Secretoglobin (SCGB) 3A2, also called UGRP1 (uteroglobin-related protein 1), is a member of the SCGB gene superfamily, consisting of cytokine-like secretory proteins of small molecular weight (~10 kDa) [
]. SCGB3A2 is highly expressed in airway Club cells, while low expression is observed at the growing tips of bronchi around embryonic day (E) 11.5 of mouse gestation [
]. SCGB3A2 plays a role in lung development as demonstrated using
embryonic lung organ cultures in the presence of SCGB3A2, and
by administration of SCGB3A2 to pregnant female mice, followed by examination of pre-term pups [
]. Recently, SCGB3A2 was shown to be an early marker for Club cells (formerly called Clara cells) in conjunction with Notch signaling. Expression of SCGB3A2 appears earlier than SCGB1A1, the founding member of the SCGB gene superfamily, also called Club cell secretory protein (CCSP) or Club cell 10 kDa protein (CC10). SCGB1A1 was previously thought to be the only definitive marker for Club cells [
SCGB1A1 is the most well-characterized SCGB protein, exhibiting anti-inflammatory, anti-fibrotic, and immunomodulatory functions [
]. SCGB1A1 possesses phospholipase A
) inhibitory activity, which was thought to be at least partially responsible for the anti-inflammatory and immunomodulatory activity of SCGB1A1 [
]. SCGB1A1 also exhibits tumor suppressor activity as demonstrated by decreased invasiveness of human lung adenocarcinoma-derived A549 cells
], and the increased incidence of tumors in chemical carcinogenesis bioassay and the increased lung metastasis of B16F10 melanoma cells using
SCGB3A2 also exhibits anti-inflammatory and anti-fibrotic activities [
]; the anti-inflammatory function was originally suggested by the fact that
mRNA levels were reduced in the lungs of fungal-induced allergic inflammation model mice, which was almost restored by dexamethasone treatment [
]. Further, in the ovalbumin (OVA)-induced airway inflammation model mice, reduced levels of lung
mRNA were inversely correlated with the increased levels of proinflammatory cytokines, IL-5 and IL-9 in bronchoalveolar lavage fluid (BALF) [
]. When OVA-induced airway inflammation model mice were intranasally administered recombinant adenovirus expressing SCGB3A2 before OVA challenge, OVA-induced airway inflammation was suppressed [
-null mice when subjected to OVA-inflammation model, showed exacerbated airway inflammation [
]. On the other hand, anti-fibrotic activity of SCGB3A2 was demonstrated by using a bleomycin (BLM)-induced mouse pulmonary fibrosis model [
]. This activity was through SCGB3A2-induced STAT1 phosphorylation and increased expression of inhibitory SMAD7, which inhibited the TGFβ signaling, resulting in reduced expression of various collagen genes and development of fibrosis [
] SCGB3A2 can also be used as a marker for pulmonary carcinomas in mice and humans [
]. Taken together, SCGB3A2 has multiple biological activities, playing a role in lung homeostasis and function, and influencing various lung diseases. Whether SCGB3A2 possesses any other activities and the mechanisms for these activities have yet to be determined.
To understand the role of SCGB3A2 in lung homeostasis and diseases, an
Scgb3a2-transgenic mouse was established that over-expresses SCGB3A2 in a lung-specific fashion under the control of the human surfactant protein C (SP-C) gene promoter. Detailed characterization demonstrated that the lungs of
Scgb3a2-transgenic mice were histologically and functionally normal as compared to wild-type. When subjected to the BLM-induced pulmonary fibrosis model, however, they exhibited increased fibrosis at 3 weeks post-BLM administration, which was more quickly resolved by 6 weeks as compared to wild-type mice. These results demonstrate that SCGB3A2 has anti-fibrotic activity and suggest a potential use of SCGB3A2 as a therapeutic agent in treating lung fibrosis.
