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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 69-79, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0111OC

Cysteine Protease Activity Is Required for Surfactant Protein B Processing and Lamellar Body Genesis

Susan Guttentag, Lauren Robinson, Peggy Zhang, Frank Brasch, Frank Bühling and Michael Beers

Division of Neonatology, Department of Pediatrics, University of Pennsylvania School of Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; Institute of Pathology, University Hospital "Bergmannsheil," Bochum, Germany; Division of Electron Microscopy, Department of Anatomy, University of Göttingen, Göttingen, Germany; Institute of Immunology, University of Magdeburg, Magdeburg, Germany; and Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania

Address correspondence to: Susan Guttentag, M.D., Abramson Research Center 416G, Children's Hospital of Philadelphia, 3516 Civic Center Blvd., Philadelphia, PA 19104-4318. E-mail: guttentag{at}email.chop.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Surfactant protein (SP)-B is essential for lamellar body genesis and for the final steps in proSP-C post-translational processing. The mature SP-B protein is derived from multistep processing of the primary translation product proSP-B; however, the enzymes required for these events are currently unknown. Recent ultrastructural colocalization studies have suggested that the cysteine protease Cathepsin H may be involved in proSP-B processing. Using models of isolated human type 2 cells in culture, we describe the effects of cysteine protease inhibition by E-64 on SP-B processing and type 2 cell differentiation. Pulse-chase labeling and Western immunoblotting studies showed that the final step of SP-B processing, specifically cleavage of SP-B9 to SP-B8, was significantly inhibited by E-64, resulting in delayed accumulation of SP-B8 without adverse effects on SP-A or glyceraldehyde phosphate dehydrogenase expression. E-64 treatment during type 2 cell differentiation mimicked features of inherited SP-B deficiency in humans and mice, specifically disrupted lamellar body genesis, and aberrant processing of proSP-C. Reverse transcriptase–polymerase chain reaction and Western immunoblotting studies showed that Cathepsin H is induced during in vitro differentiation of type 2 cells and localizes with SP-B in multivesicular bodies, composite bodies, and lamellar bodies by immunoelectron microscopy. Furthermore, Cathepsin H activity was specifically inhibited in a dose-dependent fashion by E-64. Our data show that a cysteine protease is involved in SP-B processing, lamellar body genesis, and SP-C processing, and suggest that Cathepsin H is the most likely candidate protease.

Abbreviations: isobutylmethylxanthine, DCI • Dulbecco's modified Eagle's medium, DMEM • dimethyl sulfoxide, DMSO • phosphate-buffered saline, PBS • reverse transcriptase–polymerase chain reaction, RT-PCR • surfactant protein, SP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The hydrophobic surfactant proteins (SP)-B and -C are essential to the surface tension–reducing properties of pulmonary surfactant (reviewed in Refs. 1 and 2). Human patients and transgenic mice deficient in SP-B succumb to severe respiratory failure, and both SP-B and SP-C are absent from the alveolar surfactant (3, 4). Although the absence of SP-B is due to genetic mutation, the absence of the mature 3.7-kD form of SP-C is a secondary effect. Both hydrophobic surfactant proteins are synthesized as larger proproteins and multistep post-translational processing occurs as they move through the secretory pathway (5, 6). The observations of absent lamellar bodies in SP-B deficiency indicate that SP-B plays a central role in lamellar body formation, although the mechanism by which SP-B mediates lamellar body genesis is unknown (7). However, because late events in SP-C post-translational processing occur within the lamellar body, the absence of lamellar bodies in SP-B deficiency is tied to aberrant SP-C post-translational processing that results in accumulation of 6–10 kD SP-C intermediates and deficiency of mature SP-C (8, 9).

SP-B post-translational processing is similarly a multistep phenomenon (10). PreproSP-B, the 381 amino acid primary translation product, undergoes signal peptide cleavage and glycosylation in the endoplasmic reticulum, giving rise to 42-kD proSP-B. ProSP-B is then cleaved to a 25-kD intermediate with further modification of N-linked carbohydrates. This cleavage leaves a small, residual aminoterminal propeptide fragment flanking Phe201 of the mature SP-B sequence. Proteolysis of the carboxyterminus of the 25-kD intermediate gives rise to a 9-kD intermediate and is followed by a final aminoterminus cleavage releasing the mature protein.

During human fetal lung development, no mature SP-B protein is detected until 24 wk of gestation, despite SP-B mRNA levels reaching 50% of adult levels by the end of the second trimester (11, 12). One potential mechanism for the developmental control of post-translational processing is the developmental regulation of SP-B processing enzymes. We have shown previously that the post-translational proteolysis of SP-B is induced during ex vivo maturation of type 2 cells in human lung explant culture (10). We have also shown that the kinetics of the final steps in proSP-B processing are further accelerated by the addition of glucocorticoids, suggesting that the enzyme(s) required for SP-B processing are upregulated in their expression or activated under these conditions.

The enzymes required for SP-B processing have not yet been identified with certainty. Early studies suggested that an aspartic protease mediated cleavage of the aminoterminus of proSP-B (13). More recently, Cathepsin H has been implicated in hydrophobic surfactant protein processing. Cathepsin H, a cysteine protease with unique aminopeptidase activity, has been localized to alveolar type 2 cells, specifically to multivesicular bodies (14, 15). In vitro assays have similarly shown that Cathepsin H is able to cleave both proSP-B and proSP-C recombinant proteins, resulting in protein fragments comparable in Mr to endogenous intermediates (14, 16).

