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Am J Physiol Lung Cell Mol Physiol 292: L944-L952, 2007. First published January 5, 2007; doi:10.1152/ajplung.00184.2006
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Activation of elastin transcription by transforming growth factor-beta in human lung fibroblasts

Ping-Ping Kuang, Xiao-Hui Zhang, Celeste B. Rich, Judith A. Foster, Mangalalaxmy Subramanian, and Ronald H. Goldstein

Pulmonary Center and Department of Biochemistry at Boston University School of Medicine, and the Boston Veterans Affairs Healthcare System, Boston, Massachusetts

Submitted 19 May 2006 ; accepted in final form 27 December 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Elastin synthesis is essential for lung development and postnatal maturation as well as for repair following injury. Using human embryonic lung fibroblasts that express undetectable levels of elastin as assessed by Northern analyses, we found that treatment with exogenous transforming growth factor-beta (TGF-beta) induced rapid and transient increases in levels of elastin heterogeneous nuclear RNA (hnRNA) followed by increases of elastin mRNA and protein expression. In fibroblasts derived from transgenic mice, TGF-beta induced increases in the expression of a human elastin gene promoter fragment driving a chloramphenicol acetyl transferase reporter gene. The induction of elastin hnRNA and mRNA expression by TGF-beta was abolished by pretreatments with TGF-beta receptor I inhibitor, global transcription inhibitor actinomycin D, and partially blocked by addition of protein synthesis inhibitor cycloheximide, but was not affected by the p44/42 MAPK inhibitor U0126. Pretreatment with the p38 MAPK inhibitor SB-203580 also partially attenuated the levels of TGF-beta-induced elastin mRNA but not its hnRNA. Western analysis indicated that TGF-beta stimulated Akt phosphorylation. Inhibition of phosphatidylinositol 3-kinase and Akt phosphorylation by LY-294002 abolished TGF-beta-induced increases in elastin hnRNA and mRNA expression. Treatment of lung fibroblasts with interleukin-1beta or the histone deacetylase inhibitor trichostatin A inhibited TGF-beta-induced elastin mRNA and hnRNA expression by a mechanism that involved inhibition of Akt phosphorylation. Downregulation of Akt2 but not Akt1 expression employing small interfering RNA duplexes blocked TGF-beta-induced increases of elastin hnRNA and mRNA levels. Together, our results demonstrated that TGF-beta activates elastin transcription that is dependent on phosphatidylinositol 3-kinase/Akt activity.

heterogeneous nuclear RNA; emphysema; small interfering RNA; phosphatidylinositol 3-kinase/Akt

ELASTIN IS AN ESSENTIAL STRUCTURAL component of pulmonary alveolar structures. Elastin synthesis in the parenchyma of the rodent lung is highest during the alveolarization process that usually begins in the postnatal period but decreases with maturity (6). In the adult rodent lung, elastin synthesis is reactivated during the development of pulmonary emphysema after elastase treatment or pulmonary fibrosis after exposure to bleomycin (35, 39). The regulation of elastin production is complex and not well understood. The level of tropoelastin synthesis is directly related to the expression of the elastin mRNA. Regulation of steady-state levels of elastin mRNA occurs via both transcriptional and posttranscriptional mechanisms (26, 29, 34, 70). In vitro studies reveal that elastin mRNA levels can be regulated by certain inflammatory mediators and by corticosteroids (5, 16, 29, 34, 46, 56, 70). For example, insulin-like growth factor stimulates elastin gene transcription in neonatal smooth muscle cells but not in lung fibroblasts (16, 26). Fibroblast growth factor (FGF)-18 increases elastin mRNA levels in fetal and postnatal rat lung fibroblasts (8). Elastin mRNA levels are downregulated by FGF-2, IL-1beta, and TNF-{alpha} (5, 27, 30). In certain rodent cell lines, elastin mRNA levels are regulated primarily by posttranscriptional processes (70).