An expression plasmid with the human SP-C gene promoter (3.7 kb) cloned into pUC18 vector with SV40 small T intron and poly A (0.4 kb) (SPC3.7-SV40-pUC18), was provided from Dr. Jeffrey Whitsett (University of Cincinnati, OH) [
]. The mouse
cDNA that covers the entire protein coding sequence (50–427) was inserted into the SPC3.7-SV40-pUC18 plasmid. The resultant SPC3.7-SCGB3A2-SV40-pUC18 was double-digested with restriction enzymes,
I. The linearized SPC3.7-SCGB3A2-SV40 fragment was purified before microinjection into pronuclei of C57BL/6NCr mouse eggs. Production of
-transgenic mouse lines was confirmed by Southern blotting of genomic DNAs isolated from clipped mouse-tails.
Total RNA (3 μg) isolated from adult lungs of wild type and
Scgb3a2-transgenic mice was electrophoresed on 1 % agarose gel containing 0.22 M formaldehyde and transferred onto nitrocellulose membrane (Immobilon-Ny+, Millipore, Billerica, MA). Filters were hybridized with SCGB3A2 probe obtained from
Eco RI digestion of the SCGB3A2/pCR2.1 construct. Hybridization was performed in Perfect Hybridization solution (GE Healthcare Life Sciences, Piscataway, NJ) at 68 °C overnight. The membrane was washed twice with 2 x SSC containing 0.1 % SDS at 68 °C for 30 min, followed by exposure to a phosphoimager screen (Storm 840, GE Healthcare Life Sciences, Piscataway, NJ). Data processing was carried out using ImageQuant TL 2005 software (GE Healthcare Life Sciences).
Lung from wild type and transgenic mice were frozen and crushed in 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF) with protein inhibitor cocktail (Roche Applied Science, Branford, CT). Protein concentrations were determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin (BSA) as standard, and samples were mixed with equal volume of 2 x SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4 % SDS, 20 % glycerol, 0.1 % mercaptoethanol). Ten microgram of sample was applied in each well of 20 % polyacrylamide gel and was run with running buffer of 50 mM Tris, 384 mM glycine, 2 % SDS. After electrophoresis, protein was transferred to Polyvinylidene fluoride (PVDF) membrane using a tank transfer system (Mini Trans-Blot Cell, Bio-Rad) with blotting buffer (50 mM Tris, 40 mM glycine, 20 % methanol) and electric field of 30 V for 6 h. To visualize SCGB3A2 band, PVDF membrane was treated as follows; 1 h blocking with PBST (phosphate buffered saline + 0.05 % Tween 20) +5 % BSA, 3 h incubation with 0.2 μg/ml polyclonal rabbit anti-SCGB3A2 IgG in PBST + 5 % BSA, PBST wash 3 times, incubation with 0.1 μl/ml horseradish peroxidase (HRP)-linked anti-Rabbit IgG F(ab’) fragment (GE Healthcare, NA9340) in PBST + 5 % BSA, and PBST wash 3 times before ECL plus (PerkinElmer, Waltham, MA) reaction. Polyclonal rabbit anti-SCGB3A2 antibody was produced as previously described [
]. The anti-SCGB3A2 IgG was purified using the Montage antibody purification kit (EMD Millipore, Billerica, MA) and used for all experiments. Labeled proteins were visualized using a SuperSignal West Pico Substrate (Thermo Scientific, Rockford, IL), and signals were detected using FluoChem HD2 System (ProteinSimple, San Jose, CA).
All animal studies were carried out after approval by the National Cancer Institute Animal Care and Use Committee. Mouse embryonic lungs were collected from wild-type and
-transgenic pregnant females at various embryonic days (E). Noon of the day on which a vaginal plug was found was considered as E 0.5. Branching degree of
cultured embryonic lungs were counted after 3 days of culture as previously described [
]. Breathing score assessment was performed as previously described [
] according to the criteria described by Ozdemir et al. [
]; 0, no breathing; 1, gasping; 2, gasping/labored breathing; 3, labored breathing; 4, labored breathing/unlabored breathing; 5, unlabored breathing. For the BLM-induced pulmonary fibrosis model, mice of approximately 8 weeks old (at least 5 mice per group) were intratracheally intubated and dosed with BLM (1.2 U/kg) at day 0. Mice were killed on 3, 6, and 9 weeks after BLM intubation, and bronchoalveolar lavage (BAL) fluids obtained by lavaging lungs with 1 mL PBS [
]. The collected BAL fluids were used for counting and differentiating inflammatory cell numbers with Cytospin 4 (Thermo Scientific). PBS-treated mice killed at 3 weeks were used as normal control. Experiments were repeated more than 2 times, and the combined data points were used for analysis.
