Pulmonary Center and Department of Biochemistry, Boston University School of Medicine, Boston Department of Veterans Affairs Healthcare System, Boston, Massachusetts
Submitted 27 October 2004 ; accepted in final form 30 March 2005
ABSTRACT
Elastin, a major extracellular matrix protein and the core component of elastic fiber, is essential to maintain lung structural integrity and normal physiological function. We previously found that the downregulation of elastin gene transcription by IL-1 is mediated via activation of NF-
B and CCAAT/enhancer binding protein (C/EBP)
, both targets of the ubiquitin-proteasome pathway. To further investigate the molecular mechanisms that underlie the control of elastin gene expression, we disrupted the ubiquitin-proteasome pathway with specific proteasome inhibitors. We found that specific proteasome inhibitors decreased the steady-state level of elastin mRNA in a dose-responsive manner. Run-on assay and promoter reporter study indicated that the proteasome inhibitor MG-132 repressed the rate of elastin transcription. MG-132 did not affect mRNA levels of NF-
B and C/EBP
, or the nuclear presence of NF-
B, but markedly increased C/EBP
isoforms, including liver-enriched transcriptional activating protein and liver-enriched transcriptional inhibitory protein. Addition of cycloheximide blocked these increases and the downregulation of elastin mRNA by MG-132. The MG-132-induced downregulation of elastin transcription was dependent on C/EBP
expression as assessed with small interfering RNA. These results indicate that the ubiquitin-proteasome pathway plays an essential role in maintaining elastin gene expression in lung fibroblasts. Disruption of this pathway results in the downregulation of tropoelastin transcription via posttranscriptionally induced C/EBP
isoforms.
interleukin-1; lung; fibroblasts; nuclear factor-
B; CCAAT/enhancer-binding protein
Our previous studies (26, 27) suggested that the downregulation of elastin mRNA by IL-1 is transcriptionally mediated by NF-
B and CCAAT/enhancer binding protein (C/EBP)
pathways. NF-
B and C/EBP family protein complexes are both targeted and regulated by the ubiquitin-proteasome pathway (12, 17). The ubiquitin-proteasome pathway degrades the majority of intracellular proteins via the sequential action of two ATP-dependent functional protein complexes, the ubiquitin pathway and the proteasome (37). A variety of key regulatory proteins involved in cell proliferation, differentiation, survival, and apoptosis are substrates of the ubiquitin-proteasome pathway, implicating manipulation of proteasome dysfunction as a potential therapeutic intervention in tumorigenesis, inflammation, neurodegenerative diseases, and certain genetic diseases, including cystic fibrosis (9). Proteasome inhibitors are currently under investigation as promising anticancer drugs (1).
In this study, we investigated the biological role of the ubiquitin-proteasome pathway in the control of elastin gene expression in lung fibroblasts using well-studied proteasome inhibitors (29, 30, 36, 39). Our results indicate that the ubiquitin-proteasome pathway plays an essential role in maintaining elastin gene expression, and disruption of this pathway causes downregulation of elastin gene transcription and mRNA by posttranscriptionally upregulated C/EBP proteins.
EXPERIMENTAL PROCEDURES
Cell culture.
Neonatal rat lung fibroblasts, also referred to as lung interstitial cells (LIC), were isolated from the lungs of 8-day-old Sprague-Dawley rat pups (Charles River Breeding Laboratory, Wilmington, MA) as previously described (26, 27). The protocol for handling the Sprague-Dawley rat pups in this study was reviewed and approved by the Institutional Animal Care and Use Committee of the Boston University School of Medicine. After isolation, LIC were grown in MEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS in T-75 flasks (Falcon Plastics, Los Angeles, CA) in a humidified 5% CO2-95% air incubator at 37°C for 35 days until confluence. The purity of the cultures was assessed with phase microscopy and Oil Red O staining. The first-passage cells were grown in MEM to confluence and rendered quiescent by reducing the serum content of the medium to 0.4% for 24 h before experiments. MG-132, epoxomicin, lactacystin, calpeptin, and (2S,3S)-trans-epoxysuccinyl-L-leucylamide-3-methylbutane ethyl ester were purchased from Calbiochem (La Jolla, CA). Recombinant human TGF-1 and IL-1
were purchased from R&D Systems (Minneapolis, MN). Cycloheximide (CHX) and actinomycin D (ActD) were obtained from Sigma (St. Louis, MO). Human embryonic kidney 293T cells and preadipocyte cell lines (3T3.L1-LAP and 3T3.L1-LIP, kindly provided by Dr. Stephen R. Farmer, Boston University School of Medicine, Boston, MA) were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS as described previously (27).