Elastin synthesis is essential for alveolar development and is dependent on transforming growth factor-beta (TGF-beta) signal transduction. Deficiency of Smad3 leads to repression of tropoelastin expression in lung and the development of centrilobular emphysema (10). TGF-beta increases elastin mRNA levels in both dermal and lung fibroblasts (29, 34, 70). It is also important for regulating elastin synthesis by smooth muscle cells in vascular tissues. TGF-beta colocalizes with elastin in vascular tissues during periods of active elastin synthesis (56). Mice deficient in Smad3 or the beta6-integrin develop air space enlargement (10, 44). Smad3 is a component of the TGF-beta signal transduction pathway, and the beta6-integrin is involved in TGF-beta activation. These observations suggest that lung fibroblasts under the influence of active TGF-beta are required to deposit elastin in alveolar structures. We have found that TGF-beta is upregulated in the bronchoalveolar lavage fluid after elastolytic injury to the mouse lung (7). TGF-beta mRNA levels were also found to be upregulated in the lungs of individuals with chronic obstructive lung disease (Gold criteria: stage 2) (45).

TGF-beta increases the expression of multiple matrix genes including elastin (38, 57). Many of these genes are regulated by increases in the rate of transcription or a combination of increases in the rate of transcription and increases in the half-life of the mRNA (stabilization). In contrast, much of the work to date on the regulation of elastin mRNA by TGF-beta has focused on the ability of TGF-beta to stabilize the elastin mRNA (34, 61). In the present study, we have investigated regulation of elastin expression by TGF-beta in human lung fibroblasts by examination of levels of elastin heterogeneous nuclear RNA (hnRNA) and mRNA. Our data reveal that induction of elastin mRNA and protein by TGF-beta are correlated to a large rapid increase in elastin transcription, which may require phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling and involve de novo protein synthesis and p38 MAPK pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and reagents. Cycloheximide (CHX), actinomycin D, and TGF-beta type I receptor inhibitor SB-431542 were all obtained from Sigma (St. Louis, MO). Trichostatin A (TSA; histone deacetylase inhibitor) was purchased from Upstate (Lake Placid, NY). LY-294002 (LY), U0126, and SB-203580 were obtained from Calbiochem (La Jolla, CA), and recombinant human TGF-beta1 and IL-1beta were from R&D Systems (Minneapolis, MN). Human lung fibroblasts (IMR-90) were purchased from American Type Culture Collection and maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 0.37 g sodium pyruvate/100 ml, 100 units penicillin/100 ml, and 100 µg streptomycin/100 ml in a humidified 5% CO2, 95% air incubator at 37°C. Confluent cultures were rendered quiescent by culturing in serum-free medium for 24 h before experimentation.

Total RNA isolation and Northern blot analysis. Serum-starved confluent fibroblasts were untreated or treated with TGF-beta (1 ng/ml) for various time periods. For inhibitor studies, the pretreatments were performed with CHX (10 µg/ml), actinomycin D (10 µg/ml), SB-431542 (20 µM), LY-294002 (10 µM), U0126 (10 µM), and SB-203580 (25 µM) for 1 h before treatment with TGF-beta (1 ng/ml) for 16 h. Total RNA was prepared with RNeasy (Qiagen) according to the manufacturer's protocol, followed by Northern analysis using 32P-labeled cDNA probes for human elastin and GAPDH. The same total RNA samples were also treated with RNase-free-DNase I (Qiagen) and assessed for elastin mRNA expression using TaqMan reagents in the TaqMan real-time quantitative PCR assay as described below.

Taqman real-time quantitative RT-PCR analysis. For analysis of gene expression using Taqman real-time quantitative RT-PCR analysis, DNase-treated total RNA (2 µg) was first reverse transcribed in a 50-µl volume using random primers and the High-Capacity cDNA Achieve Kit (Applied Biosystems) according to the manufacturer's protocol. Taqman reagents for detecting mRNA expression of elastin or GAPDH were purchased from Applied Biosystems (Foster City, CA). To assess elastin hnRNA expression, a set of sequence-specific 6-FAM dye-labeled probes plus forward and reverse primers flanking the first exon/intron boundaries of elastin gene were designed and synthesized using PrimerExpress software (Applied Biosystems). The TaqMan probe contained a reporter dye, 6-FAM, linked to the 5'-end of the probe, a minor groove binder (MGB), and a nonfluorescent quencher (NFQ) at the 3'-end of the probe. Primers and probe used for human elastin hnRNA analysis were: forward, 5'-TCTGAGGTTCCCATAGGTTAGGG-3'; reverse, 5'-CTAAGCCTGCAGCAG CTCCT-3'; and Taqman probe, 5'-6-FAM-AACAATGCTTTTTCT TCC-MGB-NFQ. GAPDH mRNA expression was used as the endogenous control for internal standardization and normalization of data. Taqman PCR reactions were performed in triplicate using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) according to the manufacturer's protocol.