We now show that inhibition of cysteine protease activity in cultured human type 2 cells by E-64 blocks the final step of SP-B processing, resulting in accumulation of the 9-kD intermediate. Furthermore, cysteine protease inhibition by E-64 during in vitro differentiation of human type 2 cells interrupts SP-B processing and leads to additional features of SP-B deficiency, including abnormal SP-C processing and aberrant lamellar bodies. Through the use of reverse transcriptase–polymerase chain reaction (RT-PCR)s and Western immunoblotting, we show that Cathepsin H expression is upregulated during type 2 cell differentiation and, by immunoelectron microscopy, that Cathepsin H and SP-B both localize in multivesicular bodies, composite bodies, and lamellar bodies. Together, these data suggest that cysteine protease activity, likely from Cathepsin H, plays an integral role in SP-B processing and lamellar body genesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Express Protein Labeling Mix was obtained from New England Nuclear (Boston, MA). Protein A-agarose was obtained from Life Technologies, Inc. (Gaithersburg, MD). Dexamethasone, isobutylmethylxanthine, 8-Br-cAMP, E-64, and dimethylsulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents were electrophoretic grade and were purchased from either Bio-Rad Laboratories (Hercules, CA) or Invitrogen (Carlsbad, CA). Culture media were produced by the Cell Center Facility, University of Pennsylvania School of Medicine.

Western immunoblotting and immunocytochemistry studies were performed using rabbit polyclonal antisera to human SP-A, human SP-B, NFlank (the aminoterminal propeptide of human proSP-B amino acids Gln186–Gln200), and NProSP-C (aminoterminal propeptide of rat proSP-C Met10-Glu23), which have been characterized elsewhere (5, 10), rabbit polyclonal antiserum to Cathepsin H (Athens Research and Technology, Athens, GA), and a mouse monoclonal antibody to glyceraldehyde phosphate dehydrogenase (GAPDH; Chemicon, St. Louis, MO). To identify subcellular organelles, we used the following antisera: mouse monoclonal antibody to the BiP (StressGen Biotechnologies Corp., Victoria, BC, Canada) for endoplasmic reticulum, mouse monoclonal antibody to p230 for Golgi, and mouse monoclonal antibody to EEA1 for early endosomes (Transduction Laboratories, Lexington, KY), and mouse monoclonal antibody to Lamp-1 for multivesicular body, LB, and lysosomes (H4A3 developed by J. T. August and J. E. K. Hildreth from the Developmental Studies Hybridoma Bank; developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA).

The epitope-specific antiserum CTermB was prepared as described previously (10). Based on the antigenicity index of preproSP-B, the peptide sequence Leu310–Leu329 was chosen for production of synthetic peptide by the Merrifield method by the Nucleic Acid/Protein Core, Children's Hospital of Philadelphia. Peptide purity was confirmed by mass spectroscopy. The synthetic peptide was conjugated to Keyhole Limpet Hemocyanin and injected in Freund's adjuvant into New Zealand rabbits by Strategic Biosolutions, Inc. (Ramona, CA). Antiserum was screened for reactivity against the immunizing peptide by immunodot blot and Western immunoblot assay.

Lung Explant and Primary Cell Culture
Human fetal lung obtained from second trimester therapeutic abortions (14–23 wk estimated gestational age) were obtained under protocols approved by the Committee for Human Research, Children's Hospital of Philadelphia. Tissues were frozen at –70°C for RNA and protein assays, fixed for immunohistochemistry (see below), or used for explant or cell culture as follows. Fetal lung parenchyma was dissected free of large airways, chopped into 1 mm3 explants, and cultured in Waymouth media on a rocking platform as previously described (17). After overnight culture as explants, epithelial cells were isolated by digestion with trypsin, collagenase, and DNase, followed by panning on plastic culture dishes to remove adherent fibroblasts as described previously (18). Cells were then cultured for up to 7 d in 10 nM dexamethasone, and 0.1 mM 8-Br-cAMP and 0.1 mM isobutylmethylxanthine (DCI) to induce type 2 cell differentiation. To isolate fully differentiated type 2 cells, explants were cultured in DCI for 4 d followed by isolation of epithelial cells by enzymatic digestion and panning to remove fibroblasts as previously described (19). The cells were cultured an additional 4 d in Waymouth media supplemented with DCI in 95% air, 5% CO2.

Pulse:Chase Labeling and Immunoprecipitation
We prepared fully differentiated type 2 cells from hormone-stimulated lung explants as described above. Culture medium was replaced with DCI-supplemented Waymouth medium + DMSO or E-64 at 10–100 µM for overnight culture. Cells were then incubated in Met-Cys-free Dulbecco's modified Eagle's media (DMEM; 2 ml/60 mm plate) ± E-64 for 1 h. Met-Cys–free media was replaced with Met-Cys–free DMEM ± E-64 supplemented with 200 µCi/ml of 35S Express Protein Labeling Mix that is composed of 70% methionine and 15% cysteine (New England Nuclear). After a 30-min pulse, the media was changed to complete Waymouth media with DCI ± E-64. Samples were harvested immediately after the 35S labeling and at regular intervals through 4 h post-labeling. Samples were washed in phosphate-buffered saline (PBS) with protease inhibitors (10 mM N-ethyl maleimide, 2 mM benzamidine HCl, and 80 mM phenylmethylsulfonyl fluoride) and then immunoprecipitated as previously described for SP-B (6). Electrophoresed samples were transferred to membrane and blots were visualized using the Storm phosphorimager system (Molecular Dynamics, Sunnyvale, CA).

Toxicity Studies
In addition to examining 35S-Met/Cys incorporation into TCA precipitable proteins (see above), toxicity was assessed by labeling live cells with Carboxy-SNARF-1, a long-wavelength fluorescent pH indicator (Molecular Probes, Eugene, OR). Cells were washed in PBS and then incubated with 2 µM Carboxy-SNARF-1 in PBS for 30 min, as recommended by the manufacturer. Cells were then fixed in 1% paraformaldehyde in PBS, washed in PBS followed by distilled water, dried, and mounted using Prolong (Molecular Probes) to reduce fading. Cells were examined under fluorescence microscopy using a Texas Red filter. Cytotoxicity was also assessed using an LDH assay on culture media using the Cytotoxicity Detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions.

Long Term Inhibitor Studies
Undifferentiated alveolar epithelial cells were cultured for 24 h in Waymouth medium plus either DMSO or E-64 at 10–100 µM (stock 10 mM in DMSO; final concentration DMSO 0.1%). Media ± E-64 was then supplemented with DCI for up to 7 d with daily changes of media. Cells were either frozen at –70°C for protein analysis or fixed for either electron microscopy or immunohistochemistry.

