ACTIVATION OF ELASTIN TRANSCRIPTION BY TRANSFORMING GROWTH FACTOR� IN HUMAN LUNG FIBROBLASTS

Regulation of elastin gene expression LCMP-00184-2006. R2

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ACTIVATION OF ELASTIN TRANSCRIPTION BY TRANSFORMING GROWTH

FACTOR-β IN HUMAN LUNG FIBROBLASTS

Ping-Ping Kuang*, Xiao-Hui Zhang, Celeste B. Rich, Judith A. Foster, Mangalalaxmy

Subramanian and Ronald H. Goldstein

From the Pulmonary Center and the Department of Biochemistry at Boston University

School of Medicine, and the Boston VA Healthcare System, Boston, MA 02118

*To whom correspondence should be sent:

Ping-Ping Kuang, M.D., Ph.D.

The Pulmonary Center, R 304

Boston University School of Medicine

80 E. Concord Street

Boston, MA 02118

Tel. (617) 638-4860

Fax (617) 536-8093

Email: [email protected].

Running title: Regulation of elastin gene expression

of 39Articles in PresS. Am J Physiol Lung Cell Mol Physiol (January 5, 2007). doi:10.1152/ajplung.00184.2006

Copyright © 2007 by the American Physiological Society.


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ABSTRACT

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-β (TGF-β) induced rapid and

transient increases in levels of elastin heterogeneous nuclear mRNA (hnRNA) followed

by increases of elastin mRNA and protein expression. In fibroblasts derived from

transgenic mice, TGF-β-induced increases in the expression of a human elastin gene

promoter fragment driving a chloroamphenicol acetyl transferase (CAT) reporter gene.

The induction of elastin hnRNA and mRNA expression by TGF-β was abolished by

pretreatments with TGF-β 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

SB203580 also partially attenuated the levels of TGF-β-induced elastin mRNA but not its

hnRNA. Western analysis indicated that TGF-β-stimulated Akt phosphorylation.

Inhibition of phosphatidylinositol 3-kinase and Akt phosphorylation by LY292004

abolished TGF-β-induced increases in elastin hnRNA and mRNA expression. Treatment

of lung fibroblasts with interleukin-1β or the histone deacetylase inhibitor trichostatin A

inhibited TGF-β-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 (siRNA) duplexes blocked TGF-β induced

increases of elastin hnRNA and mRNA levels. Taken together, our results demonstrated

that the TGF-β activates elastin transcription that is dependent on phosphatidylinositol 3-

kinase /Akt activity.

Keywords: heterogeneous nuclear mRNA, lung, emphysema, siRNA,

phosphatidylinositol 3-kinase /Akt.

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INTRODUCTION

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). FGF-18 increases elastin mRNA levels in fetal and postnatal rat lung

fibroblasts (8). Elastin mRNA levels are down-regulated by FGF-2, IL-1β, and TNF-α (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 TGF-β

signal transduction. Deficiency of Smad3 leads to repression of tropoelastin expression

in lung and the development of centrilobular emphysema (10). TGF-β 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-β co-

localizes with elastin in vascular tissues during periods of active elastin synthesis (56).

Mice deficient in Smad3 or the β6 integrin develop airspace enlargement (10, 44).

Smad3 is a component of the TGF-β signal transduction pathway and the β6 integrin is

involved in TGF-β activation. These observations suggest that lung fibroblasts under the

influence of active TGF-β are required to deposit elastin in alveolar structures. We have

found that TGF-β is upregulated in the bronchoalveolar lavage fluid after elastolytic injury

to the mouse lung (7). TGF-β mRNA levels were also found to be upregulated in the

lungs of individuals with chronic obstructive lung disease (COPD) (Gold criteria: stage 2)

(45).

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TGF-β 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-β has focused on the ability of TGF-β to stabilize the elastin mRNA (34,

61). In the present study, we have investigated regulation of elastin expression by TGF-

β in human lung fibroblasts by examination of levels of elastin hnRNA and mRNA. Our

data reveal that induction of elastin mRNA and protein by TGF-β are correlated to a

large rapid increase in elastin transcription, which may require phosphatidylinositol 3-

kinase (PI3-kinase)/Akt signaling and involve de novo protein synthesis and p38 MAPK

pathway.

EXPERIMENTAL PROCEDURES

Cell Culture and reagents. Cycloheximide (CHX), actinomycin D (ActD) and TGF-β

type I receptor inhibitor (TRI), SB-431542, were obtained from Sigma (St. Louis, MO).

Trichostatin A (histone deacetylase inhibitor) was purchased from Upstate (Lake Placid,

NY). LY294002 (LY), U0126, and SB2035580 were obtained from Calbiochem (La Jolla,

CA) and recombinant human TGF-β1 and IL-1β were from R&D systems, Inc.

(Minneapolis, MN). Human lung fibroblasts (IMR-90) were purchased from ATCC and

maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA)

supplemented with 10% fetal bovine serum (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 37o C. Confluent cultures were rendered quiescent by culturing in serum-

free medium for 24 h prior to experimentation.

