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Am J Physiol Lung Cell Mol Physiol 293: L790-L799, 2007. First published June 29, 2007; doi:10.1152/ajplung.00099.2007
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TGF-beta1 stimulates human AT1 receptor expression in lung fibroblasts by cross talk between the Smad, p38 MAPK, JNK, and PI3K signaling pathways

Mickey M. Martin,4 Jessica A. Buckenberger,4 Jinmai Jiang,2 Geraldine E. Malana,4 Daren L. Knoell,2,4 David S. Feldman,3,4 and Terry S. Elton1,4

Divisions of 1Pharmacology and 2Pharmaceutics, College of Pharmacy, 3Division of Cardiology, Department of Medicine, and 4Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio

Submitted 14 March 2007 ; accepted in final form 26 June 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Both angiotensin II (ANG II) and transforming growth factor-beta1 (TGF-beta1) are thought to be involved in mediating pulmonary fibrosis. Interactions between the renin-angiotensin system (RAS) and TGF-beta1 have been well documented, with most studies describing the effect of ANG II on TGF-beta1 expression. However, recent gene expression profiling experiments demonstrated that the angiotensin II type 1 receptor (AT1R) gene was a novel TGF-beta1 target in human adult lung fibroblasts. In this report, we show that TGF-beta1 augments human AT1R (hAT1R) steady-state mRNA and protein levels in a dose- and time-dependent manner in primary human fetal pulmonary fibroblasts (hPFBs). Nuclear run-on experiments demonstrate that TGF-beta1 transcriptionally activates the hAT1R gene and does not influence hAT1R mRNA stability. Pharmacological inhibitors and specific siRNA knockdown experiments demonstrate that the TGF-beta1 type 1 receptor (TbetaRI/ALK5), Smad2/3, and Smad4 are essential for TGF-beta1-stimulated hAT1R expression. Additional pharmacological inhibitor and small interference RNA experiments also demonstrated that p38 MAPK, JNK, and phosphatidylinositol 3-kinase (PI3K) signaling pathways are also involved in the TGF-beta1-stimulated increase in hAT1R density. Together, our results suggest an important role for cross talk among Smad, p38 MAPK, JNK, and PI3K pathways in mediating the augmented expression of hAT1R following TGF-beta1 treatment in hPFB. This study supports the hypothesis that a self-potentiating loop exists between the RAS and the TGF-beta1 signaling pathways and suggests that ANG II and TGF-beta1 may cooperate in the pathogenesis of pulmonary fibrosis. The synergy between these systems may require that both pathways be simultaneously inhibited to treat fibrotic lung disease.

G protein-coupled receptors; pulmonary fibrosis; angiotensin II; transforming growth factor-beta1; mitogen-activated protein kinase; c-Jun NH2-terminal kinase; phosphatidylinositol 3-kinase

IN THE CLASSIC RENIN-ANGIOTENSIN SYSTEM (RAS), circulating renal-derived renin cleaves hepatic-derived angiotensinogen to form the decapeptide angiotensin I (ANG I) (9). ANG I is subsequently converted by angiotensin-converting enzyme (ACE) in the lung to the biologically active octapeptide hormone angiotensin II (ANG II) (9). ANG II can also be generated by local tissue-specific RASs, including adrenal, blood vessel, brain, kidney, liver, and lung (8). Although ANG II was originally described as a potent vasoconstrictor, it is now recognized as a multifunctional hormone influencing many cellular processes including cell growth, apoptosis, migration, inflammation, and fibrosis (35, 37). Since ANG II is profibrotic, it is hypothesized that ANG II may, in part, play a role in the pathogenesis of pulmonary fibrosis (22, 23, 32).

The biological responses of ANG II are mediated by its interaction with two distinct high-affinity G protein-coupled receptors now designated AT1R and AT2R (9). Most of the known physiological and pathophysiological effects of ANG II are mediated via the AT1R. The multiple actions of ANG II, mediated through the AT1R, are a result of complex intracellular signaling pathways including stimulation of the PLC/inositol 1,4,5-trisphosphate/diacylglycerol cascade, MAPK/ERK, tyrosine kinases, and Rho/ROCK kinase (6, 30, 35, 37). In addition, AT1Rs mediate many of their pathophysiological effects by stimulating reactive oxygen species (ROS) generation via an NADH/NADPH oxidase-dependent mechanism (6, 37). ROS in turn influences downstream signaling molecules, including transcription factors, tyrosine kinases/phosphatases, Ca2+ channels, and MAP kinases (6, 37).