Analysis of human elastin promoter in vivo. The generation of the transgenic mouse containing –2,260 to +2 of the human elastin gene promoter fragment driving chloramphenicol acetyl transferase (CAT) has been described previously (51). Pulmonary fibroblasts were prepared from these mice as previously described (51). Total cell lysate or RNA was prepared from control and treated cells. Cell lysis samples were run on 12% SDS polyacrylamide gel. Western analysis was done using mouse monoclonal CAT antibody (ab5410, Abcam). RNA was analyzed by Northern blot and probed with rat elastin and histone cDNA (51).

Preparation of cytosol, nuclear and whole cell extract, and Western analysis. Nuclear, cytoplasmic and whole cell extracts were prepared at 4°C in the presence of protease inhibitors as previously described (28). Total protein (50 µg) was resolved in SDS-PAGE followed by Western analysis using elastin antibody (EPC, Owensville, MI) and anti-phospho-antibodies against Akt at the recommended dilution ratio (Cell Signaling, Beverly, MA). After stripping, the same blot was probed with non-phospho-antibody against Akt (Cell Signaling) to monitor the loading control.

Transfection of small interfering RNA. Validated human SMARTpool Akt1 small interfering RNA (siRNA) and Akt2 siRNA duplexes and a scrambled nontargeting control siRNA were purchased from Upstate (manufactured by Dharmacon, Lafayette, CO). Subconfluent IMR-90 cells (6 x 105 cells/100 µl) were harvested and resuspended in Nucleofector Solution (Amaxa). Nucleofection was performed in an Amaxa certified cuvette by mixing an aliquot of the cell suspension (100 µl) with the siRNA using the Nucleofector device (Amaxa) and the preoptimized program (U-23). Immediately following nucleofection, the cells were plated into six-well dishes in DMEM (Invitrogen). After 48 h, the confluent cells were placed in serum-free DMEM for 12 h followed by treatment with TGF-beta for 4 h before being harvested for total RNA isolation.

Statistics. The data for the Northern blot from the elastase experiment were normalized to the 18S signal, and fold induction of elastin mRNA was determined relative to the baseline in untreated fibroblasts. Fold induction over control was evaluated by a two-tailed Student's t-test, and a P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We investigated the effect of TGF-beta on elastin mRNA production by human lung fibroblasts. Northern blot analysis revealed that elastin mRNA was undetectable in untreated fetal fibroblast cultures (Fig. 1). Kinetic studies indicated that levels of elastin mRNAs dramatically increased between 4 and 20 h following the addition of TGF-beta (Fig. 1). Equal loading was verified by assessment of levels of the 18S ribosomal subunit.


Figure 1
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  		Fig. 1. Effect of transforming growth factor-beta (TGF-beta) on the steady-state levels of elastin mRNA expression in human lung fibroblasts. Confluent quiescent lung fibroblasts were serum starved for 24 h. The cells were untreated (C) or treated with TGF-beta1 (1 ng/ml) for the indicated time in hours (h). Cells were harvested for total RNA isolation followed by sequential Northern analysis using a 32P-labeled human elastin cDNA and a 32P-labeled oligonucleotide of the 18S ribosome subunit. Data are representative of 3 similar experiments.

 
To determine the effect of TGF-beta on the production of intact tropoelastin protein, we performed Western blot analyses (Fig. 2). Human lung fibroblasts were treated with TGF-beta, and cell lysates were harvested for analysis. We did not detect tropoelastin in untreated cultures. At 8 h following TGF-beta treatment, tropoelastin was readily detectable, and a further increase was observed in tropoelastin at 24 h following TGF-beta administration (Fig. 2). The results were similar whether we used lung fibroblasts derived from fetal or adult human lung (data not shown).


