Lung Fibrosis-associated Surfactant Protein A1 and C Variants Induce Latent Transforming Growth Factor β1 Secretion in Lung Epithelial Cells*

Background: Mutations in the genes encoding surfactant proteins have been found in patients with lung disease. Results: The relative contribution of UPR-dependent or independent induction of latent TGF-β secretion differs for surfactant variants. Conclusion: Expression of certain SP-A1 and SP-C variants leads to increased latent TGF-β secretion. Significance: Drugs that inhibit TGF-β may be especially beneficial to a molecularly defined group of patients. Missense mutations of surfactant proteins are recognized as important causes of inherited lung fibrosis. Here, we study rare and common surfactant protein (SP)-A1 and SP-C variants, either discovered in our familial pulmonary fibrosis cohort or described by others. We show that expression of two SP-A1 (R219W and R242*) and three SP-C (I73T, M71V, and L188Q) variant proteins lead to the secretion of the profibrotic latent transforming growth factor (TGF)-β1 in lung epithelial cell lines. The secreted TGF-β1 is capable of autocrine and paracrine signaling and is dependent upon expression of the latent TGF-β1 binding proteins. The dependence upon unfolded protein response (UPR) mediators for TGF-β1 induction differs for each variant. TGF-β1 secretion induced by the expression of the common SP-A1 R219W variant is nearly completely blocked by silencing the UPR transducers IRE-1α and ATF6. In contrast, the secretion of TGF-β1 induced by two rare SP-C mutant proteins (I73T and M71V), is largely unaffected by UPR silencing or by the addition of the small molecular chaperone 4-phenylbutyric acid, implicating a UPR-independent mechanism for these variants. Blocking TGF-β1 secretion reverses cell death of RLE-6TN cells expressing these SP-A1 and SP-C variants suggesting that anti-TGF-β therapeutics may be beneficial to this molecularly defined subgroup of pulmonary fibrosis patients.

Pulmonary surfactant secreted by alveolar epithelial type II cells in the lung decreases the surface tension of the alveolar air-liquid interface and is essential for the inflation of the lung at birth. Surfactant is composed of 90% phospholipids and 10% protein. The protein component includes the surfactant protein A (SP-A), 2 B, C, and D. SP-B and SP-C are very hydrophobic proteins (1). In contrast, SP-A and SP-D are hydrophilic and members of the superfamily of C-type lectins that play an important role in lung innate immunity (2).
Mutations in the gene-encoding surfactant protein C (SP-C), SFTPC, have been found in humans with a wide array of lung disease ranging from severe respiratory distress in infants to a lethal, progressive scarring lung disease in adults known as idiopathic pulmonary fibrosis (IPF). Surfactant protein C is produced in alveolar type II cells as a precursor protein consisting of a N-terminal segment (residues 1-23), a transmembrane region that represents the mature secreted peptide (residues 24 -58), a linker region (residues 59 -89), and a BRICHOS domain (residues 90 -197) (3). A mutation in this gene was first described in an affected mother and infant with a heterozygous splice site mutation resulting in skipping of exon 4 and deletion of 37 amino acids (⌬exon4) in the BRICHOS domain (4). In vitro and in vivo studies demonstrate a role of the BRICHOS domain as a molecular chaperone that impairs the formation of intracellular amyloid (5,6). The SFTPC ⌬exon 4 mutant protein forms dominant-negative perinuclear aggregates, increases ER stress, and causes disruption of lung morphogenesis (7)(8)(9)(10). Many other SP-C mutations have been described, and the lung disease associated with SP-C mutations is known collectively as type 2 surfactant metabolism dysfunction (11). Another mutation within the BRICHOS domain, the L188Q mutation, causes increased formation of insoluble aggregates, increased ER stress, cytotoxicity, and exaggerated bleomycin-induced pulmonary fibrosis in vivo (12)(13)(14). The most common missense mutation is one that substitutes a threonine for an isoleucine at amino acid position 73 (I73T) in the linker region, outside of the BRICHOS domain; this mutation alone is estimated to account for up to 30% of all SFTPC mutations (15)(16)(17). Unlike the BRICHOS domain mutations, the commonly found I73T mutant protein does not cause substantial ER stress and is mistrafficked to early endosomes (18). It is not entirely clear how this and other non-BRI-CHOS domain mutations cause lung disease.