Total RNA Isolation and Northern blot Analysis. Serum-starved confluent fibroblasts

were untreated or treated with TGF-β (1 ng/ml) for various time periods. For inhibitor

studies, the pretreatments were performed with cycloheximide (10 µg/ml), actinomycin D

(10 µg/ml), SB-431542 (20 µM), LY294002 (10 µM), U0126 (10 µM) and SB2035580 (25

µM) for 1 h prior to treatment with TGF-β (1 ng/ml) for 16 hrs. Total RNA was prepared

with RNeasy (Qiagen, CA) according to the manufacturer’s protocol, followed by

Northern analysis using 32P-labeled cDNA probes for human elastin and GAPDH. The

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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 manufacturer’s protocol. Taqman

reagents for detecting mRNA expression of elastin or GAPDH were purchased from

Applied Biosystems Inc. (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) by Applied Biosystems Inc. (Foster

City, CA). 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

triplicates 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 -2260 to +2 of the human elastin gene promoter fragment driving 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 chloroamphenicol acetyl transferase

antibody (Abcam, ab5410). RNA was run on a Northern 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 4oC in presence of protease

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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, Beverly, MA) 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 non-targeting

control siRNA were purchased from Upstate (manufactured by Dharmacon, Lafayette,

CO). Subconfluent IMR-90 cells (6x105 cells/100 µl) were harvested and resuspended

in Nucleofector Solution (Amaxa). Nucleofaction 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 pre-optimized program (U-23). Immediately

following nucleofection, the cells were plated into 6-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-β for 4 h before 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

We investigated the effect of TGF-β 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-β (Fig.

1). Equal loading was verified by assessment of levels of the 18S ribosomal subunit.

To determine the effect of TGF-β on the production of intact tropoelastin protein, we

performed Western blot analyses (Fig. 2). Human lung fibroblasts were treated with

TGF-β and cell lysates were harvested for analysis. We did not detect tropoelastin in

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untreated cultures. At 8 h following TGF-β treatment, tropoelastin was readily

detectable, and a further increase was observed in tropoelastin at 24 h following TGF-β

administration (Fig. 2). The results were similar whether we used lung fibroblasts

derived from fetal or adult human lung (data not shown).

To determine the effect of TGF-β on the transcription of the elastin gene, we examined

expression of heterogeneous nuclear RNA (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-β 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-β-induced transcriptional activation of elastin gene was

verified by using of the global transcriptional inhibitor actinomycin D and TGF-β receptor

I inhibitor, in which Northern analysis showed that pretreatment with either actinomycin

D (10 µg/ml) or TGF-β receptor I inhibitor (10 µM) completely abolished the increase of

elastin mRNA by TGF-β (Fig. 3B).

We also examined the effect of TGF-β on lung fibroblasts derived from transgenic mice

containing a 2.26 kb human elastin promoter linked to chloramphenicol acetyl

transferase (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-β 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-β-induced increase in elastin promoter

activity,

We employed protein synthesis and kinase inhibitors to gain insight into the signal

transduction pathway utilized by TGF-β to increase elastin mRNA (Fig. 5A). The

upregulation of elastin mRNA was partially dependent on protein synthesis as

cycloheximide (CHX) partially blocked the upregulation of the elastin mRNA (Fig. 5A).

Treatment with P38 inhibitor (SB203580) resulted in minimal inhibition and no inhibition

with treatment with the MEK1/2 inhibitor U0126. Real time quantitative PCR revealed

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that addition of CHX, but not p38-inhibitor inhibited TGF-β-induced elastin hnRNA

expression (Fig. 5B).

We found that inhibition of PI3-kinase with LY294002 completely blocked TGF-β-induced

elastin mRNA and hnRNA expression (Fig. 5A and B). Treatment with insulin induced

large increases in phospho-Akt but did not affect elastin mRNA levels or TGF-β-induced

increases in elastin transcription (data not shown). Western analysis confirmed that

LY294002 inhibited both basal and TGF-β-induced increased in phospho-Akt (Fig. 6).

We employed IL-1β and the histone deacetylase inhibitor trichostatin A (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-1β induced phospho-Akt

but did not affect elastin mRNA levels (Fig. 7). Interestingly, the combined treatment of

fibroblasts with IL-1β and TGF-β yielded unexpected results. IL-1β treatment inhibited

both TGF-β induced phospho-Akt (Fig. 7) and elastin mRNA and hnRNA levels (Fig. 6).

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 under-acetylation 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-β (Fig. 8B). Western

analysis revealed that TSA treatment inhibited TGF-β-induced increases in phospho-Akt

(Fig. 8C).

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. 9A and B). Downregulation of levels of Akt2 mRNA but not Akt1

mRNA markedly inhibited TGF-β-induced increases in the level of elastin mRNA (Fig.

