Surfactant proteins maintain lung function through their actions to reduce alveolar surface tension and control of innate immune responses in the lung. The ubiquitin proteasome pathway is responsible for the degradation of majority of intracellular proteins in eukaryotic cells, and proteasome dysfunction has been linked to the development of neurodegenerative, cardiac, and other diseases. Proteasome function is impaired in interstitial lung diseases associated with surfactant protein C (SP-C) mutation mapping to the BRICHOS domain located in the proSP-C protein. In this study we determined the effects of proteasome inhibition on surfactant protein expression in H441 and MLE-12 lung epithelial cells to understand the relationship between proteasome dysfunction and surfactant protein gene expression. Proteasome inhibitors lactacystin and MG132 reduced the levels of SP-A, SP-B, and SP-C mRNAs in a concentration-dependent manner in H441 and MLE-12 cells. In H441 cells, lactacystin and MG132 inhibition of SP-B mRNA was associated with similar decreases in SP-B protein, and the inhibition was due to inhibition of gene transcription. Proteasome inhibitors decreased thyroid transcription factor-1 (TTF-1)/Nkx2.1 DNA binding activity, and the reduced TTF-1 DNA binding activity was due to reduced expression levels of TTF-1 protein. These data indicated that the ubiquitin proteasome pathway is essential for the maintenance of surfactant protein gene expression and that disruption of this pathway inhibits surfactant protein gene expression via reduced expression of TTF-1 protein.
- interstitial lung diseases
- lung injury
- thyroid transcription factor-1
surfactant proteins play diverse roles in influencing the biophysical properties and the physiological function of pulmonary surfactant. To date, four distinct surfactant proteins termed surfactant protein (SP)-A, SP-B, SP-C, and SP-D have been isolated and characterized (50). SP-A and SP-D are hydrophilic proteins that belong to the collectin family of proteins, whereas SP-B and SP-C are hydrophobic in nature. A large amount of data from in vitro studies and animal models of surfactant protein deficiency have shown that SP-A and SP-D play important roles in the control of innate immune responses and inflammation in the lung (51), whereas SP-B and SP-C are necessary for the maintenance of the surface tension-lowering function of surfactant (49). SP-A, SP-B, and SP-D are expressed by the alveolar type II and bronchiolar (Clara) epithelial cells, whereas SP-C expression is restricted to alveolar type II cells. The expression of SP-A, SP-B, SP-C, and SP-D genes is induced during fetal lung development and is modulated by multiple factors such as glucocorticoids, cAMP, cytokines, and others (5).
Of the different surfactant proteins, SP-B is absolutely required for survival, because complete lack of SP-B as in the case of frame-shift mutation in codon 121 (121ins2) of SP-B gene in humans (35) and genetically engineered SP-B null mice (14) cause death due to respiratory failure. Even partial deficiency of SP-B may result in susceptibility to lung injury, suggesting that optimal SP-B levels are necessary for the maintenance of lung function (13). Adult mice engineered to be SP-B deficient developed respiratory failure within a short span of 4 days, and SP-B deficiency was associated with lung inflammation (20). Restoration of SP-B levels reversed lung dysfunction and inflammation associated with SP-B deficiency. Under conditions of SP-B deficiency, neither SP-C content nor the content or composition of surfactant phospholipids was altered, underscoring the important role for SP-B in the maintenance of lung function. Recently, several cases of interstitial lung diseases have been found to be associated with mutations in the SP-C gene (34). Although mutations are found in all of the five exons, a great majority of the mutations were found within the BRICHOS domain (exons 4 and 5) of the propeptide. Studies in cell culture (32) and transgenic mice (7) have shown that the BRICHOS domain mutation in SP-C causes accumulation of misfolded protein, leading to ER stress, apoptotic cell death, proteasome dysfunction, and disruption of lung development. These cellular abnormalities may be responsible for the development of lung dysfunction in interstitial lung diseases associated with SP-C mutation.
The ubiquitin-proteasome pathway is a highly regulated proteolytic degradation pathway that is involved in the degradation of majority of proteins in eukaryotic cells (19). The multicomplex 26S proteasome, composed of two 19S regulatory complexes and a 20S catalytic core complex, is responsible for the degradation of >90% of proteins including misfolded proteins in the cell. Although in the majority of cases proteasomes degrade proteins into small peptides, in a few cases proteasomal action results in the modulation of protein function, as in the case of NF-κB and yeast proteins SPT23 and MGA2 (38) and nuclear receptors such as progesterone receptor (15), androgen receptor (25), and glucocorticoid receptor (45). Because the ubiquitin-proteasome pathway is involved in the degradation of key cellular proteins involved in proliferation, differentiation, and apoptosis, impairment of this pathway can result in abnormalities of cellular function, leading to the development of diseases. Impairment of the proteasomal function has been implicated in the pathogenesis of neurodegenerative diseases and aging (48), (17), cardiac (37), and other diseases.