Figure 2
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  		Fig. 2. Effect of TGF-beta on tropoelastin expression by human lung fibroblasts. Serum-starved confluent fibroblasts were untreated (C) or treated with TGF-beta (T, 1 ng/ml) for 8 and 24 h as indicated. Whole cell lysates were extracted followed by Western analysis using specific human tropoelastin antibody. Equal loading was demonstrated by levels of cross-reacting proteins (CRP). The size of tropoelastin was indicated using a protein marker (M) from Invitrogen.

 
To determine the effect of TGF-beta on the transcription of the elastin gene, we examined expression of hnRNA in human lung fibroblasts. Levels of hnRNA reflect the rate of transcription of the elastin gene (6, 18). Real-time quantitative PCR was performed with a specific Taqman probe, which binds to the boundary the first exon and intron in human elastin gene and a pair of its flanking forward and reverse primers. We found that TGF-beta induced a rapid increase in hnRNA levels that was followed by an increase in elastin mRNA levels that became maximal 24 h following treatment (Fig. 3A). Elastin mRNA and hnRNA levels were normalized to levels of GAPDH mRNA. This TGF-beta-induced transcriptional activation of elastin gene was verified by using the global transcriptional inhibitor actinomycin D and TGF-beta receptor I inhibitor, in which Northern analysis showed that pretreatment with either actinomycin D (10 µg/ml) or TGF-beta receptor I inhibitor (10 µM) completely abolished the increase of elastin mRNA by TGF-beta (Fig. 3B).


Figure 3
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  		Fig. 3. Effect of TGF-beta on the transcription of the elastin gene as determined by levels of heterogeneous nuclear RNA (hnRNA). A: total RNA was isolated from serum-starved cells that were untreated (C) or treated with TGF-beta (1 ng/ml) for various time periods as indicated. Levels of elastin hnRNA were determined by TaqMan real-time PCR with a set of specific probes and primers complementary to human elastin first exon/intron boundary and expressed as fold increases. Data represent the average ± SE of 3 independent experiments performed in triplicate. B: serum-starved confluent lung fibroblasts were pretreated with either actinomycin D (ActD, 15 µg/ml) or TGF-beta receptor I inhibitor SB-431542 (TRI) (10 µM) for 1 h before addition of TGF-beta (1 ng/ml) for 16 h. Total RNA was isolated followed by Northern analysis using 32P-labeled human elastin cDNA and 32P-labeled oligonucleotide of 18S ribosome subunit. Data are representative of 3 separate experiments.

 
We also examined the effect of TGF-beta on lung fibroblasts derived from transgenic mice containing 2.26 kb of human elastin promoter linked to CAT as reporter gene (29). Previous studies revealed activation of the elastin promoter as assessed by increased expression of this transgene in the lung following elastase administration. Treatment with TGF-beta increased both the expression of CAT protein driven by human elastin promoter as well as the elastin mRNA (Fig. 4). The increase in CAT protein reflects a TGF-beta-induced increase in elastin promoter activity.


Figure 4
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  		Fig. 4. Effect of TGF-beta on transcription of the elastin gene as determined by levels of chloramphenicol acetyl transferase (CAT) protein expression. Lung fibroblasts were derived from transgenic mice expressing 2.2 kb of elastin promoter driving a CAT reporter gene. After growing 4 or 5 days in culture, fibroblasts were untreated or treated with TGF-beta (T) for 24 h. Western analysis was performed to analyze the CAT protein production (a measure of the level of elastin transcription), and Northern analysis was performed to detect levels of endogenous elastin mRNA (middle) and histone (bottom) mRNA using corresponding 32P-labeled cDNA probes.

 
We employed protein synthesis and kinase inhibitors to gain insight into the signal transduction pathway utilized by TGF-beta to increase elastin mRNA (Fig. 5A). The upregulation of elastin mRNA was partially dependent on protein synthesis as CHX partially blocked the upregulation of the elastin mRNA (Fig. 5A). Treatment with p38 inhibitor (SB-203580) resulted in minimal inhibition and no inhibition with treatment with the MEK1/2 inhibitor U0126. Real-time quantitative PCR revealed that addition of CHX, but not p38 inhibitor, inhibited TGF-beta-induced elastin hnRNA expression (Fig. 5B).