RNA isolation and Northern blot analysis.
Cells were harvested, and total RNA was prepared with a RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. RNA samples (10 µg/lane) were electrophoresed and followed by Northern blot analysis as previously described (26, 27). Hybridization was carried out in Rapid Hybridization Buffer (Amersham, Piscataway, NJ) using 32P-labeled cDNA probes for rat tropoelastin, NF-B p56 subunit, or C/EBP
. The 32P-labeled 18S ribosome oligonucleotides (IDT, Coralville, IA) were used to assess loading equivalence and transfer efficiency.
Nuclei preparation and run-on assay.
Nuclei were isolated from four P-150 dishes of cell cultures designated for each experimental group in lysis buffer [10 mM Tris-Cl (pH 7.4), 3 mM CaCl2, 2 mM MgCl2, and 1% Nonidet P-40] with a Dounce homogenizer (Thomas, Swedesboro, NJ). After centrifugation at 500 g for 5 min, nuclei were resuspended in 200 µl of nuclei storage buffer [50 mM Tris-Cl (pH 8.3), 40% glycerol, 5 mM MgCl2 and 0.1 mM EDTA]. Nuclear run-on reactions were carried out by incubation of 200 µl of nuclei with one volume of 2x reaction buffer [10 mM Tris-Cl (pH 8.0), 5 mM MgCl2, 0.3 M KCl, ATP, GTP, and CTP at 2 mM, and 100 U RNasin (Promega, Madison, WI)] and 10 µl of [-32P]UTP (3,000 Ci/mmol) at 30°C for 30 min. Reactions were terminated by sequential incubation with RNase-free DNase (100 U) at 37°C for 30 min and with protease K (200 µg/ml in 1% SDS) at 42°C for 15 min. The newly synthesized 32P-labeled RNA was isolated by phenol/chloroform extraction and ethanol precipitation. After washing with 70% ethanol, RNA was dissolved in TE buffer [10 mM Tris·HCl (pH 7.0), 0.1 mM EDTA]. For hybridization, labeled RNA was denatured in 0.2 N NaOH for 10 min on ice and neutralized in 0.2 M acid-free HEPES. The unincorporated [
-32P]UTP was removed with NucTrap probe purification columns (Stratagene, La Jolla, CA). The plasmids (10 µg each) containing cDNA coding inserts for elastin, collagen, or GAPDH and 10 µg of pBluescript II SK vector (Stratagene) were denatured in 0.3 N NaOH at 65°C for 1 h and blotted onto the nitrocellulose membrane with a Minifold II slot blotter (Schleicher & Schuell, Keene, NH). The immobilized slot blots were prehybridized in 5x Denhardt's solution, 5x SSC, 0.5% SDS, 50 mM NaH2PO4 (pH 6.5), 250 µg/ml yeast tRNA, and 50% formamide at 42°C for 24 h and hybridized with equal counts (107 counts/min) of 32P-labeled RNA at 42°C for 3 days. The slot blots were washed and exposed on X-ray films.
Reporter plasmids, transient transfection, and luciferase assay.
The rat elastin promoter luciferase reporter PGL-2/118 and the TGF-1 responsive luciferase reporter 3TP were described previously (26, 27, 38). Transient transfection and cotransfection were performed with LipofectAMINE 2000 (LF-2000; Invitrogen) according to the manufacturer's protocol. Transfection efficiency was monitored by cotransfection of pRLTK Renilla luciferase vector (0.25 µg/well) (Promega, Madison, WI). At 24 h after transfection, cells were conditioned with TGF-
1 (1 ng/ml), MG-132 (10 µM), or both for 20 h, and whole cell lysates were isolated, followed by luciferase assay as described previously (26, 27). Firefly and Renilla luciferase activity were determined with the Dual-Luciferase Reporter system (Promega) and a luminometer (TD20/20, Turner Designs, Sunnyvale, CA). Firefly luciferase values were normalized to Renilla luciferase values and expressed as relative firefly/Renilla luciferase activity. Experiments were carried out in triplicate and repeated at least three times. Statistical analyses were carried out with a two-tailed Student's t-test. In case of overexpression and cotransfection of liver-enriched transcriptional inhibitory protein (LIP) experiments, parallel experiments were carried out to prepare whole cell lysates for immunoprecipitation and Western blot analysis as previously described (26, 27).
Small interfering RNA design, preparation, and transfection.