The AT1R is expressed in a number of cell types and organs, including the lung (9). Immunohistochemical studies have demonstrated that in the lung, the AT1R is expressed on alveolar macrophages, alveolar type II cells, bronchiolar epithelial cells, vascular smooth muscle cells, endothelial cells, and fibroblasts (5, 32). Importantly, in a rat bleomycin (BLM)-induced model of pulmonary fibrosis, AT1R expression was markedly increased in the lungs of these animals (32). Furthermore, ACE inhibitors and AT1R blockers (ARBs) attenuated BLM-induced pulmonary fibrosis (21, 23, 32, 39). Together, these studies suggest that increased signaling through the AT1R may be involved in mediating pulmonary fibrosis.

Transforming growth factor (TGF)-beta1 is a multifunctional cytokine that also plays an important role in progressive lung fibrosis. Patients with idiopathic pulmonary fibrosis (4, 16) or pulmonary fibrosis associated with systemic sclerosis (7) and some animal models of pulmonary fibrosis have shown increased lung TGF-beta1 production (20). In vitro studies demonstrated that ANG II stimulation increased TGF-beta1 synthesis in cultured human lung fibroblasts (23, 24). In addition, after BLM-induced lung injury, both ANG II and TGF-beta1 expression levels were increased in lung samples, and ARB treatment subsequently attenuated TGF-beta1 levels (23, 32). Finally, recent gene expression profiling experiments demonstrated that the AT1R gene was a novel TGF-beta1 target in human adult lung fibroblasts (33). Together, these studies suggest that there is a "positive feedback loop" between ANG II and TGF-beta1 that results in the amplification of their profibrotic effects.

Currently, very little information is known regarding the mechanisms by which TGF-beta1 enhances human AT1R (hAT1R) gene expression (33). Therefore, the following study was initiated to investigate these mechanisms. We demonstrated that in primary cultured human fetal pulmonary fibroblasts (hPFBs), TGF-beta1 treatment (4 ng/ml) maximally stimulated hAT1R steady-state mRNA levels at 4 h. Maximal protein and ANG II-induced signaling occurred 8 h after TGF-beta1 treatment. Together, our data support the hypothesis that TGF-beta1 treatment enhances AT1R expression by the synergistic interaction between the Smad and specific kinase signaling pathways that are simultaneously activated by activin receptor-like kinase (ALK5).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals, reagents, and antibodies. ANG II, actinomycin D (Act D), and cycloheximide were purchased from Sigma (St. Louis, MO). 125I-[Sar1, Ile8]-ANG II was purchased from the Peptide Radioiodination Service Center (University of Mississippi, University, MS). Human TGF-beta1 was purchased from R&D Systems (Minneapolis, MN). PD-98059, LY-294002, SB 203580, R031-8425, U0126, SP6000125, and SB 431542 were purchased from Calbiochem (San Diego, CA). Tubulin, ERK1/2, phospho-ERK1/2, Smad2, phospho-Smad2, Smad3, phospho-Smad3, Smad4, MAPK1, MEK1, phosphatidylinositol 3-kinase (PI3K), p38 MAPK, and JNK antibodies were purchased from Cell Signaling (Beverly, MA). ON-TARGETplus SMARTpool human Smad2 (L-003561), Smad3 (L-020027), Smad4 (L-003902), ALK5 (L-003929), MAPK1 (MEK1, L-003555), p38 MAPK{alpha} (MAPK14, L-003512), JNK1 (MAPK8, L-003514), PI3K (PIK3CA, L-003018), and siControl (D-001810) small interference RNAs (siRNAs) were purchased from Dharmacon (Lafayette, CO). The negative control siRNA has at least four mismatches to every known human gene and was microarray tested for "off" target activity.