Expression of BRICHOS SP-C mutation in A549 lung epithelial cells resulted in many cellular abnormalities, including inhibition of proteasome activity (32), suggesting that impairment of proteasome function may be one of the factors leading to the development of respiratory dysfunction in interstitial lung diseases associated with SP-C mutations. It is possible that proteasome dysfunction in interstitial lung diseases associated with SP-C mutation could cause abnormalities of surfactant contributing to respiratory dysfunction. In this study we tested the proposal that impairment of proteasome activity results in altered expression of surfactant protein gene expression in lung epithelial cells. We studied the effects of lactacystin (16) and MG132 (46), highly specific inhibitors of proteasome activity, on the expression of SP-A, SP-B, and SP-C in H441 and MLE-12 lung epithelial cells. We found that lactacystin and MG132 inhibited SP-A, SP-B, and SP-C mRNAs in a concentration-dependent manner in H441 and MLE-12 cells. After finding that proteasome inhibitors inhibited SP-A, SP-B, and SP-C mRNA levels, we focused on understanding molecular mechanisms that mediate inhibition of SP-B gene expression. We found that lactacystin and MG132 inhibited SP-B gene expression by inhibiting gene transcription and that inhibition of SP-B mRNA levels was associated with inhibition of SP-B protein levels. Inhibition of SP-B gene expression was associated with reduced thyroid transcription factor-1 (TTF-1) DNA binding activity and that reduced TTF-1 DNA binding activity was due to decreased expression of TTF-1. These results indicate that maintenance of proteasome function is necessary for optimal expression of surfactant proteins and suggest that impairment of proteasome function in interstitial lung diseases associated with SP-C mutation may cause inhibition of surfactant protein gene expression, contributing to respiratory dysfunction.
MATERIALS AND METHODS
NCI-H441 cells (American Type Culture Collection HTB-174), a human lung adenocarcinoma cell line with characteristics of bronchiolar (Clara) epithelial cells, were grown on plastic tissue culture dishes in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml). MLE-12 cells (American Type Culture Collection CRL-2110), a SV40 transformed mouse cell line with characteristics of alveolar type II epithelial cells, were grown on plastic tissue culture dishes in HITES medium containing 2.5% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Both cell lines were maintained in a humidified atmosphere of 95% room air and 5% CO2.
Cell viability was assessed by measuring the release of lactate dehydrogenase (LDH) into medium using the CytoTox 96 nonradioactive cytotoxicity assay kit (Promega) according the instructions provided by the manufacturer. A standard curve was generated by measuring total LDH released upon lysis of known numbers of cells, and the percentage of cell death in test samples was determined from the standard curve. Cell viability also was assessed by using the Trypan blue exclusion assay.
Lactacystin and MG 132 were obtained from Calbiochem (La Jolla, CA), and actinomycin D was obtained from Sigma-Aldrich (St. Louis, MO). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA). Bronchoalveolar lavage (BAL) samples from healthy human subjects were kindly provided by Dr. David E. Griffith (Center for Pulmonary and Infectious Diseases Control, University of Texas Health Center at Tyler). The protocol for obtaining lavage was approved by the Human Subjects Committee of the University of Texas Health Center at Tyler.
RNA isolation and Northern blot analysis.
Procedures for the isolation of RNA and Northern blot analysis were as described previously (30). Briefly, total RNA was isolated using the acid-guanidinium thiocyanate-phenol method with TRI reagent (Molecular Research Center, Cincinnati, OH) and quantified by measuring absorbance at 260 nm. Equal amounts of RNAs were separated by electrophoresis on agarose gels (1%) containing 20 mM MOPS and 1.1% formaldehyde, and RNAs were transferred to HybondN+ membranes by capillary action with saline sodium citrate (20× SSC) as the transfer buffer. After transfer, membranes were UV cross-linked and hybridized to 32P-labeled cDNAs encoding human SP-A, SP-B, SP-C, IL-8, tissue factor, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RNA bands were quantified with a PhosphorImager and normalized to 18S rRNA levels to correct for variations in loading and transfer of RNA. The expression of GAPDH mRNA was routinely assessed as an internal control. The human tissue factor cDNA was provided by Dr. Usha Pendurthi (University of Texas Health Center at Tyler).