Western Immunoblotting
Western immunoblotting was accomplished using previously described procedures (10). Western blots for SP-A, SP-B, and GAPDH used NuPAGE Bis-Tris gels with MES SDS Running Buffer (Invitrogen, Carlsbad, CA) and then transferred as per the manufacturer's protocol to Duralose membranes (Stratagene, La Jolla, CA). NProSP-C immunoblots were transferred to membrane using a Tris-glycine transfer buffer at 20 mA/cm2 for 16 h.

Immunofluorescence Cytochemistry
Immunofluorescence staining was accomplished using previously described procedures (6). Briefly, cells were fixed in 1% paraformaldehyde in PBS followed by washes in PBS with 5 mM NH4Cl, and were stored at –70°C until immunostaining was performed. After incubation in sodium borohydride and blocking in primary antibody host serum, slides were incubated overnight in primary antibody at 4°C. In some cases, Triton X-100 was omitted from the blocking and antibody diluent solutions to improve antibody sensitivity. Secondary antisera were applied after washing and the slides incubated at 4°C for 2 h. We used Alexa488 and/or Alexa594 conjugated to rabbit or mouse IgG as secondary antisera (Molecular Probes). Slides were washed, air dried, and mounted using Prolong (Molecular Probes, Eugene, OR) to reduce fading. Fluorescence was examined with an Olympus 1X70 microscope and Metamorph imaging system.

Electron Microscopy
Undifferentiated epithelial cells were cultured for 5 and 7 d in DCI or DCI + 100 µM E-64. Cells were fixed in 2.5% glutaraldehyde in cacodylate buffer, postfixed in 1% osmium tetroxide, and embedded and sectioned as previously described (19). Two blocks were prepared and sectioned from each experimental group. Ultrathin sections were examined by transmission electron microscopy in the Biomedical Imaging Core of the University of Pennsylvania School of Medicine. Electron micrographs of 6–8 grid subdivisions per section were taken at x2,500, x10,000 and x30,000 magnification.

Immunoelectron Microscopy
Six single human donor lungs were fixed by instillation of a mixture of 4% formaldehyde (prepared from freshly depolymerized paraformaldehyde) and 0.1% glutaraldehyde in 0.2 M Hepes buffer. Donor lungs were used for investigation only if they could not be made available for another suitable recipient by The Eurotransplant Foundation Center (Leiden, The Netherlands). Systematic uniform random lung samples were processed for immunoelectron microscopy as previously described (14). To localize SP-B and cathepsin H, we prepared serial sections and labeled alternating sections with rabbit anti-cathepsin H or SP-B antisera (anti-SP-B, a gift of Dr. J. Whitsett, Cincinnati, OH; anti-cathepsin H, a gift of H. Kirschke, Institute of Physiological Chemistry, Halle, Germany), and immunoreactivity was visualized by incubation with a secondary 5-nm gold-coupled antibody diluted in blocking buffer. Control experiments were performed by omission of the primary antibodies. Examination of labeled ultrathin sections was conducted using an EM 900 (LEO, Oberkochen, Germany) at an accelerating voltage of 50 kV.

RT-PCR
RNA was prepared using RNA STAT (Tel-Test, Inc., Friendswood, TX) per the manufacturer's instructions. Purity was confirmed by OD 260:OD 280 ratio and by visualization of ribosomal RNA bands on a formaldehyde agarose gel. Samples were then treated with RQ1 RNase-free DNase (Promega, Madison, WI) and ethanol precipitated after phenol-chloroform extraction. cDNA was synthesized from RNA samples using the SuperScript First-Strand RT-PCR kit (Life Technologies) using manufacturer's instructions with the following exception. For multiplex RT-PCR, final concentrations of the specific primers (SP-B, Cathepsins H, P/Z, V, X) were 0.5 µM, and for GAPDH 0.1 µM. The following primers were used for RT-PCR: GAPDH sense-ACCACAGTCCATGCCATCAC, antisense-TCCACCACCCTGTTGCTGTA; SP-B sense-AGGACATCGTCCACATCCTT, antisense-GAGCAGGATGACGGAGTAGC; Cathepsin H sense-CAACAATGGGAACCACACAT, antisense-GCAAAGCTCACAGGGTTGTA; Cathepsin O sense-AGATGAAATGCCAAAAGCAC; antisense-GCTTCTGTGCTGGAGGTTTGT; Cathepsins P and Z sense-AGCTGTGGAATAATGGCAAC; antisense-GCTGACTGCATCATTGTGAG; Cathepsin V sense-GTACCAGTGGAAGGCAACAC antisense-CCTCCGTTCTCCTTGACATA Cathepsin X sense-GAAGGTAGAGCCCCAGACTC, antisense-AAACCGCATGGTTCAGATTA. Cathepsins P and Z have 99% nucleotide homology and the primers chosen do not discriminate between the two. After optimizing PCR conditions for each primer set, PCR was performed on 1 µg cDNA using the following protocol: 94°C x 5 min (94°C x 30 s, 53°C [Cathepsins P/Z, V, X] or 57°C [SP-B, Cathepsin H] x 30 s, 72°C x 30 s) x 30 cycles and 72°C x 10 min. Samples of the final reaction (5 µl) were run on 2% agarose gels with ethidium Br, and UV images obtained. Each PCR product was sequenced by the Nucleic Acid and Protein Core Facility of the Children's Hospital of Philadelphia and the sequence verified by comparison to sequences contained within the GenBank database.