In humans and higher primates, there are two oppositely oriented genes encoding surfactant protein A (SP-A1 and SP-A2), SFTPA1 and SFTPA2. Both genes are highly polymorphic, and many studies have evaluated associations FIGURE 1. Expression of certain SP-A1 and SP-C variants in human HBEC-3KT cells leads to the secretion of latent TGF-␤1. A, the amount of TGF-␤1 secreted into the medium of HBEC-3KT cells 48 h after infection with lentivirus expressing the indicated V5-epitope tagged SP-A1 proteins. B, immunoblots of HBEC-3KT cell lysates (C, 30 g) and media samples (M, 45 l) demonstrating expression of V5 epitope-tagged SP-A1 proteins, total Smad 2/3, and p-Smad2. C, the amount of TGF-␤1 secreted into the medium of HBEC-3KT cells 48 h after infection with lentivirus expressing the indicated SP-C proteins. D, immunoblots of HBEC-3KT cell lysates (30 g) demonstrating expression of SP-C, total Smad 2/3, and p-Smad2. E, co-culture PAI-luciferase activity of Mv1Lu cells expressing a PAI-luciferase reporter 48 h after mixing and growing these cells 1:1 with HBEC-3KT cells expressing WT or variant surfactant proteins. Cells not infected with any lentivirus (uninfected) and HBEC-3KT cells infected with mock virus (Mock) are shown as controls. F, co-culture PAI-luciferase activity of Mv1Lu cells expressing a PAI-luciferase reporter 48 h after mixing and culturing these cells 1:1 with HBEC-3KT cells expressing WT or variant SP-A1 and SP-C proteins in the presence or absence of 5 g/ml pan-TGF-␤ neutralizing antibody. Duplicate wells were cultured in the presence of 20 ng/ml exogenous TGF-␤. For A, C, E, and F, the mean of duplicate measurements Ϯ S.D. is shown. †, p Ͻ 0.1; *, p Ͻ 0.05; **, p Ͻ 0.005 variant versus WT. between these variants and susceptibility to various childhood and adult lung diseases such as respiratory distress syndrome, tuberculosis, and fibrosis (19). One common variant in the SP-A1 gene, rs4253527, which is found on the 6A 4 haplotype, predicts an arginine to tryptophan substitution at position 219 (R219W). This variant has been associated with an increased susceptibility to IPF (20).
Recently, we found that expression of certain SP-A2 mutant proteins leads to increased latent TGF-␤1 secretion (21). In lung epithelial cells expressing these mutant proteins, there is increased ER stress and coordinated up-regulation of TGF-␤1, LTBP-1, and LTBP-4 gene expression. The secreted latent TGF-␤1 is capable of autocrine and paracrine effects and is completely inhibited by a pan-neutralizing TGF-␤ antibody.
Here, we find that expression of certain SP-A1 and SP-C proteins can also induce latent TGF-␤1 secretion in lung epithelial cell lines. Their expression leads to variant-specific contributions of the unfolded protein response in the induction of latent TGF-␤1 secretion.

EXPERIMENTAL PROCEDURES
Human Subjects-This work was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas, and written informed consent was obtained from all subjects. Race or ethnic identity was selfreported according to a list of categories used in the U. S. census. Genomic DNA was isolated from circulating leukocytes with an Autopure LS (Qiagen).
Animal Care and Maintenance-Baboons (Papio species) were maintained at the Southwest National Primate Research Center. All procedures were approved by the University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committees. Details of housing, environmental enrichment, and feeding have been described previously (22). Cesarean sections were performed at 165 days of gestation (0.9 G) using standard techniques (23). The fetuses were removed from the uterus and euthanized by exsanguination while still under general anesthesia. Fetal lung tissue was immediately removed, flash frozen in liquid nitrogen, and stored at Ϫ80°C until use.
Genomic DNA Sequencing, Allelic Discrimination, and Quantitative Real-time PCR-The PCR primers and conditions used to sequence genomic DNA for SFTPA1 and SFTPC are listed in supplemental Table 1. Sanger sequencing was performed as described (24). The Taqman allelic discrimination oligonucleotides used to test for the SFTPA1 R242* variant in a large (n ϭ 3512) multiethnic population-based sample of Dallas County (25) are listed in supplemental Table 2. Quantitative PCR was performed as described previously (21).