9C) and hnRNA (Fig. 9D).

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DISCUSSION

In this study, we investigated the molecular mechanisms underlying TGF-β-mediated

elastin gene regulation using human lung fibroblasts. Under basal conditions, elastin

mRNA expression was undetectable by Northern analysis. Treatment with TGF-β

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 was minimally increased by exogenous

TGF-β (41). The explanation for these species-specific differences in elastin expression

is unknown.

TGF-β is a member of a subfamily of multifunctional growth factors implicated in various

physiologic and pathologic conditions, including regulation of cell proliferation,

differentiation, apoptosis, migration, and syntheses of extracellular matrix (ECM)

proteins (40). Initiation of TGF-β 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 recruits the co-mediator Smad4. The

Smads complexes translocate into the nucleus and induce or repress TGF-β targeted

gene expression (1, 58). In addition to the canonical Smads-mediated signaling, TGF-β

signals through several Smad-independent pathways (17). These pathways include

activation of extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 (3, 21), c-Jun/JNK

(23, 42), and PI3-kinase/Akt pathways (2, 55, 60). Our data indicate that this TGF-β-

induced increase of elastin transcription was markedly attenuated by inhibition of PI3-

kinase activity and Akt2 expression, but was not affected by inhibition of p38 or ERK-1/2

activity or Akt1 expression.

We detected TGF-β-induced activation of PI3-kinase/Akt signaling pathway in human

lung fibroblasts that was required for induction of elastin gene expression by TGF-β.

Similarly, PI3-kinase/Akt pathway was found to interact with Smad3-dependent TGF-β

signaling to enhance α2(I) collagen expression in mesangial cells (2, 55). This activation

of PI3-kinase occurs via a non-Smad mediated pathway (64). In some epithelial cell

lines, signaling intermediates are required for TGF-β-induced PI3-kinase activation (68).

We found that PI3-kinase activity was required but not sufficient to induce elastin

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transcription. Addition of either IL-1β or insulin increased PI3-kinase activity but failed to

increase elastin mRNA levels. PI3-kinase activity likely modulates TGF-β signal

transduction downstream of Smad phosphorylation. PI3-kinase generates PI-3, 4, 5-

triphosphate and other lipid mediators (63). Class III PI3-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 a FYVE domain and functions to recruit Smad proteins to the activated TGF-β

receptor complex (62, 65). However the activity of SARA is likely not affected by

inhibition of PI3-kinase activity. In 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 Smad 3 with low affinity that can be readily dissociated by

TGF-β 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-β.

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-β-

induced elastin transcription (data not shown).

Our studies suggest that inhibition of histone deacetylase by TSA blocked TGF-β-

induced elastin expression by decreasing levels of phospho-Akt. For certain genes,

treatment with Trichostatin A (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-β-induced increases in tissue

inhibitor of metalloproteinases-1 (TIMP-1) expression (69). Taken 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 PI3-kinase/Akt signaling pathway in cell-type specific manner.

TGF-β-induced Akt phosphorylation was downregulated by pretreatment with IL-1β,

which alone also cause the induction of Akt phosphorylation. Both IL-1β and TSA may

regulate phospho-Akt levels by modifying phosphatase 1 activity (9, 61). IL-1β is known

to increase NO synthase and production of NO that in turn may up-regulate phosphatase

activity (61). IL-1β may also induce expression and binding of inhibitory C/EBPβ

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proteins to elastin promoter to repress TGF-β-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-β 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-β-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 antiapoptotic 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-β 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 PI3-kinase/Akt signaling is mediated by increase of

transcription from VEGF promoter in a Sp1 dependent manner (48).

TGF-β increases the expression of multiple extracellular matrix genes through either

increases in the rate of transcription, or in the half-life of the mRNA (stabilization), or

both (34, 61). In rodent cells, the TGF-β-induced increase of elastin expression is

mediated primarily by posttranscriptional stabilization of elastin mRNA. In human cells,

increases in elastin mRNA was reported to occur through several signaling pathways,

including Smads, protein kinase C-δ, and p38 (33, 34, 41). The increased expression of

elastin mRNA was attributed exclusively to changes in elastin mRNA stability although

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the potential transcriptional activation of elastin was not examined. We found that

inhibition of p38 MAP kinase by addition of SB203580 partially blocked TGF-β 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 previous 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-β. These cells respond to the addition of exogenous TGF-β with a

minimal increase in elastin production (41). In contrast, TGF-β 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 respond to TGF-β may be

caused by either saturation of TGF-β receptors, binding of TGF-β 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-β is a very early event

occurring at times points not examined in previous studies. The kinetics is similar to that

observed for TGF-β-induced increases in connective tissue growth factor (CTGF) that

reaches a maximum at 6 hrs following stimulation (52). CTGF is suggested to mediate

TGF-β induced increases in collagen formation (20). These results suggest that CTGF

was likely not involved in the activation of elastin transcription. Taken together, our data

support the mechanism whereby TGF-β activates elastin transcription by a mechanism

that requires PI3-kinase activity and subsequently increases elastin stability by a p38

dependent pathway.