Enzyme Assay for Cathepsin H
Cell pellets (1–4 x 106) were sonicated on ice in phosphate buffer pH 6.8 containing 1 mM EDTA. The assay was performed at 37°C in 0.1 M phosphate buffer containing 1 mM EDTA and 10 mM cysteine at pH 6.8 with Arg-AMC as a substrate (20). For inhibition of serine, aspartic, and metallo proteinases di-isopropyl-fluorophosphate (DFP, 10 µM; Sigma, Deisenhofen, Germany), pepstatin A (1 mM; Sigma) and Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, 100 µM; Sigma) was added. To document the specificity of the enzymatic substrate cleavage, assays were performed in the presence or in absence of 5 µM trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64; Sigma), an inhibitor of cysteine proteases, which blocks the substrate cleavage. The relative cathepsin H activity was calculated from the difference in AMC release of corresponding samples with and without E64. The enzymatic activity was expressed as relative activity per total protein in the cell lysates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E-64 Inhibits SP-B Accumulation during In Vitro Differentiation of Type 2 Cells
E-64, also known as trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane, is a water-soluble inhibitor of Cathepsins B, H, L and calpain that has been used in cell culture systems (20, 21). To examine the effects of cysteine protease inhibition during type 2 cell differentiation, we cultured undifferentiated lung epithelial cells from second trimester human fetal lungs in the presence of DCI ± 10–100 µM E-64. There was no evidence of cytotoxicity by LDH assay and Carboxy-SNARF-1 fluorescence, through 7 d in culture with E-64 (not shown). Western immunoblotting of cells cultured in DCI alone showed increasing SP-A and SP-B8 production as previously described (18), and GAPDH did not change over time under these conditions (Figure 1) . The presence of E-64 during cell culture did not affect GAPDH expression, nor did it alter the accumulation of SP-A over the culture period. E-64 delayed the accumulation of SP-B8, and was associated with accumulation of a 9-kD intermediate. This intermediate was also identified by the NFlank antiserum that detects amino acids Gln186–Gln201 of the aminoterminus remnant of proSB-B (not shown). A 12-kD intermediate was also seen in small amounts on Western immunoblotting. Inhibition of SP-B processing was less effective at 10 µM than at >= 50 µM E-64 (data not shown).



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  		Figure 1. Western immunoblotting of the time course of E-64 effects on type 2 cell differentiation. Undifferentiated lung epithelial cells isolated from second trimester human fetal lung were differentiated in vitro using 10 nM dexamethasone, 0.1 mM 8-Br-cAMP, and 0.1 mM isobutylmethylxanthine in the presence of 100 µM E-64 (n = 4). A representative time course (after 24–144 h in culture, each sample 50 µg total protein) of SP-B, SP-A, and GAPDH expression by Western immunoblotting is shown. Samples obtained before the addition of hormones ± E-64 exhibited no SP-A or mature SP-B protein as previously described (not shown). GAPDH expression did not change over the time course, nor in response to E-64. SP-A increased over the time course as previously described (18), and was not perturbed by E-64. Mature SP-B increased rapidly with the addition of hormones and increased further over the 7-d culture period. E-64 treatment delayed the appearance of mature SP-B and was associated with accumulation of 9-kD and, to a lesser extent, 12-kD precursors (asterisk, nonspecific band).

 
E-64 Inhibits the Most Distal Step in SP-B Post-Translational Processing
Differentiated type 2 cells were treated with 10–100 µM E-64 for 16 h before and up to 8 h during pulse-chase labeling with 35S-cysteine/methionine. Immunoprecipitations for SP-B in cells cultured for 5 d ± 100 µM E-64 appear in Figure 2 . DMSO-exposed control cells exhibited proSP-B42 processing to mature 8 kD SP-B within 1–2 h and accumulation of labeled SP-B8, as previously described in cells cultured in the absence of DMSO (19). A 9-kD intermediate was first detected at 2 h, but did not accumulate in later samples, indicating a precursor–product relationship with rapid kinetics for conversion to SP-B8 as previously described. In the presence of E-64, proSP-B42 was cleaved to SP-B25, followed by cleavage to proteins ranging between 9 and 12 kD which accumulated over the 4-h chase period; there was delayed appearance of SP-B8 and small amounts detected at 4 and 8 h, at levels less than in DMSO-exposed cells.



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  		Figure 2. Pulse-chase labeling studies of SP-B processing ± E-64. Type 2 cells prepared from hormone-treated fetal lung explants were cultured in the presence (E-64) or absence (Control) of 100 µM E-64 for 16 h before and 8 h during pulse:chase labeling (n = 5). Representative immunoprecipitations using a polyclonal antiserum to human SP-B with phosphorimager rendering of SDS-PAGE gels transferred to membrane are shown. Cells cultured in the absence of E-64 exhibited rapid labeling of proSP-B and rapid processing to mature SP-B8 beginning at 1 h and accumulating through the 8 h chase period. Cells cultured in the presence of E-64 exhibited similar labeling of proSP-B and processing through the 25-kD intermediate, but accumulated intermediates of 9 kD (asterisk) and ~ 12 kD over the 8-h chase. Small amounts of mature SP-B were detected from 2–8 h. Samples immunoprecipitated with nonimmune serum (2NIS) exhibited no immunoprecipitable proteins.

 
To more precisely characterize the kinetics of SP-B processing ± E-64, phosphorimager data were quantified by densitometry. The densitometric units within each band were first corrected for the Cys/Met content as previously described (10). We were able to resolve and quantify 42-, 25-, 12-, 9-, and 8-kD bands (n = 3 experiments) for analysis. The total densitometric units post-labeling were determined by adding the corrected densitometric units in all 35S-labeled SP-B protein forms; this is referred to as the initial incorporated label post-pulse. Corrected densitometric units for each SP-B form were then expressed as the percent of the initial incorporated label, and the percentage change between protein intermediates was calculated. Loss of label from the 42-kD proSP-B was not different ± 100 mM E-64 (28.3 ± 5.5%/h control, 23.9 ± 4.7%/h E-64, mean ± SE; P = .46). There was a significant decrease in the rate of accumulation of mature SP-B in E-64–treated cells (16.8 ± 4.8%/h control, 0.8 ± 0.4%/h E-64, mean ± SE; P < 0.01).

E-64 Alters Type 2 Cell Phenotype and Lamellar Body Genesis during Differentiation
Nile red is a lipophilic vital stain that identifies lamellar bodies of alveolar type 2 cells (19). In Figure 3A , cells cultured in DCI alone showed intense vesicular staining with Nile red, consistent with the high lipid content of lamellar bodies. Nile red staining of E-64–treated type 2 cells displayed a more diffuse pattern of staining in contrast to the intense lamellar body pattern of staining in untreated type 2 cells. Immunostaining for markers of endoplasmic reticulum, Golgi, and early endosomes were not different in the presence of E-64 compared with control cells (Figure 3B). However, the distribution of Lamp-1, a lysosomal transmembrane protein present in the limiting membrane of lamellar bodies as well as the small vesicles within multivesicular bodies (22), was altered by E-64 treatment. Control cells showed vesicle and lamellar body staining described previously (6), whereas E-64–treated cells had more extensive, coarse Lamp-1 immunostaining.