SCGB3A2 protein levels were determined by ELISA as previously described [
]. Briefly, samples diluted with a coating solution (500 mM bicarbonate buffer, pH 9.6), were applied onto each well of 96-well plates and the plates were incubated at 4 °C overnight. Calibration curves were constructed with twelve points by serially diluting a solution of recombinant mouse SCGB3A2 (1 μg/ml). The plates were washed four times with washing solution (PBS, pH 7.4 containing 0.5 % of Tween), followed by addition of blocking buffer (PBS, pH 7.4 containing 1 % of BSA) to each well. After incubation for 2 h at 37 °C, the plates were washed four times with the washing solution. Purified anti-mouse SCGB3A2 IgG [
] was applied to each well and the plates were incubated for 4 h at 37 or 4 °C overnight. The plates were washed seven times with the washing buffer. One hundred μl of ECL anti-rabbit IgG HRP-linked F(ab’) fragment (from donkey) was added to each well and the plates were placed at 37 °C for 2 h. After further washing, the amount of SCGB3A2 was determined by addition of 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma) and was read at 450 nm after stopping the reaction by adding 1 N HCl.
Lung histological analysis
Lungs were fixed in 4 % paraformaldehyde for one day at 4 °C, dehydrated, and embedded in paraffin. Lung tissues were sectioned at 4 μm and stained with hematoxyline and eosin (H&E). For immunohistochemistry, sections were at first rinsed with 0.05 % Triton-X 100 in PBS, and non-specific binding sites were blocked using 10 % normal goat serum in PBS containing 0.05 % Tween 20. Epitope retrieval was carried out using autoclave (5 min in citrate buffer, pH 6.0 or 1X TE, pH 9.0). After cooling to room temperature, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-mouse SCGB3A2 [
] or anti-pro-surfactant protein-C (Seven Hills Bioreagents, Cincinnati, OH) primary antibodies. The sections were rinsed in distilled water, followed by treating with HRP-conjugated goat anti-rabbit IgG using the ABC method with a commercially available kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instruction. Immunovisualization was carried out with 3, 3′-diaminobenzidine as substrate (Sigma, St Louis, MO), and counterstained with hematoxylin.
For double immunofluorescence labeling of adult lungs using two primary antibodies from the same species, the sections were first incubated with anti-SCGB3A2 antibody (1:1000) at 4 °C overnight, followed by labeled goat anti-rabbit IgG (Alexa Fluor 594 or 488, 1:200, Life Technology) as the secondary antibody for 1 h at room temperature. The sections were then incubated with 5 % rabbit serum for 1 h at room temperature, followed by incubation with unconjugated Fab Fragment goat anti-rabbit IgG for 1 h at room temperature. The sections were finally incubated with the secondary primary antibody, rabbit anti-pro-SP-C antibody (1:500), followed by labeled goat anti-rabbit IgG (Alexa Fluor 488 or 594, 1:200, Life Technology). Cell nuclei were identified by counterstaining with 4,6-diamino-2-phenylindolyl-dihydrochloride (DAPI, Life Technology). Fluorescence images were obtained and processed using a Zeiss 780 laser-scanning confocal microscope. Matching confocal planes were analyzed in all co-localization studies.
Severity of fibrosis was quantified from H&E stained entire lungs using the Ashcroft scoring system [
]. The degree of fibrosis was graded from 0 (normal lung) to 8 (severe distortion of structure, large fibrous areas, and honeycomb lesions). The mean score from all fields (magnification X200, average 30 fields/animal) was taken as the fibrosis score.
