHOS traffickingmutants do not appear to act as dominant negatives (19,162).

The cellular phenotype induced by Group B SFTPC mutantsis also distinct from that of Group A1 (Table 1). Functionally,Group B SFTPC mutants have been defined by in vitro expres-sion studies (mainly using SP-CI73T), demonstrating a uniqueand markedly aberrant intracellular trafficking pattern forproSP-C first directly to the plasma membrane with subsequentinternalization and accumulation within early endosomes andLE/MVB (15, 153) (Fig. 3). This recapitulated the expressionpattern seen on a biopsy of a patient with SFTPCE66K showingproSP-C staining on the plasma membrane and in early endo-somes of AT2 cells (19). Recently, two separate preliminarystudies using knockin or transgenic expression strategies haveeach demonstrated that the distribution of mutant SP-CI73T inAT2 cells in vivo is nearly identical with the pattern noted incell lines and patients (118, 176).

The link between the mistrafficking behavior and cellulardysfunction, which does not involve ER stress (111), hasrecently been defined in vitro with cell lines expressing hSP-CI73T. As illustrated in Fig. 3, SP-CI73T induces a novel qualitycontrol phenotype punctuated by a striking disruption of mac-roautophagy characterized by the accumulation of dysmorphic,autophagic vacuoles containing organellar and proteinaceousdebris (58). The in vitro findings from hSP-CI73T cell linesphenocopied ultrastructural changes seen in AT2 cells on EMof a lung biopsy from a SFTPC I73T patient (58). Biochemi-cally, hSP-CI73T cells exhibited increased expression of Atg8/LC3, SQST/p62, and RAB7, consistent with a late block inautophagic vacuole maturation that was confirmed by au-tophagy flux studies. The observed disrupted autophagy re-sulted in a diminished functional capacity of hSP-CI73T cells todegrade model protein aggregate substrates and impairment inmitophagy, producing an accumulation of dysfunctional mito-chondria. The disruption of autophagy-dependent proteostasisand mitophagy induced by expression of SP-CI73T is reminis-cent of cellular quality control phenotypes observed in organ-

ellar storage disorders such as Niemann-Pick disease (140,146), which has been associated with a premature developmentof fibrotic lung disease.

In translational studies of IPF from two separate groups,lung tissue from IPF patients demonstrates evidence of de-creased autophagy as defined by increases in LC3, p62, andpolyubiquitinated protein expression and decreased numbers ofautophagosomes (3, 126). These findings, when combined withthe aforementioned work on aging (23), emphasize the poten-tial important role of impaired autophagy, mitophagy, andmitochondrial dysfunction in lung epithelia to IPF.

Impact of Environmental Factors on Lung Quality Control

Substantial clinical variability in the age of onset and diseaseseverity in both surfactant mutation-associated (SP-C, ABCA3,SP-A2) ILD and sporadic IPF suggests a possible role forenvironmental factors and/or for modifier genes. In addition tothe aging lung and its effects on quality control discussedabove, given the direct exposure of the lung epithelium to the“outside world,” environmental insults may participate in atwo-hit model of AT2 dysfunction to promote fibrosis. Insupport of this, IPF is found more frequently in cigarettesmokers (11, 142), and cigarette smoking is one of the stron-gest associated risk factors for the development of diffuseparenchymal lung disease (DPLD) (151). Cigarette smokeexposure induces ER stress and proteasomal dysfunction inalveolar epithelial cell lines in vitro (149, 168) and signaturesof UPR activation have been detected in the smoke-exposedhuman lung in vivo (77).

Similarly, emerging evidence also implicates human herpes-viruses (including herpesvirus-8; Epstein-Barr virus; cytomeg-alovirus) in IPF, and one or more of these viruses have beendetected in the lungs of up to 97% of tested patients with IPF(40, 86, 91, 155, 188). In a mouse model of aging, infectionwith the murine herpesvirus lead to upregulation of multiplecomponents of the UPR/ER stress pathway and accompaniedby the development of lung fibrosis (161). In human IPF,cytomegalovirus antigens and the UPR transcription factorXBP-1 were colocalized in alveolar epithelial cells from anindividual with UIP (91). More recently, sampling of asymp-tomatic relatives of patients with familial IPF showed evidenceof herpesvirus infection and ER stress (87).

In a recent clinical study, IPF exacerbations were signifi-cantly associated with antecedent 6-wk increases in air pollu-tion exposures of ozone and nitrogen dioxide, suggesting thatair pollution may contribute to the development of this clini-cally meaningful event (64, 91). Although the mechanism wasnot assessed, given the oxidative-nitrative stress applied bythese agents, it seems likely that additional proteotoxic andmitochondrial stress to the quality control network in the distallung from the enhanced generation of “challenged substrates”by these exposures may be contributing factors.