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  		Figure 3. Effects of E-64 on type 2 cell phenotype by light microscopy. Undifferentiated lung epithelial cells isolated from second trimester human fetal lung were cultured for 5 d in 10 nM dexamethasone, 0.1 mM 8-Br-cAMP, and 0.1 mM isobutylmethylxanthine ± 100 µM E-64 (n = 3). A is a representative image of live cells after staining with Nile red. Hormone treatment led to the formation of lamellar bodies, appearing as intense vesicles in a perinuclear distribution (arrow). In the presence of E-64, cells did not develop this pattern but instead exhibited a more widely distributed, flocculent pattern (bar, 50 µm). B is a composite of photomicrographs from immunohistochemistry of cells using the organelle markers BiP (ER), p230 (Golgi), early endosomes (Eea1), and lamellar bodies/multivesicular bodies (Lamp1). There was no difference in the distribution of immunostaining for BiP, p230, or Eea1 in cells cultured in the presence or absence of E-64, whereas Lamp1 immunostaining became more widespread and coarse in type 2 cells treated with E-64 compared with untreated cells (bar, 20 µm).

 
To clarify the effects of cysteine protease inhibition on lamellar body formation, we examined transmission electron microscopy of type 2 cells differentiated in the presence and absence of E-64. Ultrathin sections from each treatment group of two experiments contained from 200–600 cells each per 200 mesh grid. Cells cultured for 5 d in the presence of DCI alone (Figure 4A) demonstrated microvilli and increasing numbers of lamellar bodies as previously reported (18); 80–95% of cells within each grid subdivision examined exhibited this phenotype. The lamellar bodies were spherical with tightly packed lamellae in a concentric configuration in cross-section or with lamellae emanating from an electron-dense projection core when sectioned longitudinally, as has been described by others in human type 2 cells (14, 23). Multivesicular bodies were not readily seen. Cells cultured in DCI + E-64 (Figures 4B–4D) also exhibited microvilli and showed no ultrastructural signs of toxicity, specifically there was no evidence of mitochondrial, ER, or Golgi swelling. Cells cultured in the presence of E-64 accumulated electron-dense material in membrane-limited structures (Figures 4B and 4D, arrows). These structures occasionally contained some irregular, whorl-like, lamellar structures (Figure 4C, asterisk), but were otherwise not recognizable as lamellar bodies. Membrane-limited multivesicular bodies were evident and large numbers of small cytoplasmic vesicles seen in E-64–treated cells (Figure 4C, arrowhead). When cultured for 7 d, E-64–treated cells continued to display significant amounts of poorly organized electron-dense material but also show evidence of mature lamellar bodies (Figures 5A–5C) and secreted surfactant (Figure 5D).



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  		Figure 4. Effects of E-64 on type 2 cell ultrastructure. Undifferentiated lung epithelial cells isolated from second trimester human fetal lung were cultured for 5 d in 10 nM dexamethasone, 0.1 mM 8-Br-cAMP, and 0.1 mM isobutylmethylxanthine ± 100 µM E-64 (n = 2). Representative electron micrographs of Control cells (A) and E-64–treated cells (B–D) were taken at magnifications of x2,500, x10,000, and x30,000 (from left to right). Control cells developed both microvilli and large lamellar bodies. E-64–treated cells also exhibited microvilli; however, no lamellar bodies were seen. Numerous electron-dense organelles were observed (B and D, arrows), some with lamellar structures (C, asterisk). In addition, multivesicular bodies were observed with regularity in E-64 cells and not in Control cells (C, arrowhead). Electron-dense inclusions were all membrane-limited (D, small arrows). N, nucleus; m, mitochondrion; G, Golgi; MV, microvilli; LB, lamellar body; L, lamellae; C, projection core.

 


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  		Figure 5. Differentiating type 2 cells cultured in E-64 begin to exhibit lamellar bodies by Day 7. Undifferentiated lung epithelial cells isolated from second trimester human fetal lung were cultured for 7 d in 10 nM dexamethasone, 0.1 mM 8-Br-cAMP, and 0.1 mM isobutylmethylxanthine ± 100 µM E-64 (n = 2). Representative electron micrographs of E-64–treated cells were taken at magnifications of x10,000 (A), and x30,000 (B–D). Despite the persistence of poorly formed electron-dense structures as seen in Figure 4, cells cultured for 7 d exhibit lamellar bodies and secreted surfactant material.

 
E-64 Alters SP-C Post-Translational Processing
To examine the effects of E-64 on SP-C processing, we used the antiserum NProSP-C to detect accumulated SP-C precursors. This antiserum does not detect mature SP-C3.7. Western immunoblotting of type 2 cells cultured for 5–7 d in the presence of hormones (Figure 6 , lanes 3 and 4) showed evidence of proSP-C21, but very little SP-C intermediate, as also noted in adult lung (lane 1). Cells cultured in the presence of E-64 exhibited a 10-kD precursor (lanes 5 and 6), similar in Mr to proSP-C precursor forms which accumulate in SP-B–deficient lung (lane 2).



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  		Figure 6. SP-C processing in E-64–treated type 2 cells. Undifferentiated lung epithelial cells isolated from second trimester human fetal lung were differentiated in vitro for 5–7 d using 10 nM dexamethasone, 0.1 mM 8-Br-cAMP, and 0.1 mM isobutylmethylxanthine ± 100 µM E-64 (n = 3). Representative immunoblot using the antiserum NProSP-C, which does not recognize mature SP-C3.7. Adult lung (lane 1, 100 µg) and type 2 cells cultured for 5 and 7 d (lanes 3 and 4, respectively, 100 µg each) exhibit proSP-C at 21 kD and a ~ 16-kD intermediate only. Tissue from an SP-B–deficient patient (lane 2, 50 µg) exhibits large amounts of intermediates of 6–10 kD in addition to 21 kD proSP-C and 16 kD intermediate. E-64–treated cells (lanes 5 and 6, respectively, 100 µg each) also exhibit a 10-kD SP-C intermediate not seen in samples from untreated cells.