The design of small interfering RNA (siRNA) was according to the specific characterization of siRNA described by Elbashir et al. (14). Two 19-bp cDNA sequences were selected from the potential siRNA target sites in rat C/EBP transcript to generate siRNA expression constructs. They were synthesized as inverted repeats separated by a short loop sequence. The sense and antisense strands were annealed and inserted into pRNA-U6.1/Neo siRNA vector (Genescript, Scotch Plains, NJ). The sequences of these siRNA inserts (sense strand) were 5'-CTTCTACTACGAGCCCGACTTCAAGAGAGTCGGGCTCGTAGTAGAAG-3' (siRNA-CTT) and 5'-GCTGAGCGACGAGTACAAGTTCAAGAGACTTGTACTCGTCGCTCAGC-3' (siRNA-GCT). The corresponding siRNA constructs were named C/EBP
siRNA-CTT and -GCT constructs, respectively, and verified by DNA sequencing. To examine the effect of these siRNA constructs on C/EBP
expression, the siRNA construct (6 µg) was cotransfected with V5-tagged, liver-enriched, transcriptional activating protein (LAP) TOPO expression vectors (1 µg) (26) into 293T cells in P-60 dishes, using LF-2000 as described above. At 48 h after transfection, whole cell lysates were prepared and Western blot analysis was carried out with anti-V5 antibody (Invitrogen) as described previously (27). The effect of these siRNA constructs on IL-1
- or MG-132-induced downregulation of elastin transcription was tested by cotransfection of them (3 µg/well) with the elastin promoter luciferase reporter PGL-2/118 (0.5 µg/well) and the pRLTK Renilla luciferase vector (0.25 µg/well) into LIC in six-well plates, followed by luciferase assays and immunoprecipitation and Western blot analysis of whole cell lysates as above.
Nuclear protein extraction, immunoprecipitation, and Western blot analysis.
Preparation of nuclear proteins, immunoprecipitation of V5 from transfected whole cell extracts, and Western blot analysis were performed as previously described (26, 27). Briefly, nuclear extracts (30 µg) were resolved by 412% gradient of SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes. Immunoprecipitation was performed with 2 µg of anti-V5 antibody (Invitrogen) as previously described (26, 27). After being blocked in 5% fat-free dry milk, membranes were subjected to incubation with anti-p65 antibody (1:500 dilution, C-20; Santa Cruz Biotechnology, Santa Cruz, CA), anti-C/EBP antibody (1:500 dilution, C-19; Santa Cruz Biotechnology), anti-V5 antibody (1:1,000), or
-tubulin antibody (1:3,000; Sigma) at 4°C for 16 h with shaking. The immunoreactive signals were detected with ECL Plus Western blotting detection reagents (Amersham).
RESULTS
To examine the role of the ubiquitin-proteasome pathway in elastin gene expression, we used the specific proteasome inhibitor MG-132 (20, 29, 30, 39). Quiescent neonatal rat lung fibroblasts were treated with or without MG-132 (10 µM) for 20 h and subjected to RNA isolation and Northern blot analysis. We found that MG-132 markedly decreased the steady-state level of elastin mRNA (Fig. 1A). Densitometric analysis from six such experiments indicated that MG-132 downregulated elastin mRNA level by 85%. To verify that MG-132-induced downregulation of elastin mRNA resulted from a specific inhibition of proteasome function, we treated neonatal rat lung fibroblasts with two other structurally unrelated proteasome inhibitors that act via different mechanisms (10, 29, 35). We found that treatment with either epoxomicin or lactacystin decreased elastin mRNA in a dose-responsive manner that was consistent with their described functional activity (10, 15, 29, 35) (Fig. 1B). The expression of elastin mRNA was not altered by inhibitors of calcium-dependent calpain proteases (calpeptin, 10 µM) or lysosomal cysteine proteases (EST, 50 µM) (data not shown). These results indicated that disruption of the proteolytic function in the ubiquitin-proteasome pathway specifically downregulated elastin mRNA.
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We examined the interaction of overexpression of LIP and treatment with IL-1 or MG-132 on the activity of the elastin promoter luciferase reporter construct (PGL-2/118). We found that overexpression of LIP markedly decreased the luciferase activity of PGL-2/118 as we reported previously (27), and this decrease was further potentiated by addition of either IL-1
or MG-132 (Fig. 6C, top). Expression of LIP-V5 fusion protein and endogenous C/EBP
isoforms was demonstrated by immunoprecipitation using V5 antibody (Fig. 6C, middle) and Western blot analysis using C/EBP
antibody (Fig. 6C, bottom), respectively.