Quantitation of hydroxyproline content in lung
Hydroxyproline content was measured using hydroxyproline assay kit from Biovision (Milpitas, CA) according to the manufacture’s instruction with slight modification. In brief, whole lungs were homogenized in dH
2O, using 100 μl H
2O for every 10 mg of tissue. To 100 μl of tissue homogenate, 200 μl concentrated HCl (6 N) was added in a pressure-tight, teflon capped vial, and the mixture was hydrolyzed at 120 °C for 3 h, followed by filtration through a 45 μm syringe filter (Millipore, Bedford, MA). Ten μl of hydrolyzed sample was transferred to a 96-well plate and was evaporated to dryness under vacuum, to which 100 μl Chloramine T reagent was added per well. After incubation at room temperature for 5 min, 100 μl p-dimethylaminobenzaldehyde reagent was added to each well and further incubated for 90 min at 60 °C. Absorbance was measured at 560 nm in a microplate reader (SpectroMax Plus384, Molecular Devices, Sunnyvale, CA).
Lung total cellular RNAs were individually isolated from four mice each of no-treatment wild-type and
Scgb3a2 transgenic mice (approximately 8-weeks old) using RNeasy Mini Kit (Qiagen Science, Maryland, USA). The sample RNAs were labeled with Cy5, while pooled RNAs containing the same amount of RNA from each sample were labeled with Cy3 and used as a reference. Labeling was carried out using CyDye™ Post-Labeling Reactive Dye Pack (GE Healthcare) according to the manufacturer’s instructions. The purified Dye-coupled RNA samples were hybridized to an Agilent Whole Mouse Genome 4X44K oligo microarray kit (Agilent Techonologies, G4122F, Santa Club, CA), and were incubated for 17 h at 65 °C. The slides were washed, dried, and scanned using Agilent G26000 microarray scanner. The data were processed and analyzed by Genespring GX 11.0.2 software package (Agilent Technologies). All effective genes of microarray analysis were submitted to the Gene Expression Omnibus (GEO: ID # GSE47931).
Quantitative RT-PCR analysis
Total RNAs isolated using TRIzol (Lifetechnologies) and digested with DNase I were reverse-transcribed by Superscript II reverse transcriptase (Life Technologies). Quantitative RT-PCR (qRT-PCR) was performed with ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA) using SYBR Green master mixture. The ΔΔ Ct method was used using β-actin or 18S as normalization control. PCR condition used was 50 °C, 2 min and 95 °C, 10 min followed by 95 °C, 15 s and 60 °C, 40 s for 40 cycles with the following primers:
Sftpa (forward) 5′- ACT CCC ATT GTT TGC AGA ATC -3′, (reverse) 5′- AAG GGA GAG CCT GGA GAA AG -3′;
Sftpb (forward) 5′- ACA GCC AGC ACA CCC TTG -3′, (reverse) 5′- TTC TCT GAG CAA CAG CTC CC -3′;
Sftpc (forward) 5′- ATG AGA AGG CGT TTG AGG TG -3′, (reverse) 5′- AGC AAA GAG GTC CTG ATG GA -3′;
Sftpd (forward) 5′- GAG AGC CCC ATA GGT CCT G -3′, (reverse) 5′- GTA GCC CAA CAG AGA ATG GC -3′;
Aqp1 (forward) 5′- TGC AGA GTG CCA ATG ATC TC -3′, (reverse) 5′- GGC ATC ACC TCC TCC CTA GT −3;
Col1a1 (forward) 5′-TAG GCC ATT GTG TAT GCA GC-3′, (reverse) 5′- ACA TGT TCA GCT TTG TGG ACC-3′;
Col3a1 (forward) 5′-TAG GAC TGA CCA AGG TGG CT-3′, (reverse) 5′- GGA ACC TGG TTT CTT CTC ACC-3′;
Col4a1 (forward) 5′-CAC ATT TTC CAC AGC CAG AG-3′, (reverse) 5′- GTC TGG CTT CTG CTG CTC TT-3′;
Col5a2 (forward) 5′-CAT GGA GAA GGT TTC CAA ATG-3′, (reverse) 5′- AAA GCC CAG GAA CAA GAG AA-3′;
Col12a1 (forward) 5′-TGA GGT CTG GGT AAA GGC AA-3′, (reverse) 5′- GTA TGA GGT CAC CGT CCA GG-3′;
Acta2 (forward) 5′-GTT CAG TGG TGC CTC TGT CA-3′, (reverse) 5′-ACT GGG ACG ACA TGG AAA AG-3′;
Ctgf (forward) 5′-GCT TGG CGA TTT TAG GTG TC-3′, (reverse) 5′-CAG ACT GGA GAA GCA GAG CC-3′;
Mmp2 (forward) 5′-GGG GTC CAT TTT CTT CTT CA-3′, (reverse) 5′-CCA GCA AGT AGA TGC TGC CT-3′;
Mmp12 (forward) 5′-TTT GGA TTA TTG GAA TGC TGC-3′, (reverse) 5′-ATG AGG CAG AAA CGT GGA CT-3′; β-actin (forward) 5′-ATG GAG GGG AAT ACA GCC C-3′, (reverse) 5′-TTC TTT GCA GCT CCT TCG TT-3′; and 18S (forward) 5′- CGC GGT TCT ATT TTG TTG GT-3′, (reverse) 5′- AGT CGG CAT CGT TTA TGG TC-3′.