Cells were rinsed twice with cold phosphate-buffered saline (PBS) and incubated in lysis buffer (50 mM Tris·HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM sodium vanadate, 2.5 mM sodium pyrophosphate, 2 μg/ml leupeptin and aprotinin, 1 mM PMSF, and 15% glycerol) for 15 min on ice. The cell lysate was cleared by centrifugation at 14,000 rpm for 10 min, and the supernatant was used for Western immunoblotting analysis. SDS-PAGE separation and transfer of proteins to membrane were carried out with an XCell II Mini-Cell apparatus (Novex, San Diego, CA) according to the manufacturer's instructions. Equal amounts of cellular protein (50 μg) were separated by SDS-PAGE on 10% Bis-Tris gels with 2-morpholinoethanesulfonic acid running buffer and electrophoretically transferred to polyvinylidene difluoride membranes. Membranes were successively incubated with primary polyclonal SP-B, thyroid-specific enhancer binding protein (T/EBP), Sp1, or actin antibodies at 1:1,000 dilution overnight at 4°C, followed by goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (Cell Signaling) at 1:2,000 dilution for 1 h at room temperature. Protein bands were visualized using the enhanced chemifluorescence detection method (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's instructions. Membranes were scanned with a fluorescence scanning instrument for visualization of protein bands, and the intensity of bands was quantified using Quantity One image acquisition and analysis software (Bio-Rad). Polyclonal rabbit antibodies against human SP-B were obtained from Chemicon International (Temecula, CA), and human actin and Sp1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal rabbit antisera against the amino-terminal portion of rat T/EBP (TTF-1/Nkx2.1) was kindly provided by Dr. Shioko Kimura (National Cancer Institute, Bethesda, MD).
Transient transfection and reporter gene assay.
The construction of luciferase reporter plasmids containing −911/+41, −517/+41, −233/+41, and −80/+41 bp of human SP-B 5′ flanking DNA and plasmid containing four copies of human SP-B TTF-1 DNA binding site linked upstream of basal SP-B promoter (−59/+41 bp) has been described previously (39, 43). Plasmid DNAs were amplified in Escherichia coli Top10 strain (Invitrogen) and purified by anion exchange chromatography (Qiagen). SP-B promoter plasmids were transiently transfected along with pcDNA3.1 (Invitrogen), a β-galactosidase expression plasmid, into cells by liposome-mediated DNA transfer with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After transfection, cells were first incubated overnight and then subjected to treatments. Luciferase and β-galactosidase activities in cell extracts were measured using chemiluminescent assays (Tropix, Bedford, MA; Promega, Madison, WI), and luciferase activities were normalized to cotransfected β-galactosidase activity to correct for variations in transfection efficiency.
Preparation of nuclear extracts and electrophoretic mobility shift analysis.
Methods for the preparation of nuclear extracts (40, 42) and double-stranded oligonucleotides (28) have been described previously. The sense strand sequences of human SP-B and human IL-8 promoter oligonucleotides and those of consensus cAMP-response element (CRE) binding protein (CREB) and GATA oligonucleotides used in mobility shift analysis are as follows: (SP-B) TTF-1/Nkx2.1, 5′-GCACCTGGAGGGCTCTTCAGAGCAA-3′ (−111/−87); (SP-B) hepatocyte nuclear factor (HNF)-3, 5′-GCAAAGACAAACACTGAG-3′ (−90/−73); (SP-B) Sp1, 5′-AGCCCCCACGCCCCGCCCAGCTAT-3′ (−53/−30); (IL-8) AP-1, 5′-AGTGTGATGACTCAGGTTTG-3′ (−127/−120); (IL-8) NF-κB, 5′-AATCGTTAACTTTCCTCTGA-3′ (−81/−72); consensus CREB, 5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′; and consensus GATA, 5′-CACTTGATAACAGAAAGTGATAACTCT-3′. Double-stranded oligonucleotides were 5′-end labeled using [γ-32P]ATP and T4 polynucleotide kinase. Electrophoretic mobility shift analyses (EMSA) were performed as described previously (28) by incubating 0.5–1.0 ng (100,000 cpm) of labeled oligonucleotide with 5 μg of nuclear protein in 20 μl of binding buffer [13 mM HEPES, pH 7.9, containing 13% glycerol, 80 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, and 1 μg of poly(dI-dC) as nonspecific competitor DNA] at 30°C for 20 min. For antibody supershift assay, protein-DNA complex was first formed and then incubated with antibody or antiserum for 20 min at room temperature. After electrophoresis, the gel was dried and exposed to a storage phosphor screen or an X-ray film. Storage phosphor screens were scanned (Bio-Rad Molecular Imager), and the intensities of the bands were quantified using Quantity One image acquisition and analysis software (Bio-Rad). The images of autoradiograms were acquired and the intensities of the bands quantified using Quantity One image acquisition and analysis software (Bio-Rad).