Recombinant Lentivirus-Human SP-A1 and C, which exactly matched NM_005411 and NM_003018 was cloned into pLenti6/V5-GW/lacz (Invitrogen). An inframe V5-or Myc epitope tag was placed after the glutamic acid at amino acid 21 in the SP-A1 gene by primer extension mutagenesis and zipper PCR. Mutant constructs were constructed by site-directed mutagenesis using Pfu Ultra Taq polymerase (Agilent Technologies). Lentivirus was made as described previously (21). The construct encoding BiP-luciferase (26) and PAI-luciferase (p3TP-lux from Addgene) were subcloned into pLenti6/V5-

TABLE 2 Half-life of V5 epitope-tagged SP-A1 variants in HBEC-3KT cells by chymotrypsin-limited proteolysis
The t1 ⁄ 2 (in minutes) of protein digested with chymotrypsin was calculated using quantitative measurements of immunoblots using the Odyssey infrared imaging system from two replicate experiments. GW/lacz. Antisense shRNA expressing lentivirus targeting LTBP-1, LTBP-4, IRE-1␣, PERK, and ATF6 were described previously (21). Lentivirus infection of cells, immunoblot analysis, chymotrypsin-limited proteolysis assays, measurement of secreted TGF-␤1, BiP-luciferase assays, XBP-1 splicing, co-culture assays with Mv1Lu cells expressing a PAI-luciferase reporter, and counting and viability of RLE-6TN cells were described previously (21,26). Statistical Analysis-Data are shown as the mean (Ϯ S.D.) of duplicates and are representative of at least two independent measurements. We used a paired two-tailed Student t test to determine statistical significance.

Rare and Common SP-A1 and SP-C Variants found in Famil-
ial Pulmonary Fibrosis Kindreds-We sequenced the coding exons of the genes encoding SP-A1 (SFTPA1) and SP-C (SFTPC) in 100 probands with familial pulmonary fibrosis. The frequencies of nonsynonymous variants are listed in Table 1, and kindreds are shown in supplemental Fig. 1. Three rare SP-A1 variants were found: one predicting a change of proline at amino acid position 5 to leucine (P5L), another predicting a change of asparagine at amino acid position 9 to threonine (N9T), and a third predicting a change of leucine at amino acid 107 to isoleucine (L107I). Both affected siblings in a small kindred share the rare SP-A1 5PL variant. The rare N9T variant was found in two African American probands. This rare variant did not segregate with the pulmonary fibrosis phenotype in one kindred; we could not assess for segregation in the second kindred. The rare L107I variant did not segregate with pulmonary fibrosis in the kindred in which it was found. The other missense variants, V19A, L50V, and R219W, are more common and have observed frequencies of 0.084, 0.639, and 0.090, respectively.
Two rare nonsynonymous variants in the gene encoding SP-C were found. The variant predicting a change of isoleucine at amino acid position 46 to phenylalanine (I46F) represents a novel rare variant. We could not determine whether the variant segregated with pulmonary fibrosis as tissue and DNA were not available from the other affected individual in this family. However, three related family members, ranging in age from 51 to 89 years, also inherited the I46F variant and were asymptomatic. The rare I73T variant was found in two probands. This variant segregated with lung disease in both of these families; six individuals with the I73T variant ranging from 3 to 42 years of age were affected with pulmonary fibrosis. The I73T mutations has previously been reported as a frequently detected pathogenic mutation in both pediatric and adult patients with interstitial lung disease (15,16).
Certain SP-A1 Variants Induce TGF-␤1 Secretion-We had previously determined that mutations in the gene encoding SP-A2 lead to induction of latent TGF-␤1 secretion (21). We sought to determine whether the expression of other SP-A1 variants would induce secretion of this cytokine. We expressed all of the SP-A1 variants described above in HBEC-3KT cells as N-terminal V5-epitope-tagged proteins. We also included a SP-A1 R242* variant, which predicts a stop codon at 242 with premature truncation of the mature protein by eight amino acids. This variant had been reported to have a frequency of 2% in dbSNP (supplemental Table 3). When we genotyped this variant through a large multiethnic Dallas population by allelic discrimination, we found that it is a very rare allele with a minor allele frequency of Ͻ0.015%.