All animal studies were carried out at least twice, each using at least 5–6 mice per group. Statistical analysis was carried out between wild-type and transgenic mice of various ages using a student’s
P < 0.05 was considered significant.
This study describes the establishment of an
-transgenic mouse line that expresses SCGB3A2 in a lung-specific fashion under regulation of the human SP-C gene promoter. Since SP-C is a marker for alveolar type II cells [
], the promoter for this gene was used for transgenic over-expression of SCGB3A2 in the alveolar areas because in a previous study using the BLM-induced pulmonary fibrosis model, pulmonary fibrosis was reduced by intravenous administration of SCGB3A2 [
]. In the latter case, it is likely that SCGB3A2, an airway-specific protein, reached the alveolar areas as well as airways through blood circulation at similar levels, resulting in the suppression of fibrosis. Thus, we hypothesized that this situation may be better obtained by the use of type II-specific over-expression of SCGB3A2. Further, chose not to use the tetracycline inducible system because it is not known whether and/or how doxycycline affects formation and/or resolution of BLM-induced pulmonary fibrosis, in an otherwise well-established BLM-induced pulmonary fibrosis model.
SCGB3A2 expression faithfully mimicked the expression pattern of the lung specific SP-C gene. Over-expression of SCGB3A2 in transgenic mouse lungs was confirmed by qRT-PCR for mRNA and Western blotting for protein using lung tissues, and levels in BALF by ELISA. The difference in mRNA expression levels between wild-type and transgenic mouse lungs was observed as early as E14.5. The SCGB3A2 mRNA and protein levels in lung were highest at around two weeks of age. Immunohistochemistry further demonstrated expression of SCGB3A2 in airway epithelial cells, the endogenous expression site for SCGB3A2 as well as type II cells, the specific site for SP-C expression [
]. Since alveolar type II cells constitute ~15 % of the peripheral lung cells [
], the expression of SCGB3A2 in type II cells is likely to be a main contributor to over-expression of SCGB3A2 in the transgenic lung. Two different sizes of
mRNAs were observed by Northern blotting for
-transgenic mice that were larger than that of endogenously expressed
. We do not know the exact reason for this phenomenon. It could be due to longer polyA tails present in the transgene-derived mRNA. Alternatively, since the transgene construct contains a
cDNA that does not have an intron, the mRNA processing may have been affected, resulting in a larger mRNA. The way the transgene was inserted to a chromosome might also have affected the size of
-transgenic lungs exhibited no phenotypes with normal lung function, they showed exacerbated fibrosis at 3 weeks after subjected to the BLM-induced pulmonary fibrosis model as determined by lung histology, hydroxyproline content, inflammatory cell numbers, collagen gene expression, and inflammatory cytokine levels. Microarray analysis revealed that this was due to altered expression of genes caused by ectopic overexpression of SCGB3A2 that are involved in inflammation, cell dynamics, and/or cell trafficking-related functions as compared to wild-type. These changes in gene expression patterns may have affected metabolism and homeostasis of the transgenic lungs, which rendered the transgenic lungs more susceptible to BLM challenge. However the changes may be too subtle to affect lung homeostasis without challenge since we did not detect any gross metabolic or abnormal inflammatory phenotypes in the transgenic mice. Of most interest is that after 3 weeks, the fibrosis of
-transgenic lungs quickly resolved as compared with wild-type lungs. BLM-induced pulmonary fibrosis is known to partially resolve after weeks of BLM treatment due to unknown reasons [
]. Previously we demonstrated that SCGB3A2 exhibits anti-fibrotic activity [