H441 cells were grown on four-chamber Permanox slides (Nalge Nunc International) for 24 h and then treated with or without lactacystin (10 μM) or MG132 (5 μM) for 24 h. Cells were fixed for 1 h at 4°C in PBS containing 4% paraformaldehyde and 0.1% glutaraldehyde. After fixation, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 10% goat serum in PBS for 1 h at room temperature. Cells were incubated overnight at 4°C with rabbit polyclonal T/EBP IgG (10 μg/ml), followed by incubation with rhodamine red-conjugated goat anti-rabbit IgG for 60 min at room temperature. Nuclei were stained with SYTOX green for 5 min according to the manufacturer's instructions. The immunostained cells were observed under a Nikon Eclipse TE2000–5 inverted fluorescent microscope equipped with an UltraVIEW LCI scanning confocal system (PerkinElmer, Boston, MA). Fluorescence images were captured with a digital coupled-charged device camera (Ultra Pix; Hamamatsu Photonics, Hamamatsu, Japan) at ×20 or ×60 optical lens (oil) magnification at room temperature, using 488- and 568-nm excitation laser lines at a resolution of 1,344 × 1,024 × 12. Imaging Suite version 5.0 acquisition and processing software was used for acquisition of the images and to measure the colocalization by determining the correlation coefficient of overlap of the green and red fluorescence. The images were imported into Microsoft PowerPoint for compilation of figures.
Data are shown as means ± SD. In experiments where SP-B mRNA/protein levels were arbitrarily set at 100%, statistical significance was analyzed by one-sample t-test. For other samples, unpaired t-test was used to analyze the statistical significance. One-tailed P values <0.05 were considered significant.
Proteasome inhibitors decrease surfactant protein mRNA levels.
Little is known about the relationship between proteasome function and the surfactant system. Recent studies have shown that SP-C mutations linked to interstitial lung diseases (34) cause inhibition of proteasome activity (32), suggesting that proteasome dysfunction contributes to the development of cellular abnormalities and respiratory dysfunction. It was the objective of this study to understand the relationship between proteasome dysfunction and surfactant protein expression. To determine whether proteasome dysfunction alters surfactant protein gene expression, we studied the effects of proteasome inhibitors on surfactant protein mRNA levels in H441 and MLE-12 cells. H441 cells are of bronchiolar epithelial (Clara) cell origin expressing SP-A, SP-B and SP-D, whereas MLE-12 cells are of alveolar type II cells expressing SP-A, SP-B, and SP-C. Treatment of H441 (Fig. 1) and MLE-12 cells (Fig. 2) resulted in a concentration-dependent inhibition of SP-A, SP-B, and SP-C mRNA levels. To determine whether the inhibitory effects of lactacystin and MG132 are pleiotropic or not, we analyzed their effects on the expression of IL-8, tissue factor, and GAPDH mRNAs. Tissue factor is a cell surface protein that initiates coagulation and is expressed by lung epithelial cells. In contrast to their inhibitory effects on surfactant protein mRNA levels, lactacystin and MG132 caused a concentration-dependent induction of IL-8 and tissue factor mRNA levels and did not alter GAPDH mRNA levels (Fig. 1A). We indirectly assessed the efficacy of the lactacystin and MG132 to inhibit proteasome activity by analyzing their effects on TNF-α activation of NF-κB DNA binding activity in H441 cells. It is well established that the ubiquitin-proteasome pathway plays a crucial role in NF-κB activation by degrading IκBα. Results (data not shown) showed that lactacystin (10 μM) and MG132 (5 μM) effectively blocked NF-κB activation in TNF-α-treated cells, indicating inhibition of proteasome activity.