 
Cathepsin H Is a Candidate Protease for SP-B Processing
An extensive review of the literature indicated that only a subset of lysosomal cysteine proteases could be responsible for the cysteine protease activity within type 2 cells. Knockout models for Cathepsins B, C, K, L, and S display no pulmonary pathology nor neonatal lethality, and thus were not considered plausible candidates for SP-B proteolysis. Those cysteine protease Cathepsins known to be expressed in lung or not previously examined for pulmonary expression included Cathepsins H, O, P, Z, V, and X (14, 24–27). RT-PCR of fetal human lung, heart, kidney, and liver tissue followed by sequence analysis of the products confirmed the correct detection of Cathepsins H, O, P/Z, V, and X using our primers. We then developed multiplex RT-PCR for Cathepsins H, O, P/Z, V, X, and SP-B in conjunction with GAPDH to semiquantitatively assess Cathepsin mRNA expression. The conditions used for amplification resulted in linear increases in both products with increasing input cDNA (not shown). RNA from fetal heart, liver, and kidney (Figure 7A , upper panels; H, L, K, respectively), and samples of undifferentiated lung epithelial cells and in vitro differentiated type 2 cells from three separate fetuses (gestational ages 16–18 wk), were examined. Cathepsins H, P/Z, and X were detected in fetal heart, liver, and kidney using our RT-PCR conditions, whereas Cathepsins O and V were only detected in fetal kidney. These expression profiles were consistent with prior reports of Cathepsin expression. Small amounts of SP-B mRNA were detected in isolated undifferentiated epithelial cells (Figure 7A, lower panels, U) and SP-B mRNA increased under the influence of DCI (Figure 7A, lower panels, D). This was consistent with other reports of SP-B mRNA expression in second trimester human fetal lung (11). Importantly, no SP-B product was amplified from fetal heart, liver, or kidney. Cathepsin H was also detected in undifferentiated cells and increased in response to DCI, consistent with prior microarray studies (18). By comparison, in the samples examined, Cathepsins O, P/Z, V, and X were not detected in either undifferentiated or differentiated cells.



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  		Figure 7. Cathepsin H expression in differentiating type 2 cells ± E-64. (A) RT-PCR for Cathepsin expression. Representative multiplex RT-PCR of fetal heart, liver, kidney (upper panels, H, L, K, respectively, n = 3) and undifferentiated epithelial cells versus cells differentiated in vitro for 5 d (lower panels, U and D, respectively, n = 3). Multiplex RT-PCR correctly identified Cathepsins O (CTSO) and V (CTSV) only in fetal kidney, whereas Cathepsins P/Z (CTSP/Z) and X (CTSX) were identified in fetal heart, liver, and kidney. Only Cathepsin H (CTSH) and SP-B were detected in cells from fetal lung. RNAs for Cathepsin H and SP-B were detected in undifferentiated lung epithelial cells and increased with in vitro differentiation of type 2 cells. (B) Western immunoblotting of cells differentiated for up to 6 d ± 100 µM E-64. Expression of single chain 28-kD Cathepsin H (CTSH, upper arrowhead) increased during in vitro differentiation in both the presence and absence of E-64, whereas expression of the 22-kD heavy chain fragment accumulates to a greater degree in the presence of E-64 (n = 3). (C) Cathepsin H and SP-B localize in lamellar bodies of adult human lung tissue. Serial thin sections of adult human lung were exposed to primary antisera for SP-B (left) and Cathepsin H (right). Immunogold particles from both antisera are shown localizing over the projection core of lamellar bodies. (D) E-64 inhibits Cathepsin H activity in type 2 cells. Cathepsin H activity assay performed on freshly isolated undifferentiated alveolar epithelial cells (no DCI) and cells cultured for 3 and 5 d in DCI ± 10, 50, or 100 µM E-64. With induction of type 2 cell phenotype by DCI, Cathepsin H activity increased 2- to 3-fold (mean ± SD; n = 2 experiments). In the presence of E-64, Cathepsin H activity was inhibited in a dose-dependent fashion.

 
We also examined Cathepsin H protein expression during type 2 cell differentiation in the absence and presence of E-64 (Figure 7B). The active, single chain form of Cathepsin H was detected at 28 kD in preculture tissue (not shown), and increased over the culture period during type 2 cell differentiation. The 22-kD active heavy chain form of Cathepsin H also increased during in vitro differentiation of type 2 cells. Surprisingly, E-64 treatment resulted in further accumulation of the 22-kD protein. Immunoelectron microscopy of serial thin sections of adult human lung were used to further define the subcellular location of Cathepsin H in relation to SP-B within type 2 cells. At the ultrastructural level, SP-B and cathepsin H localized to multivesicular, composite, and lamellar bodies. In lamellar bodies of adult human type 2 cells, both localized over the projection core with weak labeling for cathepsin H also along the limiting membrane of lamellar bodies (Figure 7C).

Cathepsin H activity was assayed in cells using the substrate Arg-AMC, which is specific for Cathepsin H (20). When compared with freshly isolated undifferentiated epithelial cells, hormone-treated cells exhibited a 2- to 3-fold increase in Cathepsin H activity after 3 and 5 d in culture (Figure 7D), which was consistent with increased Cathepsin H expression noted by RT-PCR. Cells cultured in hormones with increasing concentrations of E-64 showed a dose-dependent decrease in Cathepsin H activity (n = 2 experiments).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The biosynthesis of SP-B requires extensive remodeling of the proprotein precursor, but the enzymes necessary for this process are largely undefined. In these studies, we have shown that cysteine protease inhibition altered processing of proSP-B in human type 2 cells. More specifically, the final step in SP-B processing is sensitive to inhibition by the cysteine protease inhibitor E-64. Furthermore, inhibition of this step in processing, by blocking the formation of mature SP-B, results in similar features to those previously described in both inherited SP-B deficiency in humans and in SP-B knockout mice, specifically disrupted lamellar body genesis, and aberrant SP-C processing. To begin to identify the lysosomal cysteine protease responsible for these effects, we showed that the most likely candidate is Cathepsin H. Together, these studies represent the first description of inhibition of SP-B proteolytic processing in human alveolar type 2 cells.