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Acknowledgements—This study was supported by grants from the American Lung

Association (P-P.K.), and National Institutes of Health (P01 HL46902, R.H.G; R01

HL66547, R.H.G).

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REFERENCES

1. Attisano L, and Wrana JL. Smads as transcriptional co-modulators. Curr Opin Cell

Biol 12, 235–243, 2000.

2. Asano Y, Ihn H, Yamane K, Jinnin M, Mimura Y, and Tamaki K.

Phosphatidylinositol 3-kinase is involved in alpha2(I) collagen gene expression in normal

and scleroderma fibroblasts. J Immunol 172, 7123–7135, 2004.

3. Bakin AV, Rinehart C, Tomlinson AK, and Arteaga CL. p38 mitogen-activated

protein kinase is required for TGFbeta-mediated fibroblastic transdifferentiation and cell

migration. J Cell Sci 115, 3193–3206, 2002.

4. Bashir MM, Indik Z, Yeh H, Ornstein-Goldstein N, Rosenbloom JC, Abrams W,

Fazio M, Uitto J, and Rosenbloom J. Characterization of the complete human elastin

gene. Delineation of unusual features in the 5'-flanking region. J Biol Chem 264, 8887–

8891, 1989.

5. Brettell LM, McGowen SE. Basic fibroblast growth factor decreases elastin

production by neonatal rat lung fibroblasts. Am J Resp Cell Mol Biol 10, 306–315, 1994.

6. Bruce MC, and Honaker CE. Transcriptional regulation of tropoelastin expression in

rat lung fibroblasts: changes with age and hyperoxia. Am J Physiol 274, L940–L950,

1998.

7. Buczek-Thomas JA, Lucey EC, Stone PJ, Chu CL. Rich CB, Carreras I, Goldstein

RH, Foster JA, and Nugent MA. Elastase mediates the release of growth factors from

lung in vivo. Am J Respir Cell Mol Biol 31, 344–350, 2004.

8. Chailley-Heu B, Boucherat O, Barlier-Mur AM, and Bourbon JR. FGF-18 is

upregulated in the postnatal rat lung and enhances elastogenesis in myofibroblasts Am J

Physiol 288, L43–L51, 2005.

of 39


15

9. Chen CS, Weng SC, Tseng PH, Lin HP, and Chen CS. Histone acetylation-

independent effect of histone deacetylase inhibitors on Akt through the reshuffling of

protein phosphatase 1 complexes. J Biol Chem 280, 38879–38887, 2005

10. Chen H, Sun J, Buckley S, Chen C, Warburton D, Wang XF, and Shi W.

Abnormal mouse lung alveolarization caused by Smad3 deficiency is a developmental

antecedent of centrilobular emphysema. Am J Physiol 288, L683–L691, 2005.

11. Chen WS, Xu PZ, Gottlob K, Chen ML, Sokol K, Shiyanova T, Roninson I, Weng

W, Suzuki R, Tobe K, Kadowaki T, and Hay N. Growth retardation and increased

apoptosis in mice with homozygous disruption of the Akt1 gene. Genes Dev 15, 2203–

2208, 2001.

12. Cho H, Thorvaldsen JL, Chu Q, Feng F, and Birnbaum MJ. Akt1/PKBalpha is

required for normal growth but dispensable for maintenance of glucose homeostasis in

mice. J Biol Chem 276, 38349–38352, 2001.

13. Choung J, Taylor L, Thomas K, Zhou X, Kagan H, Yang X, Polgar P. Role of EP2

receptors and cAMP in prostaglandin E2 regulated expression of type I collagen alpha1,

lysyl oxidase, and cyclooxygenase-1 genes in human embryo lung fibroblasts.

J Cell Biochem 71, 254–263, 1998.

14. Conery AR, Cao Y, Thompson EA, Townsend CM Jr, Ko TC, and Luo K. Akt

interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis.

Nat Cell Biol 6, 366–372, 2004

15. Conn KJ, Rich CB, Jensen DE, Fontanilla MR, Bashir MM, Rosenbloom J, and

Foster JA. Insulin-like growth factor-I regulates transcription of the elastin gene through

a putative retinoblastoma control element. A role for Sp3 acting as a repressor of elastin

gene transcription. J Biol Chem 271, 28853–28860, 1996.

16. Davidson JM, Zoia O, and Liu JM. Modulation of transforming growth factor-beta1

stimulated elastin and collagen production and proliferation in porcine vascular smooth

of 39


16

muscle cells and skin fibroblasts by basic fibroblast growth factor, transforming growth

factor-alpha and insulin-like growth factor-I. J Cell Physiol 155, 149–156, 1993.

17. Derynck R, and Zhang YE. Smad-dependent and Smad-independent pathways in

TGF-beta family signalling. Nature 425, 577–584, 2003.