In H441 cells, lactacystin and MG132 at the concentrations used did not cause lifting of cells or significantly affect 18S rRNA levels, indicating lack of toxicity. In MLE-12 cells, lactacystin did not cause lifting of cells or affect 18S rRNA levels. MG132 at 5 and 10 μM decreased 18S rRNA levels by 25 and 33%, respectively, indicating toxicity. We determined viability of H441 and MLE-12 cells after 24 h of treatment by measuring LDH levels in the cell medium. Spontaneous death in cells in control medium was considered as 0%. Results showed that lactacystin and MG132 had no significant effects on cell viability in H441 cells. At concentrations of 1, 5, and 10 μM, lactacystin caused 0.37 ± 0.64, 2.32 ± 2.7, and 1.2 ± 1.28% cell death, respectively, in H441 cells (means ± SD, n = 4). At concentrations of 1, 5, and 10 μM, MG132 caused 1.22 ± 1.28, 2.57 ± 0.9, and 1.9 ± 2% cell death, respectively, in H441 cells (means ± SD, n = 4). Gross morphological changes in the appearance of H441 cells were observed at 10 μM MG132. MLE-12 cells were found to be more susceptible to cell death than H441 cells, especially at higher concentrations of lactacystin and MG132. At concentrations of 1, 5, and 10 μM, lactacystin caused 0, 4.5 ± 2.9, and 15 ± 1.69% cell death, respectively, in MLE-12 cells (means ± SD). MG132 at concentrations higher than 1 μM was toxic. At concentrations of 1, 5, and 10 μM, MG132 caused 14.7 ± 3.8, 32 ± 4.5, and 53 ± 1.8% cell death, respectively, in MLE-12 cells (means ± SD). Together, our results showed that lactacystin and MG132 inhibited SP-A and SP-B mRNAs in H441 cells without causing toxicity. On the other hand, in MLE-12 cells, lactacystin (10 μM) and MG132 (1 μM) inhibition of SP-B and SP-C mRNA levels was associated with low-level toxicity, suggesting that toxicity alone does not contribute to the inhibition. Other studies have found that proteasome inhibitors, including lactacystin and MG132, did not have significant toxic effects on a variety of lung cells (18, 21).
Proteasome inhibitors decrease SP-B mRNA and SP-B protein levels.
After our initial findings of the inhibition of surfactant protein mRNAs by proteasome inhibitors, we focused our studies on understanding molecular mechanisms that mediate inhibition of SP-B mRNA. We were interested to determine whether lactacystin and MG132 also inhibited dexamethasone induction of SP-B mRNA. Dexamethasone is a strong inducer of surfactant synthesis and SP-B gene expression (5). Results (Fig. 3) showed that as in the case of H441 cells maintained in control medium, lactacystin and MG132 inhibited dexamethasone induction of SP-B mRNA, indicating that proteasome inhibitors have dominant inhibitory effects on SP-B mRNA expression. Time-course effects of the inhibitors showed that lactacystin and MG132 inhibited SP-B mRNA to a significant extent after 12 h and at 24 h of treatment in H441 cells (data not shown).
To determine whether inhibition of SP-B mRNA is associated with inhibition of protein expression, we analyzed the effects of proteasome inhibitors on SP-B levels by immunoblotting in dexamethasone-treated H441 cells. To facilitate identification of SP-B immunoreactive protein in H441 cell lysates, we subjected human BAL and A549 lung cell lysate samples to immunoblotting along with H441 cell lysates. BAL contains significant amounts of surfactant, making it positive for SP-B, whereas A549 cells do not express any of the surfactant proteins, including SP-B. Results (Fig. 4) showed that immunoreactive bands of ∼8 kDa were detected in H441 cell lysates and lung lavage but not in A549 cell lysate. The size of the band detected in H441 cell lysates appeared to be identical to the band detected in the BAL sample. An additional band of molecular weight (Mr) ∼62,000 reactive to SP-B antibody, whose identity remains to be determined, was detected in the BAL sample. The intensity of the ∼8-kDa band was significantly increased in dexamethasone-treated cells but was reduced to control levels in cells treated with dexamethasone in the presence of lactacystin or MG132. The changes in the intensities of the ∼8-kDa band closely parallel those for SP-B mRNA in H441 cells treated with dexamethasone in the presence of lactacystin or MG132. Together, these data strongly identify the 8-kDa band detected in immunoblots of H441 cells as the SP-B mature protein. A band of Mr ∼42,000 whose intensity was much weaker than the Mr 8,000 band was also detected in H441 cell lysates, and its levels were altered in a manner similar to the Mr 8,000 band, suggesting that it could be the SP-B precursor. Immunohistochemical experiments (data not shown) showed that in cells treated with dexamethasone in the presence of lactacystin and MG132, staining for SP-B was reduced to levels found in control cells, confirming the results of the immunoblotting experiments.
Our results on the identification of mature SP-B protein differ from previously published studies (36), which could not detect processing of SP-B precursor to mature SP-B protein of Mr 8,000 in H441 cells. The reasons for the differences between our study and the previously published studies on the identification of mature SP-B protein in H441 cells are not clear. A major difference between our study and the previously published studies is that whereas our studies investigated changes in the intracellular SP-B levels, the published studies were focused mostly on the secreted forms of SP-B. The low levels of SP-B and the different antibodies used for detection also could have contributed to the observed discrepancy.
Proteasome inhibitors inhibit SP-B mRNA at the transcriptional level.