HBEC-3KT cells are immortalized human bronchial epithelial cell lines that do not require serum for growth (28), thus allowing measurement of secreted cytokines. We find that HBEC-3KT cells expressing the common R219W variant and the rare R242* variant demonstrate secretion of latent TGF-␤1 and autocrine activation of the TGF-␤1 signaling cascade with serine phosphorylation of endogenous Smad2 (Fig. 1, A and B). All of the SP-A1 variants except for R242* are detected in cell lysates and are secreted into tissue culture medium. The R242* variant is not secreted into the culture medium, similar to the SP-A2 G231V and F198S mutant proteins (29). The R219W and R242* variants induce TGF-␤1 secretion to levels of ϳ2000 pg/ml, 4 -5-fold higher than levels induced by the pathogenic SP-A2 mutant proteins (21). We also utilized a co-culture assay to measure the ability of secreted TGF-␤1 to directly activate a PAI-luciferase reported expressed in co-cultured MvLu1 cells. As shown in Fig. 1, E and F, there is robust luciferase activity in cells co-cultured with HBEC-3KT cells expressing the R219W or R242* variants. The luciferase activity is completely abolished by culturing the cells in the presence of a pan-TGF-␤ neutralizing antibody. None of the other SP-A1 proteins induce secretion of latent TGF-␤1. The increase in TGF-␤1 correlates with increased expression of de novo TGF-␤1 mRNA expression, which is seen to increase by 6 h after lentiviral infection of the various surfactant variants (supplemental Fig. 2).
Biochemical Features of SP-A1 Variants-We sought to determine whether the R219W and R242* variants demonstrated any unique biochemical features. We used chymotrypsin limited proteolysis to estimate the relative stability of the proteins in comparison with wild type SP-A1. In these experiments, the V5 epitope-tagged proteins were expressed in HBEC-3KT cells, the entire cell lysate was subject to proteolysis with chymotrypsin for different incubation times, the V5-tagged proteins were quantitated using the Odyssey infrared imaging system of immunoblots, and the t1 ⁄2 of the variant was estimated from its rate of disappearance. We find that most variants demonstrate a stability similar to wild type SP-A1 (Table 2). Three variants, L107I, R219W, and R242*, demonstrate less stability than the wild type protein.
SP-A monomers trimerize through interactions of the collagen-like domain and the coiled-coil domain proximal to the carbohydrate recognition domain (30). To determine the effect of the variants on the oligomerization of SP-A, we analyzed SDS-PAGE gels of reduced and non-reduced HBEC-3KT cell lysates. Immunoblot analysis of reduced samples revealed a single major band at 28 -36 kDa for all variants, corresponding to protein monomers ( Fig. 2A). Immunoblot analysis of nonreduced samples revealed additional major bands for the wild type SP-A1 protein at ϳ60 and ϳ90 kDa, corresponding to the expected size of dimer and trimer species (Fig. 2B). Most of the variants formed oligomers such as the wild type SP-A1 protein.
However, more monomers, fewer dimers, and an aberrant pattern of higher molecular weight species were seen in the nonreduced R219W and R242* lysates.
To determine whether the V5 epitope tag causes altered protein folding and thus contributes to the induction of TGF-␤1, we expressed the SP-A1 variant proteins without the N-terminal epitope tag and measured protein production using a rabbit polyclonal antibody raised against purified human SP-A (29). The untagged SP-A variants induced the secretion of TGF-␤1 to similar levels (supplemental Fig. 3, A and B). The level of TGF-␤1 secretion did not change when the variants were coexpressed with the reciprocal SP-A paralog (supplemental Fig.  3, C and D). To determine the relative expression of the SP-A1 and SP-A2 from fetal to adult life, we isolated RNA from individual baboon fetal lung samples nearing the end of gestation (on day 165 of a total 184-day gestation) and from adult baboons ranging in age from 6 to 10 years. Using quantitative PCR primers that specifically amplify these two genes, we find that both paralogs are expressed in fetal and adult lung samples (supplemental Fig. 4).