]. The anti-fibrotic activity of SCGB3A2 was due to suppression of the TGFβ-induced differentiation of fibroblasts to myofibroblasts, a hallmark of the fibrogenic process through increased phosphorylation of STAT1 and expression of SMAD7, and decreased phosphorylation of SMAD2 and SMAD3 [
]. The BLM-induced injury includes damage to and involvement of alveolar and bronchial epithelium, and decreased SCGB1A1 expression in the airway epithelial cells after BLM treatment was described following BLM [
]. In the current study, SCGB3A2 overexpression predisposed mice to a more severe phenotype 3 weeks post-BLM, at which time SCGB3A2 expression was almost at the level of wild-type mice, suggesting that changes in gene expression patterns in transgenic mice predominate in determination of the phenotype. However by 9 weeks post-BLM, the expression of SCGB3A2 in transgenic lungs recovered to levels found in pre-BLM lungs, which was significantly higher than that of wild-type lungs. Ectopic expression of SP-C was also observed in the bronchial epithelial cells, which may have contributed to the more rapid increase in SCGB3A2 expression in the transgenic lungs. Previously it was shown that after BLM administration, SP-C is co-expressed in bronchial epithelial cells with SCGB1A1, the marker for Club cells that is the site for SCGB3A2 expression [
]. The temporary SP-C-positive, SCGB1A1-positive cells were suggested to eventually differentiate into type II cells during the repair after severe pulmonary injury [
]. In the current study, the resolution of fibrosis coincided with the rapid increase of SCGB3A2 expression in lungs of
-transgenic mice, suggesting the anti-fibrotic activity of SCGB3A2. How SCGB3A2 promotes natural resolution of BLM-induced pulmonary fibrosis requires further studies.
In the single dose BLM model, pulmonary fibrosis develops through sequential events after BLM administration; first in inflammation phase (≤7 days), followed by fibrosis phase (≥7 days) [
]. Many compounds were reported as anti-fibrotic agents, however most of them (over 220 compounds) were given ≤7 days of BLM administration and thus considered as preventive agents. Only a handful compounds were administered in the fibrosis phase as therapeutic agents. The present studies demonstrated that expression of SCGB3A2 markedly increased in transgenic mice after the severity of fibrosis reached peak levels, and thus the situation may resemble that of SCGB3A2 being administered in the therapeutic phase, which likely resulted in the rapid decrease of fibrosis. These results are in good agreement with the previous reports using BLM-induced pulmonary fibrosis model mice with intravenously administered SCGB3A2 that SCGB3A2 possesses anti-fibrotic activity and may be used as a therapeutic agent in treatment of pulmonary fibrosis [
We thank Jeffrey Whitsett (Cincinnati, OH) for the human SP-C gene promoter plasmid and Poonam Mannan, Langston Lim, and Susan H. Garfield (CCR Confocal Microscopy Core Facility, Laboratory of Cancer Biology and Genetics, NCI) for their help in confocal microscopy. This study was funded by the Intramural Research Program of the National Cancer Institute.
The authors declare that they have no competing interests.
YC conceived of the study, designed the experiments, characterized
Scgb3a2-transgenic mouse lungs, carried out BLM study, and wrote a draft of the manuscript. TT established and characterized the
Scgb3a2-transgenic mouse line. MY, RK, TK participated in characterization of the transgenic mouse lungs. MY, RK, MO carried out immunohistochemistry/immunofluorescence studies, and analyzed the data. HA carried out electron microscopy analysis. WM intellectually and technically contributed to morphometric analysis of mouse lungs, AG carried out characterization of mouse embryonic lungs, SK designed and integrated this study, wrote and revised the manuscript. All authors read and approved the final manuscript.