To gain insights into the mechanisms of inhibition, we determined the effects of the transcriptional inhibitor actinomycin D on lactacystin and MG132 inhibition of SP-B mRNA. Results (Fig. 5) showed that prior blockage of transcription with actinomycin D significantly reversed lactacystin and MG132 inhibition of SP-B mRNA, indicating that continued transcription and transcriptional mechanisms are necessary for the inhibition. The use of another transcriptional inhibitor, 5,6-dichloro-1-b-d-ribofuranozyl-benzimidazole, also produced similar results (data not shown) consistent with the involvement of transcriptional mechanisms in the inhibition. We further studied the role of transcription in the inhibition of SP-B gene expression by investigating the effects of lactacystin and MG132 on SP-B promoter activity in H441 and MLE-12 cells. Transient transfection experiments showed that lactacystin and MG132 caused a concentration-dependent inhibition of luciferase expression from the human SP-B promoter fragment containing −911/+41 bp of 5′ flanking DNA (Fig. 6). Lactacystin was less effective than MG132 to inhibit SP-B promoter activity in MLE-12 cells.
SP-B promoter −233/+41 contains DNA elements necessary for lactacystin and MG132 inhibition.
To map SP-B genomic region(s) responsible for lactacystin and MG132 inhibition of promoter activity, we determined the effects of lactacystin and MG132 on luciferase expression from SP-B genomic fragments containing deletions at the 5′ end. Results (Fig. 7) showed that deletion of the SP-B 5′ flanking DNA from −991 to −233 bp had no effect on lactacystin and MG132 inhibition of SP-B promoter, and further deletion to −80 bp rendered the promoter somewhat less sensitive to inhibition. Deletion of SP-B 5′ flanking DNA caused gradual loss of SP-B promoter activity with the greatest effect on deletion from −233 to −80 bp. The −80/+41-bp region had ∼10% of the activity of the −233/+41-bp region.
Deletion mapping experiments indicated that lactacystin and MG132 inhibited SP-B promoter containing −233/+41-bp DNA to the same extent as the −911/+41-bp DNA, indicating that the −233/+41-bp fragment contains cis-DNA elements necessary for inhibition. Previous studies from our laboratory and others have identified TTF-1/Nkx2.1, HNF-3, Sp1/Sp3, and ATF/CRE DNA elements to be functionally important for the activity of SP-B promoter (3, 6, 29). In particular, in the human SP-B promoter, HNF-3 and TTF-1/Nkx2.1 are necessary for the activity of the promoter. As in the case of rabbit SP-B promoter (29), a DNA element with a high degree of similarity to consensus Sp1 sequence is located at −43/−35 bp in the human SP-B promoter. Our studies have shown that the Sp1 element in the human SP-B promoter is functionally important (Boggaram V, unpublished observations).
We determined the effects of lactacystin and MG132 on the DNA binding activities of TTF-1/Nkx2.1, HNF-3, and Sp1 elements to understand their role in the inhibition of SP-B promoter activity. Dexamethasone induces SP-B expression predominantly via stabilization of SP-B mRNA (5) and does not induce TTF-1 binding activity or levels (27). We therefore determined the effects of lactacystin and MG132 on TTF-1 binding activity and levels in untreated H441 cells. EMSA experiments showed that lactacystin and MG132 decreased the DNA binding activity of TTF-1/Nkx2.1 by ∼50% without significantly affecting the DNA binding activities of HNF-3 and Sp1 (Figs. 8, A and C). Antibody-supershift EMSA with TTF-1/Nkx2.1 antibodies, in agreement with the data of EMSA, showed that lactacystin and MG132 caused a concentration-dependent decrease in TTF-1 bound to the SP-B promoter oligonucleotide (Fig. 8B). We also determined the effects of lactacystin and MG132 on the binding activities of AP-1, CREB, GATA, and NF-κB to clarify whether the effects of the inhibitors are pleiotropic or not. Jun and CREB have been shown to regulate SP-B promoter (3, 41), and GATA-6 has been shown to be important for SP-A (9) and SP-C promoter (26) activities. Because inhibition of proteasome function is associated with the generation of reactive oxygen species (ROS), we were interested to know what effects proteasome inhibitors would have on NF-κB activity. Analysis of the binding activities of AP-1, NF-κB, CREB, and GATA in lactacystin- and MG132-treated cells showed that whereas AP-1 activity was increased, the activities of NF-κB, CREB, and GATA did not change (Fig. 8D), indicating that lactacystin and MG132 have unique effects on the DNA binding activities of transcription factors.
Proteasome inhibitors decrease TTF-1/Nkx2.1 expression levels.