Cell culture. hPFBs were established in primary culture from enzyme-dispersed tissue fragments adhered to Primaria culture plates of human neonatal lung tissue obtained at autopsy and were the generous gift of D. L. Knoell. Pulmonary fibroblasts were maintained in a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% FBS (HyClone), 80 U/ml penicillin, and 80 µg/ml streptomycin and Fungizone TM (Invitrogen). Cells were rendered quiescent by a 24-h exposure to HEPES-buffered DMEM containing 0.5% FBS and penicillin-streptomycin-Fungizone TM. Cells were used for ~7–10 passages before replacement with fresh early passage stocks. All cells were maintained in a humidified atmosphere of 5% CO2 at 37°C.

Adenovirus constructs and infection. Constitutively active ALK5 (caALK5) fused to green fluorescent protein (GFP) and empty adenovirus were kindly provided by Dr. Joey Barnett (Vanderbilt University). Confluent hPFBs were transduced with virus [multiplicity of infection 100] expressing empty vector or caALK5-GFP. Following 48 h of incubation, the hPFBs were harvested and utilized for Northern blot analyses.

Northern blot hybridization. hPFB cells were plated onto 100-mm dishes and grown to 70–80% confluence, washed once, and serum-starved for 24 h. The cells were subsequently incubated with TGF-beta1 for the times and concentrations indicated. Alternatively, after serum starvation, the cells were preincubated with various inhibitors or siRNAs for the times and concentrations indicated. Subsequently, total RNA was isolated from hPFB cells, and Northern blot hybridization experiments were performed as previously described (25, 26). To normalize the data for loading and transferring variations, the membrane was stripped and reprobed with a radiolabeled glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA. The hAT1R mRNA expression levels were normalized to the GAPDH values obtained by laser densitometry. All autoradiographs were calculated in the linear range of the signal.

AT1R radioligand binding studies. hPFB cells were plated on six-well dishes, grown to 70–80% confluence, washed once with PBS, and serum-starved for 24 h. The cells were pretreated with various pharmacological inhibitors and subsequently stimulated with 4 ng/ml TGF-beta1 for 0, 2, 4, 8, 12, or 24 h, or the cells were only stimulated with TGF-beta1. Alternatively, hPFBs were grown to 30–60% confluence and transiently transfected with control, ALK5, Smad2, Smad3, Smad4, MAPK1, p38 MAPK, JNK1, PI3K, or negative control siRNAs (25 nM final concentrations). Forty-eight hours after transfection, hPFBs were serum-starved for an additional 24 h and subsequently stimulated with TGF-beta1 (4 ng/ml, 8 h). Whole cell AT1R binding was measured as previously described (25, 26). Values presented represent specific (total minus nonspecific, i.e., total nontreated hPFBs ~8,000 cpm, and nonspecific ~500–800 cpm) binding and have been normalized with protein content. To ensure that TGF-beta1 treatment did not modulate the Kd of the AT1Rs, radioligand saturation isotherm experiments and Scatchard analysis were also performed as previously described (25, 26).

Western blot analyses. hPFB cells were plated and treated as described in Cell culture. These cells were subsequently lysed with RIPA buffer with freshly added protease and phosphatase inhibitors. Equal quantities (10 µg/well) of cell lysate were separated by 10% SDS-PAGE. Following transfer to nitrocellulose membrane and blocking with 5% nonfat milk, the blot was incubated with the appropriate antibody. The immunoblots were then incubated with a secondary antibody conjugated with horseradish peroxidase and visualized with enhanced chemiluminescence, and the autoradiograph was quantitated by densitometric analysis.

Nuclear run-on assays. hPFB cells were plated on six-well dishes, grown to 70–80% confluence, washed once with PBS, and serum-starved for 24 h. The cells were then stimulated with, or without, 4 ng/ml TGF-beta1 for 4 h. The cells were lysed, and nuclei were isolated by centrifugation. The nuclei (~2 x 107/reaction) were utilized to perform the in vitro transcription in a reaction mixture containing 40% glycerol, 50 mM Tris·HCl, pH 8.3, 5 mM MgCl2, 0.1 mM EDTA, and 25 mM of CTP, GTP, ATP, and UTP at 30°C for 30 min. RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's protocol. The RNA was subsequently treated with RNase-free DNase I, and cDNA was synthesized from the "run-on" transcripts using oligo(dT) (for hAT1R amplification) or 18S gene-specific primer. The expression of hAT1R mRNA relative to 18S rRNA was determined using SYBR green real-time quantitative PCR assay as described (26). Relative gene expression was calculated as Formula and was multiplied by 10–6 to simplify data presentation. The hAT1R-specific primers used were as follows: sense primer, 5'-CACCATGTTTTGAGGTTGACTGAC-3'; antisense primer, 5'-CAGGCTAGGGAGATTGCATTTCTG-3'.

