Lost after translation: insights from pulmonary surfactant for understanding the role of alveolar epithelial dysfunction and cellular quality control in fibrotic lung disease

Abstract

Dating back nearly 35 years ago to the Witschi hypothesis, epithelial cell dysfunction and abnormal wound healing have reemerged as central concepts in the pathophysiology of idiopathic pulmonary fibrosis (IPF) in adults and in interstitial lung disease in children. Alveolar type 2 (AT2) cells represent a metabolically active compartment in the distal air spaces responsible for pulmonary surfactant biosynthesis and function as a progenitor population required for maintenance of alveolar integrity. Rare mutations in surfactant system components have provided new clues to understanding broader questions regarding the role of AT2 cell dysfunction in the pathophysiology of fibrotic lung diseases. Drawing on data generated from a variety of model systems expressing disease-related surfactant component mutations [surfactant proteins A and C (SP-A and SP-C); the lipid transporter ABCA3], this review will examine the concept of epithelial dysfunction in fibrotic lung disease, provide an update on AT2 cell and surfactant biology, summarize cellular responses to mutant surfactant components [including endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and intrinsic apoptosis], and examine quality control pathways (unfolded protein response, the ubiquitin-proteasome system, macroautophagy) that can be utilized to restore AT2 homeostasis. This integrated response and its derangement will be placed in the context of cell stress and quality control signatures found in patients with familial or sporadic IPF as well as non-surfactant-related AT2 cell dysfunction syndromes associated with a fibrotic lung phenotype. Finally, the need for targeted therapeutic strategies for pulmonary fibrosis that address epithelial ER stress, its downstream signaling, and cell quality control are discussed.

Keywords: alveolar type 2 cells; cell quality control; epithelial ER stress; fibrotic lung disease; surfactant proteins.

Fig. 1.

Schematic representation of structure, biosynthetic processing, and life cycle of 3 surfactant system components associated with interstitial lung disease in 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, and ABCA3. 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 the endoplasmic reticulum (ER), is sorted in the Golgi, and enters the regulated secretory pathway where it is processed by 4 endoproteolytic cleavages of NH₂ and COOH 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 include NH₂-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 α-helical transmembrane hydrophobic region; linker domain, site for the common I73T mutant variant (yellow pentagon); BRICHOS domain with the exon4 deletion region (depicted in gray), and L188Q mutation (yellow triangle). 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 proximal NH₂-terminal region at distal post-Golgi compartments. Inset, bottom right: predicted topological structure of the ABCA3 transporter. ABCA3 comprises 12 putative membrane-spanning helices where the NH₂- and COOH-terminal domains and the 2 ABC binding cassettes (ABC1 and ABC2) face the cytosol. The NH₂ 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 assembled form of SP-A is secreted by both regulated and constitutive pathways. Posttranslational modifications of SP-A include signal peptide cleavage prior to entering the ER, hydroxylation of proline residues, and N-linked glycosylation. Inset, top right: structural representation of a native SP-A. The primary structure of SP-A protein consists of 4 domains: a short amino-terminal domain, a collagenous domain, a neck domain, and a carbohydrate recognition domain (CRD). Six of the SP-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 proline residue in the collagenous domain of SP-A primary structure provides a flexible kink that causes the trimers to bend outward in different directions, giving the native 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 by regulated 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 for degradation or recycled back to LBs via endosomes and MVBs.

Fig. 2.

Quality control machinery utilized by cells in response to the expression of misfolded or unassembled proteins and dysfunctional organelles. Unfolded protein 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 initiate the downstream signaling of the UPR. These sensors are typically maintained in an inactive state as they are bound to the molecular chaperone, Bip/GRP78. The presence of increasing load of misfolded SP-C proteins within the ER requires the availability of Bip/GRP78 to bind to the misfolding proteins and consequently Bip/GRP78 dissociates from the sensors sites to meet this requirement and to allow 3 downstream signaling pathways to activate the following. 1) ATF6: when Bip is released from ATF6p90, this transmembrane sensor is first translocated to Golgi and subjected to cleavage by S1 and S2 protease generating a soluble cytosolic form (ATF6p50) that traffics to the nucleus where it binds to ER-stress-responsive elements (ERSE) to upregulate chaperone production. 2) IRE1, in the absence of Bip binding, undergoes phosphodimerization and acquires specific endonuclease activity for cytosolic splicing of XBP1 mRNA. The translated XBP1, a transcription factor, also binds to ERSEs in the nucleus. 3) PERK activation results in both phosphorylation of cytosolic eIF2α leading to a generalized translation repression plus selective upregulation of transcription factors such as CHOP/GADD153 and ATF4 to promote a specific subset of gene expression involved in expanding proteostatic capacity. ER-associated protein degradation (ERAD) involves a multistep process consisting of recognition of misfolded cargo by ER-resident proteins [e.g., EDEM, protein disulfide isomerase (PDI), Bip/Grp78] followed by their retrotranslocation out of the ER through translocons and subsequent polyubiquitination mediated by cytosolic E1/E2/E3 ubiquitin ligases. Targeting of ubiquitin-decorated coformers to the 26S proteasome results in eventual 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 a phagophore. The Atg5-Atg12-Atg16L complex and atg8 (LC3-I) phosphatidylethanolamine (PE) conjugate recruit and through elongation envelop cytosolic ubiquitinated cargos including protein aggregates and mitochondria, leading to the formation of autophagosome. Subsequently, the autophagosome fuses with lysosomes, resulting in an acidified and functional autophagolysosome (“autolysosome”), which also contains lysosome-derived acid hydrolases, to promote degradation of internalized cytosolic content. Aggresome formation: as a last coping mechanism, failure of UPR/ubiquitin-proteasome system (UPS)/autophagy network to control mutant protein levels results in transport of aggregate forms of misfolded protein in a microtubule-dependent manner to the microtubule organizing center (MTOC) near the nucleus, resulting in the formation of aggresomes.

Fig. 3.

Pathways to AT2 cell injury/dysfunction defined by using mutant proSP-C isoforms as prototype substrates. The expression of proSP-C mutants can produce 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 longer maintain ER homeostasis because of overwhelming expression of misfolded SP-C, leading to a cascade of cellular disruption and injury: 1) Dysfunction of ERAD due to an overloaded and defective UPS. This event can be further exacerbated by second hits such as cigarette smoke and viruses. 2) ER stress-dependent recruitment of TRAF2 by IRE1 to activate JNK that promotes upregulation and release of cytokines. 3) Induction of apoptotic pathways by ER stress-induced activation of either the PERK/EIF2α/ATF4 network to trigger CHOP activation, IRE1/TRAF2 activation of caspase 4/12, and/or cytochrome c release from dysfunctional mitochondria. All 3 intersect downstream at the activation of caspase 3. 4) A failure of aggresomal compartmentalization or autophagy also contributes to the eventual accumulation of toxic macroaggregates. Mistrafficked Linker (non-BRICHOS) proSP-C (Group B): mutations in the COOH-terminus outside BRICHOS domain of proSP-C reported to date appear to be initially mistargeted to the plasma membrane via a constitutive pathway. Because of proper processing failure, proSP-C in the plasma membrane can be reinternalized and trafficked through early endosomes to late endosomes (LE)/MVB. The enhanced presence of aberrantly trafficked and misprocessed proSP-C in these compartments leads to a functional disruption of normal endosome/amphisome/lysosome turnover within the autophagic machinery that ultimately results in a distal block in autophagy. Such a block leads to the accumulation of abnormally large autophagic 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 expression of mistrafficked proSP-C.