Reduced TTF-1 DNA binding activity in lactacystin- and MG132-treated H441 cells could be due to decreased DNA binding activity per se or to decreased expression of TTF-1 or a combination of both. We determined the effects of lactacystin and MG132 on nuclear TTF-1 levels by immunoblotting. Results (Figs. 9, A and B) showed that lactacystin and MG132 treatment significantly reduced the content of TTF-1. When the same blot was probed for Sp1 and β-actin, it was found that their levels were not similarly affected. Next, we determined the effects of lactacystin and MG132 on luciferase reporter activity controlled by multiple TTF-1 binding sites placed upstream of basal SP-B promoter. Results (Fig. 9C) showed that in agreement with the results of immunoblotting studies, lactacystin and MG132 reduced luciferase expression from the TTF-1 reporter plasmid. We further analyzed the effects of lactacystin and MG132 on the nuclear content of TTF-1 by immunofluorescence microscopy. Results (Fig. 10) showed that in agreement with the immunoblotting data, TTF-1 detection was restricted to the nuclei and the nuclear content of TTF-1 was significantly reduced in lactacystin- and MG132-treated cells. Staining for TTF-1 could not be detected in the cytosol of control or lactacystin- and MG132-treated cells. Consistent with the results of immunofluorescence microscopy, immunoblotting experiments showed that lactacystin and MG132 treatment did not cause accumulation of TTF-1 protein in the cytosolic fraction (data not shown), indicating that the decreased nuclear content of TTF-1 is not due to redistribution of TTF-1.
Our study has shown for the first time that proteasome inhibitors specifically decrease the levels of SP-A, SP-B, and SP-C mRNAs in lung epithelial cells, suggesting a link between proteasome dysfunction and surfactant protein gene regulation. Proteasome inhibitors did not have adverse effects on H441 cell viability and had low-level toxicity on MLE-12 cells at lower concentrations, indicating that the inhibition of surfactant protein gene expression cannot be due to toxicity. Proteasome inhibitors decreased the expression of SP-A, SP-B, and SP-C mRNAs, indicating a common mechanism of inhibition. Our experiments also showed that the proteasome inhibitors acted at the transcriptional level to inhibit SP-B gene expression. Deletion mapping experiments showed that SP-B promoter region −233/+41 bp contains DNA elements necessary for inhibition of promoter activity. Further deletion to −80/+41 bp, although removing TTF-1 and HNF-3 binding sites, did not render the promoter insensitive to inhibition by lactacystin and MG132. This could be due to the presence of a functional Sp1 site at −35 bp. Although our experiments showed that Sp1 levels and binding activity were not reduced by lactacystin and MG132, its function in the activation of SP-B promoter could be subject to modulation by posttranslational modifications such as phosphorylation, glycosylation, and others. It is known in case of certain genes that the regulation of transcription activation by Sp1 is not accompanied by changes in DNA binding but via changes in the phosphorylation status (11). Whether lactacystin and MG132 modulate transcription activation by Sp1 via phosphorylation or other posttranslational modifications remains to be determined.
Lactacystin and MG132 decreased the DNA binding activity of TTF-1/Nkx2.1 without significantly altering the binding activities of HNF-3 and Sp1/Sp3 elements. The decrease in the DNA binding activity of TTF-1/Nkx2.1 in lactacystin- and MG132-treated cells was associated with a decrease in the expression levels of TTF-1/Nkx2.1, indicating that proteasome inhibitors decrease the expression of TTF-1/Nkx2.1 to reduce its DNA binding activity. Consistent with the results of immunoblotting experiments, immunofluorescence microscopy and TTF-1 reporter assays showed that proteasome inhibitors decreased the expression of TTF-1/Nkx2.1 levels. TTF-1/Nkx2.1 is a lung (23)- and thyroid-restricted (12) homeodomain-containing transcription factor that is a key activator of surfactant protein gene expression (31). Molecular mechanisms underlying reduced expression of TTF-1/Nkx2.1 in proteasome inhibitor-treated H441 cells remain to be investigated. Whether proteasome inhibitors influence the DNA binding activity of TTF-1/Nkx2.1 via posttranslational modifications such as phosphorylation and oxidation also remains to be investigated. Posttranslational modifications by phosphorylation (24, 53, 54) and oxidation (47) are known to influence the activity of TTF-1. SP-B promoter function is sensitive to changes in the helical phasing and orientation of cis-DNA elements (1) and is dependent on the combinatorial interactions between TTF-1, HNF-3, Sp1/Sp3, and other transcription factors (6, 29). It is possible that in proteasome inhibitor-treated cells, decreased levels of TTF-1/Nkx2.1 disrupt the formation of the transcriptional complex that is necessary for SP-B promoter activity, leading to reduced SP-B gene expression. Proteasome dysfunction is associated with endoplasmic reticulum (ER) stress response (52), oxidative stress (17), and activation of JNK MAP kinase (18). Whether such mechanisms are involved in lactacystin and MG132 decrease of TTF-1/Nkx2.1 levels and inhibition of surfactant protein gene expression in H441 cells remains to be determined. The inhibition of SP-B mRNA levels in cells treated with TNF-α (4), ceramide (44), and phorbol esters (22) is associated with reduced DNA binding activity of TTF-1, indicating that TTF-1 is a target for inhibition of SP-B gene expression.