hAT1R mRNA half-life assays. hPFB cells were plated on six-well dishes, grown to 70–80% confluence, washed once with PBS, and serum-starved for 24 h. The cells were then stimulated with or without 4 ng/ml TGF-beta1 for 4 h. The cells were then either collected (time 0, control) or treated with 3 µg/ml Act D to further block transcription of mRNA, and the cells were subsequently harvested at 1, 4, and 8 h. Total RNA was isolated and subjected to Northern blot analyses as described above. Alternatively, total RNA was utilized for real-time PCR experiments utilizing hAT1R and 18S gene-specific primers as described in Nuclear run-on assays.

Transfection. siRNAs (i.e., Smart Pool siRNAs from Dharmacon) were transfected into hPFBs by using magnet-assisted transfection (IBA, Gottingen, Germany) as instructed by the manufacturer (magnets were purchased separately from Engineered Concepts, Birmingham, AL). hPFB cell transfection conditions were optimized using a fluorescein-labeled double-stranded RNA oligomer designated BLOCK-iT fluorescent oligo (Invitrogen). siRNA transfection efficiency approached 100%. Forty-eight hours after transfection, hPFBs were serum-starved for an additional 24 h and subsequently stimulated with TGF-beta1 (4 ng/ml, 4 or 8 h) and either used for Northern blot analysis or subjected to AT1R radioligand binding assays as described above. Alternatively, hPFBs were transfected with a pGFP construct by using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. The cells were subsequently treated with or without cycloheximide (10 µg/ml), and the cells were imaged by confocal microscopy for GFP expression.

Statistical analysis. All data are means ± SE. When comparisons were made between two different groups, statistical significance was determined using Student's t-test. When multiple comparisons were made, statistical significance was determined using one-way ANOVA followed by Tukey's posttest. All statistical analyses were performed using the software package Prism 4.0b (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TGF-beta1 upregulates hAT1R steady-state mRNA and protein levels in human primary lung fibroblasts. Gene expression profiling of adult human lung fibroblasts treated with TGF-beta1 demonstrated that the hAT1R gene was an important target (33). To begin to investigate the mechanisms by which TGF-beta1 upregulates hAT1R gene expression, hPFBs were incubated with this stimulus for the times indicated (Fig. 1A). Northern blot experiments demonstrated that hAT1R steady-state mRNA levels were maximally increased at 4 h (i.e., ~6- to 8-fold vs. nonstimulated level) and returned to basal conditions by 24 h (Fig. 1, A and B). To determine the optimal dose for TGF-beta1-induced hAT1R mRNA upregulation, hPFBs were treated with increasing concentrations of TGF-beta1 for 4 h and total RNA was isolated. Northern blot analyses demonstrated that 4 ng/ml TGF-beta1 maximally stimulated hAT1R mRNA levels (Fig. 2, A and B).


Figure 1
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  		Fig. 1. Transforming growth factor-beta1 (TGF-beta1) stimulation upregulates human angiotensin II type 1 receptor (hAT1R) steady-state mRNA levels in human fetal pulmonary fibroblasts (hPFBs) in a time-dependent manner. hPFBs were grown to 70–80% confluence, washed twice, and serum-starved for 24 h. A: total RNA was isolated from TGF-beta1-treated (4 ng/ml) fibroblasts at the times indicated. RNA (20 µg) was fractionated, blotted, and probed with a radiolabeled cDNA specific for hAT1R mRNA. The Northern blot was stripped and reprobed with a GAPDH control cDNA to ensure that the relative quantity of the total RNA present in each lane was approximately equal. Data are representative of 3 separate experiments. B: TGF-beta1 time course of hAT1R and GAPDH Northern hybridization signal intensity was quantitated by densitometric analysis. Each point represents the relative hybridization signal (±SE) normalized to 0-h treatment with vehicle (100%) from 3 separate experiments. *P < 0.01, TGF-beta1-treated vs. nontreated hPFBs.