18. Elferink CJ, and Reiners JJ Jr. Quantitative RT-PCR on CYP1A1 heterogeneous

nuclear RNA: a surrogate for the in vitro transcription run-on assay. Biotechniques 20,

470–477, 1996.

19. Garofalo RS, Orena SJ, Rafidi K, Torchia AJ, Stock JL, Hildebrandt AL,

Coskran T, Black SC, Brees DJ, Wicks JR, McNeish JD, Coleman KG. Severe

diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice

lacking Akt2/PKB beta. J. Clin. Invest 112, 197–208, 2003.

20. Grotendorst GR, and Duncan MR. Individual domains of connective tissue growth

factor regulate fibroblast proliferation and myofibroblast differentiation. FASEB J 19,

729–738, 2005

21. Hartsough MT, and Mulder KM. Transforming growth factor beta activation of

p44mapk in proliferating cultures of epithelial cells. J Biol Chem 270, 7117–7124, 1995.

22. Heegaard AM, Xie Z, Young MF and Nielsen KL. Transforming growth factor beta

stimulation of biglycan gene expression is potentially mediated by Sp1 binding factors. J

Cell Biochem 93, 463–475, 2004.

23. Hocevar BA, Brown TL, and Howe PH. TGF-ß induces fibronectin synthesis

through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway. EMBO J

18, 1345–1356, 1999.

24. Inagaki Y, Truter S, and Ramirez F. Transforming growth factor-beta stimulates

alpha 2(I) collagen gene expression through a cis-acting element that contains a Sp1-

binding site. J Biol Chem 269, 14828–14834, 1994.

of 39


17

25. Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N,

Natesan S, and Brugge JS. Distinct roles of Akt1 and Akt2 in regulating cell migration

and epithelial-mesenchymal transition. J Cell Biol 171, 1023–1034, 2005

26. Jensen DE, Rich CB, Terpstra AJ, Farmer SR, and Foster JA. Transcriptional

regulation of the elastin gene by insulin-like growth factor-I involves disruption of Sp1

binding. Evidence for the role of Rb in mediating Sp1 binding in aortic smooth muscle

cells. J Biol Chem 270, 6555–6563, 1995.

27. Kahari VM, Chen YQ, Bashir MM, Rosenbloom J, and Uitto J. Tumor necrosis

factor-alpha down-regulates human elastin gene expression. Evidence for the role of

AP-1 in the suppression of promoter activity. J Biol Chem 267, 26134–26141, 1992

28. Kahari VM, Fazio MJ, Chen YQ, Bashir MM, Rosenbloom J, and Uitto J. Deletion

analyses of 5'-flanking region of the human elastin gene. Delineation of functional

promoter and regulatory cis-elements. J Biol Chem 265, 9485–9490, 1990.

29. Kahari VM, Olsen DR, Rhudy RW, Carillo P, Chen YQ, and Uitto J. Transforming

growth factor–beta upregulates elastin gene expression in human skin fibroblasts.

Evidence for a post transcriptional modulation Lab Invest 66, 580–588, 1992.

30. Kuang PP, Berk JL, Rishikof DC, Foster JA, Humphries DE, Ricupero DA, and

Goldstein RH. NF-κB induced by IL-1β inhibits elastin transcription and the

myofibroblast phenotype. Am J Physiol Cell Physiol 283, C58–C65, 2002.

31. Kuang PP, and Goldstein RH. Regulation of elastin gene transcription by

interleukin-1 beta-induced C/EBP beta isoforms. Am J Physiol Cell Physiol 285, C1349–

C1355, 2003.

32. Kuang PP, Joyce-Brady M, Zhang XH, Jean JC, and Goldstein RH. Fibulin-5

gene expression in human lung fibroblasts is regulated by TGF-beta and

phosphatidylinositol 3-kinase activity. Am J Physiol Cell Physiol. 291, C1412-C1421,

2006.

of 39


18

33. Kucich U, Rosenbloom JC, Abrams WR, Bashir MM, and Rosenbloom J.

Stabilization of elastin mRNA by TGF-beta: initial characterization of signaling pathway.

Am J Respir Cell Mol Biol 17, 10–6, 1997.

34. Kucich U, Rosenbloom JC, Abrams WR, and Rosenbloom J. Transforming

growth factor-beta stabilizes elastin mRNA by a pathway requiring active Smads, protein

kinase C-delta, and p38. Am J Respir Cell Mol Biol 26, 183–188, 2002.

35. Kuhn C, Yu SY, Chraplyvy M, Linder HE, and Senior RM. Induction of

emphysema with elastase. II Changes in connective tissue. Lab Invest 34, 372–380,

1976.

36. Kwak HJ, Park MJ, Cho H, Park CM, Moon SI, Lee HC, Park IC, Kim MS, Rhee

CH, and Hong SI. Transforming growth factor-beta1 induces tissue inhibitor of

metalloproteinase-1 expression via activation of extracellular signal-regulated kinase and

Sp1 in human fibrosarcoma cells. Mol Cancer Res 4, 209–220, 2006.