We previously demonstrated (27) that the truncated C/EBP protein, LIP, decreases the rate of elastin transcription. To further define the relationship between C/EBP
expression and proteasome dysfunction on the elastin transcription, we used siRNA techniques. The siRNA sequences were selected according to Elbashir et al. (14), and the double-stranded siRNA was inserted into pRNA-U6.1/Neo siRNA vector. The silencing effect of these siRNA constructs on C/EBP
expression was tested by cotransfection with LAP-V5 TOPO expression vector into 293T cells and analyzed by Western blot analysis. C/EBP
siRNA-CTT construct suppressed the expression of LAP-V5, whereas C/EBP
siRNA-GCT construct had no effect on LAP-V5 expression (Fig. 7A). The effect of siRNA-CTT construct on MG-132- or IL-1
-induced elastin promoter inhibition was examined by cotransfection of elastin promoter luciferase reporter PGL-2/118 and the siRNA construct into neonatal rat lung fibroblasts. Overexpression of C/EBP
siRNA-CTT construct completely blocked the decrease of luciferase activities by MG-132 or IL-1
, whereas C/EBP
siRNA-GCT construct had only minimal effect (Fig. 7B, top). The effect of these siRNA constructs on endogenous C/EBP
proteins was demonstrated by Western blot analysis of whole cell lysates with C/EBP
antibody (Fig. 7B, bottom).
|
We demonstrate herein that proteasome inhibitors specifically decrease the steady-state level of elastin mRNA in neonatal rat lung fibroblasts. Treatment with MG-132 decreased the rate of elastin transcription as determined by nuclear run-on assay and promoter analysis. Inhibition of protein synthesis by CHX and inhibition of transcription by ActD blocked the downregulation of elastin mRNA by MG-132. The level of C/EBP proteins, but not mRNA, was strongly increased by MG-132, which was abolished by CHX. Overexpression of an effective C/EBP
siRNA abrogated the inhibition of elastin promoter activity by MG-132. These results indicate that disruption of the proteolytic function in the ubiquitin-proteasome pathway inhibits tropoelastin gene transcription via upregulation of C/EBP
proteins and suggest that the ubiquitin-proteasome pathway plays an essential role during maintenance of elastin homeostasis.
In vivo, C/EBP proteins dimerize among themselves or with other C/EBP family proteins and exist as homo- or heterodimers. Self-dimerization or dimerization with other C/EBP family members increases the stability of C/EBP
proteins and protects them from ubiquitin-proteasome degradation (17). Treatment with MG-132 did not increase the level of ectopic expressed C/EBP
or generate any higher-molecular-weight form of C/EBP
(ubiquitin linked), whereas the levels of ectopic expressed Ig/EBP and C/EBP homologous protein (CHOP), which lack self-dimerization and exist as monomers, were markedly increased and ubiquitin-marked by MG-132 treatment (17). These findings indicate that the proteasome dysfunction does not upregulate C/EBP
proteins in lung fibroblasts via decreases in degradation. The molecular mechanisms underlying turnover of the functional dimeric C/EBP
complexes and regulation of their availability are not understood. During transcription initiation, DNA-bound C/EBP
complexes may be specifically subjected to various modifications, such as phosphorylation, acetylation, or methylation by coregulators such as p300/CBP or other components in general transcription machinery. Such modifications may lead to a conformational change of C/EBP
that causes loss of their DNA binding activity or dimerization ability. Inhibition of DNA binding activity may temporarily disable dimeric C/EBP
complexes to function as transcriptional regulators, whereas permanent loss of dimerization property could be an essential step for irreversible termination by the ubiquitin-proteasome pathway.
Our results showed that the expression pattern of C/EBP protein induced by MG-132 was similar to that induced by IL-1
, except for the appearance, in MG-132-treated nuclear extracts, of a unique anti-C/EBP
reactive complex (LIP*) with a slightly higher molecular weight than LIP. There is no additional potential in-frame translation start codon ATG located between ATG of LAP and ATG of LIP. We did not detect any ubiquitin-marked C/EBP
proteins (higher molecular weight), suggesting that the turnover of C/EBP
proteins is not mediated by the ubiquitin-proteasome pathway and indicating that certain posttranslational modification may specifically occur to LIP during proteasome dysfunction. The 5' UTR of C/EBP
gene is critical for the differential expression of C/EBP
proteins (2, 7). The expression of slow-migrating LIP* may be controlled by the components of 5' UTR in the C/EBP
gene, because MG-132 failed to induce such a slow-migrating LIP*-V5 complex from rat neonatal lung fibroblasts that were transfected with a CMV-driven and V5-tagged LIP expression vector. The biological properties of this modification are not clear and are currently under investigation in this laboratory. It has been reported that the addition of the small ubiquitin-like modifier (SUMO) to both C/EBP
and C/EBP
LAPs can specifically alter their functional roles during regulation of gene transcription (24, 43). It is possible that LIP can also be subjected to sumoylation to modulate its gene transcriptional regulation.