 

Figure 2
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  		Fig. 2. TGF-beta1 stimulation upregulates hAT1R steady-state mRNA levels in hPFBs in a dose-dependent manner. hPFBs were grown to 70–80% confluence, washed twice, and serum-starved for 24 h. A: hPFBs were treated with TGF-beta1 at the concentrations indicated for 4 h, and total RNA was isolated. RNA samples (20 µg) were fractionated, blotted, and probed with a radiolabeled hAT1R cDNA. The Northern blot was stripped and hybridized with a labeled GAPDH cDNA probe. Data are representative of 3 separate experiments. B: TGF-beta1 dose response of hAT1R and GAPDH Northern hybridization signal intensity was quantitated by densitometric analysis. Each point represents the relative hybridization signal (±SE) normalized to untreated hPFBs from 3 separate experiments. *P < 0.01, TGF-beta1-treated vs. nontreated hPFBs.

 
To investigate whether the increase in hAT1R steady-state mRNA levels equated to enhanced receptor expression, hAT1R density was quantitated by performing whole cell radioreceptor binding assays. AT1R binding assays demonstrated that TGF-beta1 (4 ng/ml) stimulation of hPFBs maximally increased hAT1R density (~5-fold) at 8 h after treatment (Fig. 3A). To ensure that TGF-beta1 treatment did not modulate the affinity of ANG II for the hAT1R, saturation isotherm experiments were performed and Scatchard analysis was conducted. These experiments demonstrated that although the maximal binding capacity (Bmax) values differed in the TGF-beta1-treated hPFB cells (i.e., 8 h) compared with the nontreated cells (Bmax: 584 ± 52 vs. 97 ± 23 fmol/mg protein), the Kd value (1.25 ± 0.31 nM) did not change (data not shown). To determine whether the enhanced hAT1R protein expression levels also resulted in augmented ANG II-induced signal transduction, TGF-beta1-treated or nontreated hPFBs were activated with 0.1 µM ANG II for 5 min, and phospho-ERK1/2 levels were determined. These results demonstrated that hPFBs treated with TGF-beta1 exhibited an approximately fourfold increase in ANG II-induced phospho-ERK1/2 levels (Fig. 3, B and C). Together, these experiments demonstrated that TGF-beta1 treatment not only increased hAT1R expression but also enhanced ANG II-induced signaling via the hAT1R.


Figure 3
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  		Fig. 3. TGF-beta1 stimulation increases AT1R protein levels and ANG II-induced ERK1/2 activation. hPFBs were grown to 70–80% confluence, washed twice, and serum-starved for 24 h. A: hPFBs were subsequently incubated with TGF-beta1 (4 ng/ml) for the times indicated, and AT1R radioreceptor binding assays were performed as described in EXPERIMENTAL PROCEDURES. Data are expressed as relative increase over nontreated (i.e., 0 h) hPFBs. Error bars represent SE of 3 independent experiments. *P < 0.01, TGF-beta1-treated vs. nontreated hPFBs. B: hPFBs were serum-starved for 24 h, incubated with TGF-beta1 (4 ng/ml, 8 h), and further activated with 0.1 µM ANG II for 5 min, and phospho-ERK1/2 activation was determined by Western blot analysis. The blot was stripped and reprobed with an ERK1/2-specific antibody. Data are representative of 3 separate experiments. C: each autoradiograph was quantitated by densitometric analysis, and ERK1/2 phosphorylation (p) was normalized with ERK1/2 protein levels and plotted as relative increase of ANG II-induced pERK1/2 over non-ANG II-induced pERK1/2 values. Error bars represent SE of 3 independent experiments. *P < 0.01, TGF-beta1-treated vs. nontreated hPFBs.

 
TGF-beta1 transcriptionally upregulates the hAT1R gene. Accumulation of hAT1R mRNAs within 4 h of TGF-beta1 treatment suggested that a transcriptional mechanism may be involved. To test this hypothesis, hPFBs were pretreated with the transcriptional inhibitor Act D (5 µg/ml) for 1 h before a 4-h combined TGF-beta1/Act D treatment. The data shown in Fig. 4A demonstrate that Act D pretreatment prevented TGF-beta1 activation of the hAT1R gene. This inhibition was not a result of Act D toxicity, since GAPDH steady-state mRNA levels were not influenced by this reagent (Fig. 4A).