37. Lai CF, Feng X, Nishimura R, Teitelbaum SL, Avioli LV, Ross FP, and Cheng

SL. Transforming growth factor-beta up-regulates the beta 5 integrin subunit expression

via Sp1 and Smad signaling. J Biol Chem 275, 36400–36406, 2000.

38. Leask A, and Abraham DJ. TGF-beta signaling and the fibrotic response. FASEB J

18, 816–27, 2004.

39. Lucey EC, Ngo HQ, Agarwal A, Smith BD, Snider GL, and Goldstein RH.

Differential expression of elastin and α1(I) collagen mRNA in mice with bleomycin-

induced pulmonary fibrosis. Lab Invest 74,12–20, 1996.

40. Massague J. TGF-beta signal transduction. Annu Rev Biochem 67, 753–791, 1998.

41. McGowan SE, Jackson SK, Olson PJ, Parekh T, and Gold LI. Exogenous and

endogenous transforming growth factors-beta influence elastin gene expression in

cultured lung fibroblasts. Am J Respir Cell Mol Biol 17, 25–35, 1997.

of 39


19

42. Engel ME, McDonnell MA, Law BK, and Moses HL. Interdependent SMAD and

JNK Signaling in Transforming Growth Factor- -mediated Transcription. J Biol Chem

274, 37413–37420, 1999.

43. Monneret C. Histone deacetylase inhibitors. Eur J Med Chem 40, 1–13, 2005.

44. Morris DG, Huang X, Kaminski N, Wang Y, Shapiro SD, Dolganov G, Glick A,

and Sheppard D. Loss of integrin alpha(v)beta6-mediated TGF-beta activation causes

MMP12-dependent emphysema. Nature 422, 169–173, 2003.

45. Ning W, Li CJ, Kaminski N, Feghali-Bostwick CA, Alber SM, Di YP, Otterbein

SL, Song R, Hayashi S, Zhou Z, Pinsky DJ, Watkins SC, Pilewski JM, Sciurba FC,

Peters DG, Hogg JC, and Choi AM. Comprehensive gene expression profiles reveal

pathways related to the pathogenesis of chronic obstructive pulmonary disease. Proc

Nat Acad Sci USA 101, 14895–14900, 2004.

46. Pierce RA, Mariencheck WI, Sandefur S, Crouch EC, and Parks WC.

Glucocorticoids upregulate tropoelastin expression during late stages of fetal lung

development. Am J Physiol 268, L491–L500, 1995.

47. Poncelet AC, and Schnaper HW. Sp1 and Smad proteins cooperate to mediate

transforming growth factor-beta 1-induced alpha 2(I) collagen expression in human

glomerular mesangial cells. J Biol Chem 276, 6983–6992, 2001.

48. Pore N, Liu S, Shu HK, Li B, Haas-Kogan D, Stokoe D, Milanini-Mongiat J,

Pages G, O'Rourke DM, Bernhard E, and Maity A. Sp1 is involved in Akt-mediated

induction of VEGF expression through a HIF-1-independent mechanism. Mol Biol Cell

15, 4841-4853, 2004.

49. Qureshi HY, Sylvester J, Mabrouk ME, and Zafarullah M. TGF-beta-induced

expression of tissue inhibitor of metalloproteinases-3 gene in chondrocytes is mediated

by extracellular signal-regulated kinase pathway and Sp1 transcription factor. J Cell

Physiol 203, 345–352, 2005.

of 39


20

50. Reddy SA, Huang JH, and Liao WS. Phosphatidylinositol 3-kinase in interleukin 1

signaling. Physical interaction with the interleukin 1 receptor and requirement in NF

kappa B and AP-1 activation. J Biol Chem 272, 29167–29173, 1997.

51. Rich CB, Carreras I, Lucey EC, Jaworski JA, Buczek-Thomas JA, Nugent MA,

Stone P, and Foster JA. Transcriptional regulation of pulmonary elastin gene

expression in elastase-induced injury. Am J Physiol Lung Cell Mol Physiol 285, L354–

L362, 2003.

52. Ricupero DA, Rishikof DC, Kuang PP, Poliks CF, and Goldstein RH. Regulation

of connective tissue growth factor expression by prostaglandin E(2). Am J Physiol 277,

L1165–L1171, 1999.

53. Rolz W, Xin C, Ren S, Pfeilschifter J, and Huwiler A. Interleukin-1 inhibits

angiotensin II-stimulated protein kinase B pathway in renal mesangial cells via the

inducible nitric oxide synthase. Eur J Pharmacol 442, 195–203, 2002.

54. Roy R, Polgar P, Wang Y, Goldstein RH, Taylor L, and Kagan HM. Regulation of

lysyl oxidase and cyclooxygenase expression in human lung fibroblasts: interactions

among TGF-beta, IL-1 beta, and prostaglandin E. J Cell Biochem 62, 411-417, 1996.