Figure 5
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  		Fig. 5. Dissection of signaling pathways that may be involved in TGF-beta-induced upregulation of elastin expression. A: serum-starved confluent fibroblasts were untreated (C), treated with TGF-beta1 (1 ng/ml) (T), IL-1beta (IL, 250 pg/ml), or both (T/IL), or pretreated with cycloheximide (CHX) (10 µg/ml), LY-294002 (LY) (10 µM), SB-203580 (SB) (25 µM), or U0126 (10 µM) for 1 h before addition of TGF-beta1 for 16 h. Total RNA was isolated and followed by Northern analysis using 32P-labeled human cDNA probes for human elastin and a 32P-labeled oligonucleotide of the18S ribosome subunit. Data are representative of 3 similar experiments. B: fibroblasts were similarly treated with TGF-beta1 (T) for 12 h followed by total RNA isolation and treatment with DNase I. The expression profiles of elastin hnRNA were assessed by TaqMan real-time PCR with a set of specific probe and primers complementary to the first human elastin exon/intron boundary. Data were normalized to GAPDH mRNA values and expressed as the relative fold increases of average ± SE of 3 independent experiments performed in triplicate. *Significant difference from untreated control, **significant difference from TGF-beta-treated fibroblasts (P < 0.05).

 
We found that inhibition of PI 3-kinase with LY-294002 completely blocked TGF-beta-induced elastin mRNA and hnRNA expression (Fig. 5, A and B). Treatment with insulin induced large increases in phospho-Akt but did not affect elastin mRNA levels or TGF-beta-induced increases in elastin transcription (data not shown). Western analysis confirmed that LY-294002 inhibited both basal and TGF-beta-induced increases in phospho-Akt (Fig. 6). We employed IL-1beta and the histone deacetylase inhibitor TSA to further investigate the role of phospho-Akt in the regulation of elastin mRNA levels as both are known to modulate Akt signaling (9, 50, 59). Activation of phospho-Akt was not sufficient to induce elastin expression since treatment with IL-1beta induced phospho-Akt but did not affect elastin mRNA levels (Fig. 7). Interestingly, the combined treatment of fibroblasts with IL-1beta and TGF-beta yielded unexpected results. IL-1beta treatment inhibited both TGF-beta-induced phospho-Akt (Fig. 7) and elastin mRNA and hnRNA levels (Fig. 6).


Figure 6
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  		Fig. 6. Effect of LY-294002 on TGF-beta-induced Akt phosphorylation. Serum-starved confluent cells were untreated (control) or treated with TGF-beta1 (1 ng/ml) (TGF), LY-294002 (10 µM) (LY), or both for 16 h. Cytosolic (C) and nuclear (N) extracts were isolated and followed by Western blot analysis using antibodies against phospho-Akt (P-AKT; Ser473). The Akt antibody against total Akt protein was used as a loading control. Data are representative of 3 similar experiments.

 

Figure 7
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  		Fig. 7. Effect of IL-1beta on TGF-beta-induced Akt phosphorylation. Serum-starved confluent cells were untreated (control) or treated with IL-1beta (250 pg/ml) (IL), TGF-beta1 (1 ng/ml) (TGF), or both for 16 h. Cytosolic extracts were isolated and followed by Western blot analysis using antibodies against phospho-Akt (Ser473). The Akt antibody against total Akt protein was used as a loading control. Data are representative of 3 similar experiments.

 
Treatment with TSA increases the acetylation of chromatin histones and upregulates the transcriptional activity of specific genes (43). It is also known to affect Akt phosphorylation in certain cell lines (9). We first examined whether the low basal levels of elastin transcription resulted from underacetylation of the elastin promoter. We found that treatment with TSA did not increase elastin mRNA levels (Fig. 8A). In contrast, TSA treatment decreased the levels of basal elastin transcription and completely blocked the activation of elastin transcription by TGF-beta (Fig. 8B). Western analysis revealed that TSA treatment inhibited TGF-beta-induced increases in phospho-Akt (Fig. 8C).