Certain SP-C Variants Induce TGF-␤1 Secretion-We expressed all four discovered nonsynonomous SP-C variants ( Table 1) and 13 additional rare SP-C mutations described in the literature (supplemental Table 3) in HBEC-3KT cells. These variants include 11 missense variants, the ⌬exon 4 mutation (4), and a deletion of c.424delC (17) that predicts a frameshift (H142fs) resulting in a protein predicted to be 13 amino acids smaller than the wild type protein. The expression of three SP-C variants (I73T, M71V, and L188Q) led to the secretion of TGF-␤1 as detected by ELISA (Fig. 1C). The amount of secreted TGF-␤1 induced by the SP-C variants (200 -400 pg/ml) is much less than the levels induced by the SP-A1 or SP-A2 variants. The secreted TGF-␤1 is capable of autocrine Smad2 phosphorylation and paracrine activation of a PAI-luciferase reporter that is abolished by culturing the cells in the presence of a pan-TGF-␤ neutralizing antibody (Fig. 1, D-F).
Secretion of Induced TGF-␤1 Dependent upon Expression of LTBPs-Immunoblots of media samples from cells expressing the variant SP-A1 and SP-C proteins that induce TGF-␤1 secretion demonstrate that the cytokine is secreted with latent TGF-␤1 binding proteins (LTBPs). Under non-reduced condi-tions, the LTBPs migrate in SDS-PAGE gels at 250 -300 kDa (Fig. 3A), representing a complex between LTBP, the N-terminal remnant of the TGF-␤ precursor protein, and TGF-␤. (Fig. 3B). Using lentiviruses that express shRNA constructs that specifically silence LTBP-1 and LTBP-4, we find that decreased expression of these genes leads to a reduction of secreted latent TGF-␤1 by Ͼ90% (Fig. 3, C and  D). Expression of LTBP-1 and LTBP-4 cDNAs with changes engineered into each shRNA target site so that it is resistant to gene silencing restores LTBP expression, LTBP secretion into the culture medium, and secretion of TGF-␤1. Silencing of the LTBPs leads to a reduction of secreted TGF-␤1 by Ͼ90%, regardless of the expressed SP-A1 or SP-C variant protein.

Expression of LTBP-1 and LTBP-4 is greater than expression of LTBP-2 and LTBP-3 in cells expressing the SP-A1 and SP-C variant proteins
Secretion of Induced TGF-␤1 Dependent upon UPR-Expression of the mutant SP-A2 proteins lead to ER stress and activation of a BiP-luciferase reporter (21). We used this reporter system to determine whether the variant SP-A1 and SP-C proteins that induce TGF-␤1 secretion also demonstrate increased cellular ER stress. In comparison with wild type SP-A1 protein, the expression of both the SP-A1 R219W and R242* variants lead to more BiP-luciferase activity (Fig. 4A) and splicing of XBP-1 (Fig. 4B). In contrast, the expression of the SP-C I73T and L188Q variants induces less BiP-luciferase or XBP-1 splicing than the ⌬exon4 mutant protein. To determine whether signaling through the UPR is necessary for secretion of TGF-␤1, we used lentivirus that express shRNAs targeted to IRE-1␣, PERK, and ATF6. We find that silencing the UPR genes completely abolishes TGF-␤1 secretion induced by the common SP-A1 R219W but leads to a ϳ50% decline in secreted TGF-␤1 induced by the SP-A1 R242* variant (Fig. 4C). Silencing IRE-1␣ and ATF6 individually has a dramatic effect of reducing the R219W-mediated TGF-␤1 secretion. There is little effect of UPR silencing upon TGF-␤1 secretion induced by the SP-C M71V and I73T variants. We find a ϳ50% decline in secreted cytokine levels induced by the SP-C L188Q mutant protein (Fig.  4D). In general, silencing IRE-1␣ and ATF6 alone have a greater effect than silencing PERK alone (supplemental Fig. 5). We find that the genes encoding TGF-␤1, LTBP-1, and LTBP-4 are coordinately down-regulated for the SP-A1 R219W, R242*, and SP-C L188Q variants in response to the UPR silencing shRNAs (supplemental Fig. 6). Consistent with the secreted TGF-␤1 ELISA results, the expression of the TGF-␤1, LTBP-1, and LTBP-4 RNAs in cells expressing the common SP-A1 R219W variant are suppressed to wild type levels upon silencing of the UPR transducer genes.