55. Runyan CE, Schnaper HW, and Poncelet AC. The phosphatidylinositol 3-

kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in

response to transforming growth factor-beta1. J Biol Chem 279, 2632–2639, 2004.

56. Sauvage M, Hinglais N, Mandet C, Badier C, Deslandes F, Michel JB, and Jacob

MP. Localization of elastin mRNA and TGF-beta1 in rat aorta and caudal artery as a

function of age. Cell Tissue Res 291, 305–314, 1998.

57. Schiller M, Javelaud D, and Mauviel A. TGF-beta-induced SMAD signaling and

gene regulation: consequences for extracellular matrix remodeling and wound healing. J

Derm Sci 35, 83–92, 2004.

of 39


21

58. Shi Y, and Massague J. Mechanisms of TGF-beta signaling from cell membrane to

the nucleus. Cell 113, 685–700, 2003.

59. Sizemore N, Leung S, and Stark GR. Activation of phosphatidylinositol 3-kinase in

response to interleukin-1 leads to phosphorylation and activation of the NF-kappa B

p65/RelA subunit. Mol. Cell Biol 19, 4798–4805, 1999.

60. Song K, Cornelius SC, Reiss M, and Danielpour D. Insulin-like growth factor-I

inhibits transcriptional responses of transforming growth factor-beta by

phosphatidylinositol 3-kinase/Akt-dependent suppression of the activation of Smad3 but

not Smad2. J Biol Chem 278, 38342–38351, 2003.

61. Swee MH, WC Parks, and Pierce RA. Developmental regulation of elastin

production. Expression of tropoelastin pre-mRNA persists after downregulation of

steady-state mRNA levels. J Biol Chem 270, 14899–14906, 1995.

62. Tsukazaki T, Chiang TA, Davison AF, Attisano L, and Wrana JL. SARA, a FYVE

domain protein that recruits Smad2 to the TGFbeta receptor. Cell 95, 779–791, 1998

63. Vanhaesebroeck B and Waterfield MD. Signaling by distinct classes of

phosphoinositide-3-kinases. Exp Cell Res 253, 239–254, 1999.

64. Wilkes MC, Mitchell H, Penheiter SG, Dore JJ, Suzuki K, Edens M, Sharma DK,

Pagano RE, and Leof EB. Transforming growth factor-beta activation of

phosphatidylinositol 3-kinase is independent of Smad2 and Smad3 and regulates

fibroblast responses via p21-activated kinase-2. Cancer Res 65, 10431–10440, 2005.

65. Wurmser AE, Gary JD, and Emr SD. Phosphoinositide 3-kinases and their FYVE

domain-containing effectors as regulators of vacuolar/lysosomal membrane trafficking

pathways. J Biol Chem 274, 9129–9132, 1999.

66. Yamada E, Okada S, Saito T, Ohshima K, Sato M, Tsuchiya T, Uehara Y,

Shimizu H, and Mori M. Akt2 phosphorylates Synip to regulate docking and fusion of

GLUT4-containing vesicles. J Cell Biol 168, 921-928, 2005

of 39


22

67. Yang ZZ, Tschopp O, Hemmings-Mieszczak M, Feng J, Brodbeck D, Perentes

E, and Hemmings BA. Protein kinase B alpha/Akt1 regulates placental development

and fetal growth. J Biol Chem 278, 32124–32131, 2003.

68. Yi JY. Shin I. and Arteaga CL. Type I transforming growth factor beta receptor

binds to and activates phosphatidylinositol 3-kinase. J Biol Chem 280,10870–10876,

2005.

69. Young DA, Billingham O, Sampieri CL, Edwards DR, Clark IM. Differential effects

of histone deacetylase inhibitors on phorbol ester- and TGF-beta1 induced murine tissue

inhibitor of metalloproteinases-1 gene expression. FEBS J 272, 1912-1926, 2005.

70. Zhang M, Pierce RA, Wachi H, Mecham RP, and Parks WC. An open reading

frame element mediates posttranscriptional regulation of tropoelastin and

responsiveness to transforming growth factor beta1. Mol Cell Biol 19, 7314–7326, 1999.

FOOTNOTES

The abbreviations used are: Smad, son of mothers against dpp; hnRNA, heterogeneous

nuclear RNA; TGF-β, transforming growth factor-β; IL-1β, interleukin-1β; GAPDH; TSA,

Trichostatin A; C/EBPβ, CAAT-enhancer binding protein β; CHX, cycloheximide; PI3-

kinase, Phosphatidylinositol kinase; CTGF, connective tissue growth factor.

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FIGURE LEGENDS

Figure 1. Effect of TGF-β 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-β1 (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 three similar experiments.

Figure 2. Effect of TGF-β on tropoelastin expression by human lung fibroblasts. Serum-

starved confluent fibroblasts were untreated (C) or treated with TGF-β (T, 1 ng/ml) for 8h

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.