Figure 8
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  		Fig. 8. Effect of inhibition of histone deacetylase by trichostatin A (TSA) on TGF-beta-induced regulation of elastin mRNA, hnRNA, and Akt phosphorylation. A: serum-starved confluent fibroblasts were untreated (C) or treated with IL-1beta (250 pg/ml) (IL), TGF-beta1 (1 ng/ml) (T), their combination (T/IL), TSA (10 µM), TSA + TGF-beta (T/TSA), U0126 (10 µM), or U0126 + TGF-beta (T/U0126) for 16 h. Cells were harvested for total RNA isolation and followed by Northern analysis using a 32P-labeled human elastin cDNA and a 32P-labeled oligonucleotide of the 18S ribosome subunit. Data are representative of 3 separate experiments. B: fibroblasts were untreated (C) or treated with TGF-beta (TGF, 1 ng/ml) alone, TSA (10 µM) alone, or both (TGF/TSA) for various time periods as indicated. Total RNA was isolated and treated with DNase I. The expression profiles of elastin hnRNA were assessed by TaqMan real-time PCR with a set of specific probe and primers complementary to the first human elastin exon/intron boundary. Data were normalized to GAPDH mRNA values and expressed as the relative fold increases of average ± SE of 3 independent experiments performed in triplicate. C: cytosolic extracts were isolated from serum-starved confluent fibroblasts that were untreated (control) or treated with TGF-beta1 (1 ng/ml), TSA (10 µM), or both (TSA/TGF-beta). Western analysis was sequentially performed using antibodies against phospho-Akt (Ser473) and the Akt antibody against total Akt protein. Data are representative of 3 similar experiments.

 
We employed siRNA techniques to downregulate Akt1 and Akt2 mRNA expression. Treatment with siRNA duplexes directed against Akt1 or Akt2 mRNA resulted in greater than 80% reduction in Akt1 and Akt2 mRNA levels as assessed using Taqman real-time PCR (Fig. 9, A and B). Downregulation of levels of Akt2 mRNA but not Akt1 mRNA markedly inhibited TGF-beta-induced increases in the level of elastin mRNA (Fig. 9C) and hnRNA (Fig. 9D).


Figure 9
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  		Fig. 9. Effect of downregulation of Akt1 and Akt2 mRNA on elastin mRNA and hnRNA levels. Fibroblasts were treated with a scrambled nontargeting small interfering RNA (siRNA; si-Control) and validated human SMARTpool Akt1 specific siRNA (si-Akt1) and Akt2 specific siRNA (si-Akt2) at the indicated concentrations. At 48 h after nucleofection, the cells were untreated (C) or treated with TGF-beta (T) for 4 h before being harvested for total RNA isolation. The levels of Akt1 mRNA (A), Akt2 mRNA (B), elastin mRNA (C), and elastin hnRNA (D) were assessed using TaqMan reagents and real-time PCR. N = 3, *significant difference from untreated control, **significant difference from TGF-beta-treated fibroblasts. (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we investigated the molecular mechanisms underlying TGF-beta-mediated elastin gene regulation using human lung fibroblasts. Under basal conditions, elastin mRNA expression was undetectable by Northern analysis. Treatment with TGF-beta induced a dramatic increase in elastin transcription resulting in increased elastin mRNA and protein expression. The regulation of elastin in human lung fibroblasts is markedly different from that observed in rodent neonatal lung fibroblasts. Rat lung fibroblasts express high basal levels of elastin mRNA that were minimally increased by exogenous TGF-beta (41). The explanation for these species-specific differences in elastin expression is unknown.