The small molecule, 4-phenylbutyric acid (PBA), reduces ER stress through its actions as a molecular chaperone. Consistent with a previous study (31), culturing HBEC-3KT cells express- ing the SP-A2 G213V or F198S or SP-A1 R242* variant proteins in the presence of 1 to 5 mM PBA facilitates the secretion of these mutant proteins from the cell lysate to the culture medium (Fig. 5A). PBA reduces the levels of intracellular ER stress as measured by the BiP-luciferase reporter close to a level achieved by gene silencing of the UPR transducers (Figs. 5B and 6A). Adding 5 mM PBA to cells in which the UPR transducers are silenced further reduces the amount of secreted TGF-␤1

SP-A1 and SP-C Variants Induce Latent TGF-␤ Secretion
induced by the SP-A2 G231V and F198S mutant proteins or by the SP-A1 R242* variant protein (Fig. 5C), suggesting that the presence of these mutant proteins in an intracellular secretory pathway is required for maximal latent TGF-␤ secretion. We find that the addition of PBA and UPR transducer silencing lentivirus to cells expressing the SP-C L188Q protein has a more modest effect of reducing latent TGF-␤1 secretion (Fig.  6B). PBA has no effect on reducing the amount of latent TGF-␤1 secreted from cells expressing the SP-C I73T or M71V proteins, either in the absence or presence of the UPR transducer shRNAs.

Inhibition of TGF-␤ Prevents Cell Death of RLE-6TN Cells Expressing Certain Variant SP-A1 and SP-C Proteins-We
have previously found that rat lung epithelial RLE-6TN cells are especially susceptible to the downstream effects of latent TGF-␤ secreted in response to the expression of the SP-A2 G231V or F198S mutant proteins, leading to morphologic and transcriptional alterations consistent with epithelial to mesenchymal transition and cell death after 4 days (21). RLE-6TN cells expressing the SP-A1 R219W and R242* variants and the SP-C M71V, I73T, and L188Q variants demonstrate more TGF-␤1 expression than cells expressing the wild type proteins. Addition of a pan-TGF-␤ neutralizing antibody leads to the reduction of secreted latent TGF-␤1 and reversal of cell death over 4 days. Fig. 7A shows the rate of decline of secreted TGF-␤1 in cells expressing the SP-A1 R219W and R242* variants in comparison with the SP-A2 G231V mutant protein.
Similarly, the addition of the neutralizing antibody to cells expressing the SP-C mutant I73T, M71V, and L188Q proteins reduces the amount of secreted latent TGF-␤1 (Fig. 7B). The TGF-␤ neutralizing antibody leads to the rescue of cell death as shown by cell counting, viability, and morphology studies (Fig.  7, C-H). The expression of LTBP-1 and LTBP-4 are coordinately regulated with TGF-␤1; addition of the TGF-␤ neutralizing antibody leads to a reduction in all three genes (supplemental Fig. 7).

DISCUSSION
Type II alveolar epithelial cells play an important role in the pathogenesis of interstitial lung disease. These cells produce abundant amounts of phospholipids and proteins that constitute the surfactant secreted into the alveolar space. In the present study, we show that the expression of mutant surfactant proteins in two different lung epithelial cell lines (HBEC-3KT and RLE-6TN) leads to increased secretion of latent TGF-␤1. This pluripotent cytokine is synthesized and secreted as an inactive precursor, termed the large latent complex. Its activation in the extracellular space by different pathways such as acidic pH, proteolysis, or activation by integrins can lead to autocrine and paracrine signaling and lung fibrosis.
In an earlier study, we found that the expression of two different pathogenic SP-A2 proteins leads to increased secretion of latent TGF-␤1 through both ER stress-dependent and ER stress-independent mechanisms (21). We have now investigated whether the SP-A1 and SP-C variants are able to induce the expression and secretion of latent TGF-␤. We find that, unlike the SP-A2 G231V or F198S mutant proteins, the secretion of TGF-␤1 induced by the expression of SP-A1 R219W protein is completely dependent upon the expression of two transducers of the unfolded protein response, IRE-1␣ and ATF6. Also in contrast to the SP-A2 mutant proteins, secretion of TGF-␤1 from cells expressing the SP-C M71V or I73T proteins is largely unaffected by UPR gene silencing. The secretion of TGF-␤1 mediated by these two mutant SP-C proteins appears to be dependent upon a UPR-independent pathway. Cells expressing either the SP-C L188Q or SP-A1 R242* protein secrete latent TGF-␤1 through a combination of UPR-dependent and UPR-independent pathways, similar to the mutant SP-A2 proteins. Silencing of the UPR genes leads to the coordinated down-regulation of the TGF-␤1 and LTBP genes. Thus, all of these surfactant proteins have in common the ability to induce secretion of the profibrotic cytokine TGF-␤, but the relative contribution of the UPR transducers is specific for each variant. Blocking the TGF-␤ autocrine positive feedback loop (32) through the addition of TGF-␤ neutralizing antibody leads to the coordinated down-regulation of the TGF-␤1 and the LTBP genes. A schematic overview of the mechanism for TGF-␤ secretion for each variant is outlined in Fig. 8.