Figure 3. Effect of TGF-β on the transcription of the elastin gene as determined by

levels of hnRNA. A. Total RNA was isolated from serum-starved cells that were

untreated (C) or treated with TGF-β (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 ± S.E. of 3 independent

experiments performed in triplicates. B. Serum-starved confluent lung fibroblasts were

pretreated with either actinomycin D (Act D, 15 µg/ml) or TGF-β receptor I inhibitor SB-

431542 (TRI) (10 µM) for 1 h prior to addition of TGF-β (1 ng/ml) for 16 h. Total RNA

was isolated followed by Northern analysis using 32P-labeled human elastin cDNA and

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32P-labeled oligonucleotide of 18S ribosome subunit. Data are representative of three

separate experiments.

Figure 4. Effect of TGF-β on transcription of the elastin gene as determined by levels of

CAT protein expression. Lung fibroblasts were derived from transgenic mice expressing

a 2.2 kb elastin promoter driving a CAT reporter gene. After growing 4 or 5 days in

culture, fibroblasts were untreated or treated with TGF-β (T) for 24 hrs. 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 panel) and histone (bottom panel) mRNA using corresponding

32P-labeled cDNA probes.

Figure 5. Dissection of signaling pathways that may be involved in TGF-β-induced

upregulation of elastin expression. A. Serum-starved confluent fibroblasts were

untreated (C) or treated with TGF-β1 (1 ng/ml) (T), or IL-1β (IL, 250 pg/ml) or both (T/IL),

or pretreated with cycloheximide (CHX) (10 µg/ml), LY294002 (LY) (10 µM), SB2035580

(SB) (25 µM), or U0126 (10 µM) for 1 h before addition of TGF-β1 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 three similar experiments. B. Fibroblasts were similarly treated

with TGF-β1 (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 ± S.E. of 3 independent experiments performed in

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triplicates. (*denotes significant difference from untreated control. **denotes significant

difference from TGF-β-treated fibroblasts. (P<0.05))

Figure 6. Effect of LY294002 on TGF-β-induced Akt phosphorylation. Serum-starved

confluent cells were untreated (Control) or treated with TGF-β1 (1 ng/ml) (TGF),

LY294002 (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

(Ser473). The Akt antibody against total Akt protein was used as a loading control.

Data are representative of three similar experiments.

Figure 7. Effect of IL-1β on TGF-β-induced Akt phosphorylation. Serum-starved

confluent cells were untreated (Control) or treated with IL-1β (250 pg/ml) (IL), TGF-β1 (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 three similar

experiments.

Figure 8. Effect of inhibition of histone deacetylase by TSA on TGF-β-induced regulation

of elastin mRNA, hnRNA and Akt phosphorylation. A. Serum-starved confluent

fibroblasts were untreated (C) or treated with IL-1β (250 pg/ml) (IL), TGF-β1 (1 ng/ml)

(T), their combination (T/IL), TSA (10 µM), TSA plus TGF-β (T/TSA), U0126 (10 µM) or

U0126 plus TGF-β (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 three separate

experiments. B. Fibroblasts were untreated (C) or treated with TGF-β (TGF, 1 ng/ml)

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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 ± S.E.

of 3 independent experiments performed in triplicates. C. Cytosolic extracts were

isolated from serum-starved confluent fibroblasts that were untreated (Control) or treated

with TGF-β1 (1 ng/ml), TSA (10 µM), or both (TSA/TGF-β). Western analysis was

sequentially performed using antibodies against phospho-Akt (Ser473) and the Akt

antibody against total Akt protein. Data are representative of three similar experiments.

Figure 9. Effect of downregulation of Akt1 and Akt2 mRNA on elastin mRNA and

hnRNA levels. Fibroblasts were treated with a scrambled non-targeting 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-β (T) for 4 h before 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, *denotes

significant difference from untreated control. **denotes significant difference from TGF-β-

treated fibroblasts. (P<0.05))

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Figure 1

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Figure 2

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Figure 3A

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Figure 5B

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Figure 6

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Figure 7

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Figure 8A

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Figure 8B

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Figure 8C

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Figure 9

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Fo

ld c

han

ges

of A

kt2

mR

NA

100 nm

100 nm

100 nm

50 nm

50 nm

50 nm

50 nm

- -

- -

-

-

-

-

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

C T C T C T C T C T

si-Control

si-Akt1

si-Akt2

Fo

ld c

han

ges

of A

kt2

mR

NA

100 nm

100 nm

100 nm

50 nm

50 nm

50 nm

50 nm

- -

- -

-

-

-

-

* ** *

of 39

ACTIVATION OF ELASTIN TRANSCRIPTION BY TRANSFORMING GROWTH FACTOR� IN HUMAN LUNG FIBROBLASTS

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Transcript of ACTIVATION OF ELASTIN TRANSCRIPTION BY TRANSFORMING GROWTH FACTOR� IN HUMAN LUNG FIBROBLASTS