SP-B proteolytic processing involves multiple steps, resulting in release of the mature 79 amino acid SP-B protein from the larger 381-kD preproprotein. Early studies suggested that proSP-B underwent complete cleavage of the aminoterminus followed by cleavage of the carboxyterminus to release mature SP-B (reviewed in Ref. 1). Our prior studies expanded upon the original processing scheme, showing that proteolysis of the aminoterminus occurred in two steps within distinct compartments of the type 2 cell (10). Our current studies lend support to the processing scheme (depicted in Figure 8) in which a residual portion of the aminoterminus is exposed in proximal compartments within the type 2 cell and cleaved in a prelamellar body compartment. It is well known that the first cleavage of the aminoterminus is a non–type 2 cell–specific process occurring in cell lines (28, 29), whereas the latter steps, specifically cleavages of the carboxyterminus and residual aminoterminal propeptide, are type 2 cell–specific. It is the cell-type–specific steps in processing that occur in more distal compartments of the type 2 cell, in closer proximity to the lamellar body (6). The aminoterminus remnant in association with mature SP-B constitutes the 9-kD intermediate that accumulates upon E-64 inhibition during pulse-chase studies in differentiated type 2 cells and during differentiation of type 2 cells in vitro (Figures 1 and 2). Our previous studies indicated that cleavage of the residual aminoterminus occurred in a post-Golgi, pre-lamellar body compartment (6). E-64 treatment caused no gross disruption of ER, Golgi, or early endosomes by immunocytochemistry, although more detailed functions of these compartments were not explored. The compartment most significantly affected was a Lamp1-positive compartment, which by immunostaining appeared engorged within the E-64–treated type 2 cells when compared with untreated cells. Lamp1 is a lysosomal transmembrane protein that has been localized to both multivesicular bodies and lamellar bodies in type 2 cells (22). Electron microscopy demonstrated increased numbers of multivesicular bodies and no normal lamellar bodies (Figure 4), and these structures evolved into lamellar bodies after sufficient mature SP-B accumulated (Figure 5), indicating that the inhibited cleavage event occurred within multivesicular bodies. Thus, our studies show that the terminal cleavage of the aminoterminus remnant of SP-B resulted from the action of a cysteine protease within multivesicular bodies.



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  		Figure 8. Schematic diagram of SP-B processing and inhibition of E-64. The diagram depicts the multistep processing of SP-B and subcellular locations of the various intermediates generated. Based upon the data presented, cysteine protease inhibition occurs before the release of mature SP-B8, resulting in accumulation of the intermediate SP-B9 in multivesicular bodies.

 
The family of cysteine proteases is large and includes both lysosomal and nonlysosomal proteases (21). Because SP-B processing has been localized within lysosomal/pre-lamellar body compartments, specifically the multivesicular body, we focused on the family of lysosomal cysteine proteases. The largest group of lysosomal cysteine proteases is the Cathepsin family, although not all cathepsins are cysteine proteases. Of the 14 well-characterized cysteine protease Cathepsins (specifically, B, C, F, H, K, L, M, O, P, Q, S, W, X, and Z), many could be excluded from consideration. Knockout models for Cathepsins B, C, K, L, and S do not display pulmonary pathology (30–34). Tissue surveys by Western or Northern blot analysis have shown that Cathepsins F, W, M, and Q are also absent from adult lung tissue (35–38). This narrowed the list of candidate proteases to Cathepsins P and Z (which share 99% DNA sequence homology), H, O, V, and X, all of which have been described previously in adult lung tissue (24–27, 39, 40) and/or specifically in alveolar type 2 cells (14, 16).

Our recent report of microarray analysis of in vitro differentiation of type 2 cells (18) showed a 2.7-fold induction of Cathepsin H and no significant expression of Cathepsin O in type 2 cells; Cathepsins P, Z, V, and X were not represented on the arrays used. We have now demonstrated by multiplex RT-PCR no expression of Cathepsins O, P, Z, V, and X in second trimester human fetal lung epithelial cells, and inducible expression of only Cathepsin H during differentiation of type 2 cells, suggesting that Cathepsin H is the source of cysteine protease activity in our studies. Prior studies demonstrating these cathepsins in lung tissue may have been confounded by the presence of alveolar macrophages or other cells containing cysteine proteases.

The advantages of our model system include the enrichment of type 2 cells during isolation and the absence of contaminating macrophages in fetal lung tissue. Cathepsin H displayed a similar time course of expression as SP-B during type 2 cell differentiation and localized with SP-B in multivesicular body, composite bodies, and lamellar bodies within type 2 cells by immunoelectron microscopy. Furthermore, Cathepsin H activity in type 2 cells, as determined by cleavage of the specific substrate Arg-AMC, was inhibited by the addition of E-64 to the culture media. We therefore conclude that the most likely cysteine protease participating in SP-B processing and inhibited by E-64 was Cathepsin H.

Two additional observations from protease inhibition require further investigation: the presence of a second intermediate of ~ 12 kD in pulse chase studies and the partial inhibition of this step in SP-B processing. Cathepsin H is a cysteine protease with unique aminopeptidase activity (20, 41). It behaves as both an endopeptidase, like Cathepsins B, L, and K, but also as an aminopeptidase (42, 43). Cathepsin H has preference for cleavages proximal to amino acids with bulky side chains, such as proline and phenylalanine (41, 44). Mature human SP-B amino acid sequence starts with the amino acids Phe201-Pro-Ile-Pro (reviewed in Ref. 1), fulfilling the sequence criteria for Cathepsin H cleavage, but the propeptide aminoterminus flanking the mature SP-B sequence also contains prolines in close proximity, i.e., Pro189-Gly-Pro and Pro179-Val-Pro. Alternative cleavages at these sites could generate 9- to 12-kD proteins detectable by our antisera. In fact, the region of proSP-B from Cys143 to Gln200, lying distal to the previously described saposin-like structural arrangement of the aminoterminus, contains 13 prolines often in Pro-X-Pro configuration. It is therefore reasonable to speculate that additional cleavage sites for Cathepsin H proteolysis are contained within this region and that additional intermediates may exist that as yet have not been detected. Additional studies will be required to confirm this, including sequencing of these 9- and 12-kD intermediates from E-64–treated cells.