Although ER stress plays a key role in the pathogenesis of other SP-C mutant proteins (33)(34)(35), the expression of two SP-C variants, I73T and M71V, lead to minimal or no increase in BiP-dependent gene expression. Both of these missense mutations affect residues outside the BRICHOS domain and thus would be unlikely to interfere with the role of SP-C as an anti-amyloid molecular chaperone (5,6). These data suggest that the pathogenesis of these mutant SP-C proteins may involve induction of latent TGF-␤1 secretion. Both of these variants and the BRICHOS-domain SP-C L188Q variant have been described in kindreds with adult-onset pulmonary fibrosis (12,16), suggesting that the mechanism of induced latent TGF-␤ secretion may be especially relevant in patients with a delayed presentation of lung disease.
All of the SP-A variants studied so far that have the ability to induce secretion of latent TGF-␤ from cell lines involve amino acid residues within the carboxyl-terminal carbohydrate recognition domain. The SP-A1 R219W variant has a minor allele frequency of 8%, far greater than the estimated frequency of 13-40/100,000 cases of IPF in the United States (36). It was previously found to be associated with IPF (20), although the association was not replicated in a more recent genome-wide association study (37). It is currently unclear how the R219W variant, which has a weak genetic effect, strongly induces TGF-␤1 in vitro. Perhaps the R219W protein transiently interacts with an intracellular protein that facilitates TGF-␤1 processing and/or secretion. One candidate protein is BiP (Grp78) itself, which can associate with intracellular and surface forms of TGF-␤ (38) as well as misfolded surfactant proteins (39). It is possible that modulation of R219W expression perhaps through imbalanced expression of particular SP-A alleles (40) or the change in the subcellular localization of TGF-␤ through its interactions with intracellular sequestering proteins may ultimately regulate latent TGF-␤1 secretion in vivo.
We find a wide range of secreted latent TGF-␤1 levels induced by the expression of variant surfactant proteins. The levels induced by the SP-C mutant proteins are less than those induced by mutant SP-A2 proteins, which are, in turn, less than those induced by variant SP-A1 proteins. These levels do not correlate with disease latency or severity of disease at presentation (12,15,16,29). Disease presentation in humans may be related to in vivo levels of surfactant variant expression and the cellular regulation, timing, and activation of the latent TGF-␤ secreted from the type II alveolar epithelial cells and Clara cells expressing these proteins. It is interesting to speculate that environmental exposures to inhaled fibrogenic stimuli or infections with respiratory viruses that potentiate TGF-␤ production (41) or activation may lead to increased TGF-␤ activity, thus triggering disease onset. It is not known whether individuals for these alleles have elevated levels of TGF-␤ in bronchoalveolar lavage samples.
Limitations of this study include the lack of human tissue or bronchoalveolar lavage samples from individuals with these SP-A1 and SP-C variants that can induce the secretion of TGF-␤ in cell lines. We have previously seen that adults who have inherited the SP-A2 G231V mutation have 2-3-fold more TGF-␤1 in concentrated bronchoalveolar lavage samples than controls (21). Another limitation is the lack of clinical data for the SP-A1 R242* variant. This variant was previously reported by dbSNP and the clinical details of the person in which it was discovered are unknown.
For all of the variants studied here, we find that the addition of the pan-TGF-␤ neutralizing antibody to cells can effectively reduce levels of secreted latent TGF-␤, regardless of the contribution of UPR-dependent or UPR-independent pathways. The chemical chaperone PBA, which facilitates the transit of mutant surfactant proteins through the intracellular secretory pathway, alleviates ER stress and can also reduce levels of secreted latent TGF-␤ induced by certain mutant proteins. It is predicted that therapeutic interventions directed at reducing TGF-␤ production or activation may be especially beneficial to a subset of molecularly defined group of IPF patients whose  pathogenesis includes gain-of-function induction of latent TGF-␤ secretion.