TGF-beta is a member of a subfamily of multifunctional growth factors implicated in various physiological and pathological conditions, including regulation of cell proliferation, differentiation, apoptosis, migration, and syntheses of extracellular matrix proteins (40). Initiation of TGF-beta signaling occurs following ligand binding and activation of its cognate receptor complex. The activated receptor kinase phosphorylates receptor-associated Smads, Smad2 and Smad3, which then recruit the comediator Smad4. The Smad complexes translocate into the nucleus and induce or repress TGF-beta-targeted gene expression (1, 58). In addition to the canonical Smads-mediated signaling, TGF-beta signals through several Smad-independent pathways (17). These pathways include activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 (3, 21), c-Jun/JNK (18a, 23), and PI 3-kinase/Akt pathways (2, 55, 60). Our data indicate that this TGF-beta-induced increase of elastin transcription was markedly attenuated by inhibition of PI 3-kinase activity and Akt2 expression but was not affected by inhibition of p38 or ERK1/2 activity or Akt1 expression.

We detected TGF-beta-induced activation of PI 3-kinase/Akt signaling pathway in human lung fibroblasts that was required for induction of elastin gene expression by TGF-beta. Similarly, PI 3-kinase/Akt pathway was found to interact with Smad3-dependent TGF-beta signaling to enhance {alpha}2(I) collagen expression in mesangial cells (2, 55). This activation of PI 3-kinase occurs via a non-Smad-mediated pathway (64). In some epithelial cell lines, signaling intermediates are required for TGF-beta-induced PI 3-kinase activation (68). We found that PI 3-kinase activity was required but not sufficient to induce elastin transcription. Addition of either IL-1beta or insulin increased PI 3-kinase activity but failed to increase elastin mRNA levels. PI 3-kinase activity likely modulates TGF-beta signal transduction downstream of Smad phosphorylation. PI 3-kinase generates PI 3,4,5-triphosphate and other lipid mediators (63). Class III PI 3-kinases generate constitutive levels of PI3 phosphate. This lipid mediator binds to the FYVE and pleckstrin homology domains of proteins (65). Interesting, the Smad anchor for receptor activation (SARA) contains an FYVE domain and functions to recruit Smad proteins to the activated TGF-beta receptor complex (62, 65). However, the activity of SARA is likely not affected by inhibition of PI 3-kinase activity. In the human Hep3B cell line, Akt interacts with Smad3 to sequester it outside the nucleus (14). These results suggested a model whereby unphosphorylated Akt binds to Smad3 with low affinity that can be readily dissociated by TGF-beta, resulting in nuclear translocation (14). In the presence of insulin, phosphorylated Akt forms a high affinity complex with Smad3 that is resistant to dissociation by TGF-beta. Our results indicate that this mechanism is not functional in human lung fibroblasts. Insulin induced large increases in phospho-Akt that did not affect basal or TGF-beta-induced elastin transcription (data not shown).

Our studies suggest that inhibition of histone deacetylase by TSA blocked TGF-beta-induced elastin expression by decreasing levels of phospho-Akt. For certain genes, treatment with TSA, a deacetylase inhibitor, resulted in an increase in histone acetylation and an increase in gene transaction. In contrast, we found that addition of TSA did not activate the elastin transcription, suggesting that acetylation of histones and the uncoiling of chromatin were not required to activate the elastin promoter. Similarly, TSA was shown to repress TGF-beta-induced increases in tissue inhibitor of metalloproteinases-1 (TIMP-1) expression (69). Together, these observations suggest that TSA blocked elastin gene expression through a mechanism other than induction of histone acetylation of the elastin gene. One potential mechanism involves inhibition of PI 3-kinase/Akt signaling pathway in a cell type-specific manner.

TGF-beta-induced Akt phosphorylation was downregulated by pretreatment with IL-1beta, which alone also causes the induction of Akt phosphorylation. Both IL-1beta and TSA may regulate phospho-Akt levels by modifying phosphatase 1 activity (9, 61). IL-1beta is known to increase nitric oxide synthase and production of nitric oxide that in turn may upregulate phosphatase activity (61). IL-1beta may also induce expression and binding of inhibitory C/EBPbeta proteins to elastin promoter to repress TGF-beta-induced elastin transcription as we have previously demonstrated in neonatal rat lung fibroblasts (36). Inhibition of protein synthesis with CHX markedly blocked upregulation of elastin hnRNA and mRNA expression, suggesting that TGF-beta requires de novo protein synthesis to synthesize or activate an important transcriptional activator to switch on elastin promoter for maximal activity.