Proteasome inhibitors increased levels of C/EBP isoforms and their DNA binding activities in human intestinal epithelial cells (19). It was reported that proteasome dysfunction caused endoplasmic reticulum (ER) stress (6, 23), a cellular response to accumulation of unfolded and denatured proteins that are normally degraded by the ubiquitin-proteasome pathway. Interestingly, C/EBP
gene transcription was induced via an unfolded protein response element harbored in its 3' UTR (8). However, our data showed that MG-132 did not alter C/EBP
mRNA but markedly increased the levels of LAPs and induced LIP expression that were CHX sensitive. These results suggest that, in these rat lung fibroblasts, ER stress induced by MG-132 may increase C/EBP
mRNA translation or its stability. The LIP isoform lacks most of the NH2-terminal transactivation domain, enabling it to act as a dominant-negative isoform for LAP isoform and other bZIP transcription factors. LIP is generated by alternative translational initiation at the third in-frame AUG during a variety of important cellular processes in response to physiological and pathological stimulations (2, 7, 13, 42, 45). The specific RNA binding protein CUG-BP1, which binds to CUG repeats within the 5' UTR of C/EBP
mRNA, may function as a key regulator of LIP expression (2). In addition, two short open reading frames located in this 5' UTR may also be essential for induction of truncated C/EBP isoforms (7). It is possible that these molecular mechanisms may be utilized to initiate LIP translation under the inhibition of proteolytic function of proteasome.
We previously demonstrated (26, 27) that IL-1-induced upregulation of C/EBP
proteins LAPs and LIP and downregulation of tropoelastin transcription are dependent on NF-
B activation. Proinflammatory stimuli failed to induce C/EBP
mRNA in p65/ fibroblasts (25). Interestingly, upregulation of C/EBP
proteins by MG-132 appears to be independent of the NF-
B pathway, because MG-132 increased neither the steady-state level of p65 mRNA nor the nuclear NF-
B level (Fig. 4). Moreover, MG-132 blocked the activation of NF-
B by IL-1
, as indicated by abrogation of increases of C/EBP
and p65 mRNA (Fig. 5, lane 4). Activation of NF-
B pathway by cytokines, such as IL-1
and TNF-
, occurs via ubiquitin-proteasome pathway-dependent degradation of its cytoplasmic inhibitory protein I
B family (22).
We have also shown (27) that IL-1 inhibits elastin transcription by increasing C/EBP
isoforms and binding of the inhibitory complexes LIP-LAP and LIP-LIP to a GCAAT element located within the proximal elastin promoter at 56 to 62 bp. In the present study, we used an RNA interference technique and further demonstrated that downregulation of elastin gene transcription by MG-132 or IL-1
is dependent on the expression of C/EBP
proteins. The molecular basis of this C/EBP
-dependent inhibition of elastin transcription is not known. Both transactivation and leucine zipper domains of C/EBP
are required for cooperative activation of rat CYP2D5 gene by Sp1 and C/EBP
LAP isoforms (29). Therefore, LIP-containing inhibitory complexes may recruit other mediator complexes to change DNA structure in the elastin promoter to disrupt the association of Sp1 enhancesome or indirectly block its transactivation of transcriptional apparatus. In summary, for the first time, we demonstrate here that the proteolytic function of the ubiquitin-proteasome pathway is essential for normal elastin gene expression. Disruption of this pathway may cause an ER stress response to upregulate C/EBP
proteins LAPs and LIP and downregulate elastin biosynthesis.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute Grants P01-HL-46902 and R01-HL-66547, a Research Enhancement Award Program grant from the Department of Veterans Affairs Research Service, and a Research Grant Award (to P.-P. Kuang) from the American Lung Association.
FOOTNOTES
Address for reprint requests and other correspondence: P.-P. Kuang, Pulmonary Center, R 304, Boston Univ. School of Medicine, 80 E. Concord St., Boston, MA 02118 (E-mail: pkuang{at}lung.bumc.bu.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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