Despite the use of 100 µM E-64, we consistently observed gradual accumulation of SP-B8 due to incomplete enzyme inhibition. In our enzyme activity assays, there was a wider variation in activity at 5 d compared with 3 d, suggesting that residual activity remained that could explain the accumulation of SP-B8. Although E-64 is highly specific for cysteine proteases and a very effective inhibitor of Cathepsin B, it is not as effective an inhibitor of Cathepsin H, as indicated by a higher IC50 and lower KI, compared with other cysteine proteases of the Cathepsin B class (21). E-64 is also a polar molecule that crosses membranes poorly and has been shown in other cell types to be taken up by endocytosis (45). Although this was advantageous for our experiments because the endocytic pathway of type 2 cell intersects with both multivesicular bodies and lamellar bodies (46–48), it may also explain the incomplete inhibition of SP-B processing in our studies, especially over time. We also observed a dose- and time-dependent increase the expression of the 22-kD form of Cathepsin H, which also has enzymatic activity. Studies aimed at specifically eliminating Cathepsin H expression to further clarify these issues are in progress.

There has been much speculation over the rationale for multistep processing of SP-B. The most popular theory is that multistep processing allows for caging of this extremely hydrophobic, fusogenic protein. The secretory pathway and specifically the lipid-rich lamellar body provide the appropriate organellar environment for liberating the mature protein. Although the mechanisms governing lamellar body formation, concentration of surfactant-specific lipids, and accumulation of surfactant protein components are poorly understood, current theories of lamellar body genesis (2, 49) and experimental evidence of SP-C proteolytic processing (5, 50) suggest that the lamellar body is derived from maturation of multivesicular bodies enriched in surfactant phospholipid. The interior vesicles of multivesicular bodies contain the integral membrane protein intermediate of proSP-C oriented so that the aminoterminus is within the lumen of these multivesicular body vesicles. Activation of the fusogenic properties of SP-B would then allow for disruption of the small vesicles and exposure of the proSP-C aminoterminus to proteases within the multivesicular body matrix. Our model system provides a powerful tool for dissecting the process of lamellar body genesis. There are no cell lines that faithfully duplicate the processing of SP-B and the formation of lamellar bodies. In addition, adult type 2 cells do not maintain their differentiated phenotype in culture to permit manipulation of lamellar body genesis, and it is difficult to distinguish lamellar bodies generated de novo from pre-existing lamellar bodies. In beginning with a naive cell devoid of lamellar bodies, we are able to manipulate and closely monitor this process in a manner not possible with transgenic fetal animals.

Our current data and prior publications establish that liberation of mature SP-B is required to facilitate the process of lamellar body genesis. Cathepsin H has been localized to the matrix surrounding the small vesicles of multivesicular bodies, and has been shown in vitro to cleave the aminoterminus of proSP-C (14). We previously showed that the distal step in SP-B processing occurred in a prelamellar body compartment (6). We have now shown ultrastructural localization of both SP-B and Cathepsin H in multivesicular bodies, composite bodies, and lamellar bodies. Furthermore, inhibition of the distal processing step by E-64 impaired lamellar body formation, favoring accumulation of multivesicular bodies, and altered cleavage of the SP-C aminoterminus. Large collections of electron-dense material coalesced in membrane-limited structures within E-64–treated cells were suggestive of attempted formation of lamellae, which may reflect the presence of some SP-B8 detectable by Western immunoblotting in E-64–treated cells. In fact, with increasing time in culture, we saw increasing numbers of lamellar bodies in E-64–treated cells in association with an escape from E-64 inhibition of SP-B processing (Figures 2 and 5), suggesting that a critical mass of SP-B8 is required for lamellar body genesis. These results correlate well with prior in vitro characterization of the dose dependency of lipid membrane fusion on SP-B8 concentration (51). Because sequential processing of SP-B followed by SP-C is achieved through the accessibility of their protein intermediates to Cathepsin H, inhibition of this SP-B processing step would impair lysis of small vesicles within multivesicular bodies and thus inhibit cleavage of SP-C intermediates. Alternatively, aberrant SP-C processing might be due simply to E-64 inhibition of Cathepsin H activity.

In conclusion, our data are consistent with participation of a cysteine protease in the processing of SP-B and lamellar body genesis. We have demonstrated for the first time that inhibition of SP-B processing produces a type 2 cell phenotype similar to that of inherited SP-B deficiency, including absent lamellar bodies, increased numbers of multivesicular bodies, and aberrant processing of SP-C. We have also shown that the most likely candidate cysteine protease is Cathepsin H. Although our data are compelling, they are not yet conclusive. Given the number of cysteine proteases and the broad inhibition profile of E-64, it is possible that an as yet unknown, type 2 cell–enriched cysteine protease is being inhibited by E-64. We must also consider the possibility that a cysteine protease is critical for some other proteolytic cleavage of an as yet unidentified protease or protein necessary for SP-B processing or trafficking or for lamellar body genesis. Nonetheless, the induction of cathepsin H during type 2 cell maturation, and its ultrastructural localization with SP-B, suggest a critical role for cathepsin H during lung development and maturation.


   Acknowledgments
 
The authors acknowledge the assistance of Linda Gonzales and Sree Angampalli in the preparation of type 2 cells, Marlene Strayer in the design of multiplex PCR, Neelima Shah and the Biomedical Imaging Core of the University of Pennsylvania School of Medicine, and Dr. Philip Ballard for editorial assistance. These studies were supported by NIH HL59959 (S.H.G.), HL 19737 (M.F.B.), and P50 HL56401 (S.H.G., M.F.B.).

Received in original form July 10, 2002

Received in final form August 6, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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