We employed siRNA techniques to assess the role of the Akt1 and Akt2 isoforms. Downregulation of Akt2 but not Akt1 expression inhibited TGF-beta-induced upregulation of elastin hnRNA and mRNA levels. Akt1 and Akt2 were detectable by Western analysis in these human lung fibroblasts. These isoforms selectively activate downstream targets. Studies from Akt1- and Akt2-null mice indicate that both Akt1 and Akt2 are required for optimal animal growth and adipogenesis (11, 12, 19, 67). In breast epithelial cells, downregulation of Akt2, but not Akt1, inhibited proliferation and anti-apoptotic activity induced by activation of the insulin-like growth factor-I receptor (25). Akt2, but not Akt1, phosphorylates a serine residue in the protein Synip, resulting in disruption of Synip-Syntaxin4 interactions (66).

The downstream effectors required for Akt2-mediated elastin gene expression and regulation by TGF-beta are not yet defined. Phospho-Akt2 may be required to enhance the activity of Sp1. The elastin promoter contains a cluster of highly GC-rich sequences that function as Sp1/Sp3 binding cis-elements (4, 28, 30). Binding of Sp1/Sp3 to these sequences results in activation of the elastin promoter (15, 26, 28, 30). In other systems, levels of phospho-Akt correlate with Sp1 phosphorylation and binding. Induction of VEGF expression by PI 3-kinase/Akt signaling is mediated by increase of transcription from VEGF promoter in a Sp1-dependent manner (48).

TGF-beta increases the expression of multiple extracellular matrix genes through increases in the rate of transcription, or in the half-life of the mRNA (stabilization), or both (34, 61). In rodent cells, the TGF-beta-induced increase of elastin expression is mediated primarily by posttranscriptional stabilization of elastin mRNA. In human cells, increases in elastin mRNA were reported to occur through several signaling pathways, including Smads, protein kinase C{delta}, and p38 (33, 34, 41). The increased expression of elastin mRNA was attributed exclusively to changes in elastin mRNA stability, although the potential transcriptional activation of elastin was not examined. We found that inhibition of p38 MAP kinase by addition of SB-203580 partially blocked TGF-beta-induced increases in elastin mRNA but did not affect increases in hnRNA expression, confirming a role for p38 MAPK in stabilization of elastin mRNA.

The discrepancy between the previously reported data and our results likely resulted from both experimental design and utilization of different cell types. Rat primary lung fibroblast cultures produce tropoelastin that is crosslinked into elastin fibers without the addition of TGF-beta. These cells respond to the addition of exogenous TGF-beta with a minimal increase in elastin production (41). In contrast, TGF-beta markedly increases the transcription of the elastin gene in human lung fibroblasts. We do not yet know whether the tropoelastin is crosslinked into mature elastin fibers in this system. These cells produce both lysyl oxidase and fibulin-5 that are important components of elastogenesis (13, 32, 54). The failure of rat lung fibroblasts to marked response to TGF-beta may be caused by saturation of TGF-beta receptors, binding of TGF-beta to the extracellular matrix, or other undefined processes.

Our kinetic studies employing Taqman quantitative PCR measured the dynamic changes of elastin mRNA and hnRNA levels at very early time points. These studies demonstrate that activation of elastin transcription by TGF-beta is a very early event occurring at times points not examined in previous studies. The kinetics are similar to that observed for TGF-beta-induced increases in connective tissue growth factor (CTGF) that reach a maximum at 6 h following stimulation (52). CTGF is suggested to mediate TGF-beta-induced increases in collagen formation (20). These results suggest that CTGF was likely not involved in the activation of elastin transcription. Together, our data support the mechanism whereby TGF-beta activates elastin transcription by a mechanism that requires PI 3-kinase activity and subsequently increases elastin stability by a p38-dependent pathway.


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This study was supported by a grant from the American Lung Association (P.-P. Kuang) and National Heart, Lung, and Blood Institute Grants P01-HL-46902 and R01-HL-66547 (R. H. Goldstein).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P.-P. Kuang, The Pulmonary Center, R304, Boston Univ. School of Medicine, 80 E. Concord St., Boston, MA 02118 (email: ppkuang{at}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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