There is limited information on the relationship between proteasome function and regulation of gene expression. The association between proteasome dysfunction and diseases such as neurodegenerative diseases and aging (48) and cardiac diseases (37) suggests a possible link between aberrant gene expression and pathogenesis of the diseases. In a variety of lung epithelial cell lines, proteasome inhibitors were found to increase IL-8 levels (18). In A549 lung epithelial cells, induction of IL-8 was due to both transcriptional and mRNA stabilization mechanisms (18). DNA binding activity of AP-1 but not of NF-κB was increased in A549 cells treated with proteasome inhibitors. In neonatal lung fibroblasts, disruption of proteasome function resulted in the inhibition of elastin gene expression due to upregulation of CCAAT/enhancer binding protein β proteins (21). Proteasome function is necessary for the transcriptional activities of androgen (25), glucocorticoid (45), and progesterone receptors (15). Inhibition of proteasome activity was found to suppress androgen receptor activity by inhibiting androgen receptor transactivation, androgen receptor nuclear translocation, and interactions between androgen receptors and androgen receptor coregulators (25). Inhibition of proteasome blocks progesterone receptor-dependent transcription via failed recruitment of RNA polymerase II (15). Proteasome function is necessary for rapid glucocorticoid receptor exchange at a promoter, suggesting yet another role for proteasomes in the regulation of gene transcription (45).
In neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease and in diseases such as cystic fibrosis and the recently discovered interstitial lung diseases associated with SP-C gene mutations, intra- and/or extracellular aggregation of abnormal proteins is linked to ER stress, proteasome dysfunction, and apoptosis. Expression of polyglutamine repeat fragments derived from proteins associated with expanded polyglutamine diseases such as Huntington's disease and Machado-Joseph disease, a folding mutant of cystic fibrosis transmembrane conductance regulator and human SP-Cexon4 mutant, caused inhibition of the ubiquitin-proteasome system (2, 32) and apoptosis signal-regulating kinase-1-mediated cell death (33). These data indicate that proteasome dysfunction due to protein aggregates contributes to the pathogenesis of disease process. Expression of SP-Cexon4 in HEK-293 embryonic kidney cells resulted in enhanced cell survival via induction of NF-κB activity and susceptibility to virus-induced cell death (8). It is not known whether abnormalities of surfactant metabolism and/or surfactant protein expression occur in interstitial lung diseases associated with SP-C gene mutations. In a recent study of the association of SP-C I73T mutation with lung disease, immunohistochemical analysis of lung tissue from one of seven patients showed normal staining patterns for proSP-B, SP-B, and proSP-C (10). Additional studies are necessary to determine whether SP-C mutations alter the levels of surfactant lipids and proteins in interstitial lung diseases. Recent studies have shown that inhibition of proteasomes is sufficient to induce mitochondrial dysfunction and increased generation of ROS (17), suggesting that proteasomes serve as regulators of oxidative stress. It is therefore possible that in diseases wherein proteasome function is impaired, oxidative stress contributes to cellular abnormalities, leading to the pathogenesis of the disease. Whether oxidative stress due to proteasome dysfunction is the underlying cause of surfactant protein inhibition in H441 and MLE-12 lung epithelial cells found in our study remains to be investigated.
In summary, our studies have shown that inhibition of proteasome activity reduces surfactant protein gene expression in lung epithelial cells. Proteasome inhibitors decreased SP-B gene expression by inhibiting gene transcription through reduction of expression levels of TTF-1/Nkx2.1. Our studies suggest that proteasome dysfunction that occurs in interstitial lung diseases associated with SP-C mutations may contribute to lung dysfunction through downregulation of surfactant protein gene expression. Whether proteasome dysfunction occurs in other lung diseases and its role in the development of disease process are not known.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-48048.
We thank James B. McKnight for technical assistance and Dr. Samir K. Mandal for help with immunofluorescence microscopy. We thank Dr. Shioko Kimura, National Cancer Institute, for providing T/EBP antibodies and Dr. David E. Griffith, University of Texas Health Center at Tyler, for providing human bronchoalveolar lavage samples.
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