CONCLUSIONS, UNANSWERED QUESTIONS, ANDOPPORTUNITIES

Over the past 15 years much progress has been made inunderstanding the pathogenesis of IPF. There is now substan-tial evidence to show that the AT2 epithelium is altered in thefibroblastic foci of IPF lungs and that the AT2 cell is a pivotalregulator of lung injury, inflammation, and repair. Although




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SFTPC, SFTPA, and ABCA3 mutations are rare (2–20% inmost ILD cohorts) (92, 167), understanding both the spectrumof clinical phenotypes and the molecular pathogenesis of theAT2 cell dysfunction caused by these mutations adds value tounderstanding the broader mechanisms that underlie the patho-genesis of sporadic IPF.

The data summarized here generated by using surfactantprotein mutations as a platform has revealed the role for bothER stress and alterations in cellular quality control pathways inepithelial cells in IPF pathogenesis (Fig. 3). The fact thatcharacteristic cellular phenotypes are induced by distinctclasses of surfactant component mutations and that these con-verge at the epithelial cell should spur further focus on effortsto understand AT2 biology in health and disease. Coupled withthe emergence of confounding factors in IPF pathogenesis suchas age, environmental exposures, and infection, the findingssummarized in this review strongly support a two-hit (ormultiple-hit) model whereby the repetitively injured AT2 cellpopulation serves as a driver of disordered repair and injury.Although not discussed here, the well-known associations oftelomerase mutations with familial IPF (4), which can also bethought of as a “quality control disorder” (for telomeres), alsoconverge on this epithelial-centric model of IPF and highlightthe importance for aging-associated processes and senescencethat were not previously linked to IPF. In this context, the roleof the recently described Muc5B promoter polymorphism inIPF remains to be clarified (127, 143).

Given the above model, it should come as no surprise thatthe therapies to date have done little to stabilize or reverse thefibrotic process (18). Analogous to cancer, it is likely thatfuture therapeutic options for IPF should involve directedtherapies aimed at engaging multiple targets including theepithelial cell dysfunction, fibroblast activation, and matrixdeposition. This approach is probably not amenable to singledrug therapy. Furthermore, experience with the surfactant pro-tein component mutations reveals that the final common his-topathological pathway seen as UIP or chILD likely reflectsdistinct combinations of several different upstream molecularevents. Identifying the specific pathogenic pathway signaturesin patients will be critical to identify personalized therapiesbased on pathogenesis not histology.

Finally, given the protracted period for drug developmentand the overlap of many of the pathways discussed above (ERstress, misfolding, aggregation, mistrafficking, autophagy, ap-optosis, and MAP kinase signaling) with those seen in otherchronic degenerative diseases (Alzheimer’s disease, Hunting-ton’s disease, cystic fibrosis, and �1-antitrypsin deficiency), itmay be worth considering a cross-fertilization of IPF research/treatment strategies with these disease areas by adopting ex-perimental paradigms and reagents, coupled with readily avail-able compound libraries, and approved drugs that alreadytarget some of these signatures in an attempt to open newtherapeutic avenues for treatment of this devastating disease.

ACKNOWLEDGMENTS

The authors acknowledge the help and support of many mentors, collabo-rators, technical staff, and trainees over the years including Aron B. Fisher,M.D.; Philip L. Ballard, M.D., Ph.D.; Susan H. Guttentag, M.D.; Sandra R.Bates, Ph.D.; Frank Brasch, M.D.; Paul N. Stevens, M.D.; Larry Nogee, M.D.;Aaron Hamvas, M.D.; Michael Koval, Ph.D.; Sheldon I. Feinstein, Ph.D.;Linda Gonzalez, Ph.D.; Albert Kabore, Ph.D.; Chi Y. Kim, M.D.; Catherine A.Lomax, Scott J. Russo, Amy L. Johnson, V.M.D.; and Wen-Jing Wang, M.D.

We apologize to the many additional colleagues in advance whose work, forthe limits of space, could not be cited.

GRANTS

This research was supported by VA Merit Award BX001176 (M. F. Beers);NIH HL 119436 (M. F. Beers), NIH P30 ES013508 (M. F. Beers), andAmerican Heart Association Research Grant 13GRNT17070104 (M. F. Beers).

M. F. Beers is an Albert M. Rose Established Investigator of the PulmonaryFibrosis Foundation.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

S.M., S.-I.N., and M.F.B. conception and design of research; S.M. andS.-I.N. performed experiments; S.M. prepared figures; S.M. and M.F.B.drafted manuscript; S.M., S.-I.N., and M.F.B. edited and revised manuscript;S.M., S.-I.N., and M.F.B. approved final version of manuscript.

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