Figure 4
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  		Fig. 4. TGF-beta1-enhanced hAT1R mRNA expression requires transcription but does not require protein synthesis. hPFBs were serum-starved for 24 h and then pretreated for 1 h with PBS or 5 µg/ml actinomycin D (Act D) to block transcription of mRNA (A) or with 10 µg/ml cycloheximide to block protein synthesis (B). Cells were subsequently treated with TGF-beta1 (4 ng/ml, 4 h), total RNA was isolated, and Northern blot analysis was performed as described. Data are representative of 3 separate experiments.

 
To investigate whether protein synthesis was required for TGF-beta1 induction of the hAT1R gene, hPFBs were pretreated with the protein translation inhibitor cycloheximide 1 h before a 4-h combined TGF-beta1/cycloheximide (10 µg/ml) treatment. These experiments demonstrated that hAT1R steady-state mRNA levels increased in a TGF-beta1-dependent manner irrespective of cycloheximide pretreatment, indicating that new protein synthesis is not required for TGF-beta1 induction of hAT1R gene expression (Fig. 4B). To ensure that the concentration of cycloheximide utilized did inhibit protein synthesis, control experiments were performed utilizing GFP-transfected hPFBs treated with or without cycloheximide for 4 h. Imaging of the transfected cells revealed no reporter gene expression in hPFBs treated with cycloheximide (data not shown). These data suggest that the hAT1R gene is a direct TGF-beta1 transcription target and that new protein synthesis is not necessary to activate the hAT1R gene.

To validate this hypothesis, hPFBs were incubated for 4 h with 4 ng/ml TGF-beta1 or vehicle before nuclear hnRNA de novo synthesis was assessed using nuclear run-on assays. These experiments demonstrated that TGF-beta1 induced an approximately threefold increase in the transcription rate of the hAT1R gene (Fig. 5A). In addition, hAT1R mRNA stability was also assessed following TGF-beta1 stimulation of hPFBs and subsequent incubation with Act D (Fig. 5B). The half-life of the hAT1R mRNA in TGF-beta1-treated and nontreated hPFBs was ~5.5 h (Fig. 5C). To verify the autoradiographic data, the hAT1R mRNA half-life was also calculated using RNA isolated from TGF-beta1-treated and nontreated hPFBs by utilizing RT-PCR. The values obtained with this procedure gave similar results (data not shown). It was concluded from these experiments that TGF-beta1 treatment augments hAT1R expression by increasing the transcription rate of the hAT1R gene and not by modulating hAT1R mRNA stability.


Figure 5
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  		Fig. 5. TGF-beta1 stimulation of hPFBs increases hAT1R mRNA levels through a transcriptional mechanism and does not affect hAT1R mRNA stability. A: hPFBs were serum-starved for 24 h and incubated with TGF-beta1 (4 ng/ml, 4 h), nuclei were isolated and subjected to nuclear run-on analyses, and hAT1R mRNA levels were quantitated utilizing RT-PCR as described in EXPERIMENTAL PROCEDURES. Error bars represent SE of 3 independent experiments. *P < 0.01, TGF-beta1-treated vs. nontreated hPFBs. B: hPFBs were serum-starved for 24 h and then incubated with TGF-beta1 (4 ng/ml, 4 hr). Cells were then either collected (time 0, control) or treated with 5 µg/ml Act D, to further block transcription of mRNA, and then harvested at 1, 4 and 8 h thereafter. AT1R and GAPDH mRNAs were detected by Northern blot analysis. Data are representative of 3 separate experiments. C: hAT1R and GAPDH mRNA levels were quantitated by densitometric analysis. hAT1R mRNA values were normalized to GAPDH expression at each time point. To obtain hAT1R values for the nontreated hPFBs, autoradiography was performed for 7 days (data not shown). The half-life of hAT1R mRNA in TGF-beta1-treated and nontreated cells was calculated as the time required for a given transcript to decrease to 50% of its initial abundance. Error bars represent SE of 3 independent experiments.

 
TGF-beta1 stimulation of hPFBs augments hAT1R mRNA expression through ALK5 and a Smad-dependent transcriptional mechanism. The physiological actions of TGF-beta are mediated by specific receptor complexes that are assembled upon ligand binding. TGF-beta1 binding to the type II TGF-beta receptor (TbetaRII) leads to recruitment, phosphorylation, and activation of the type I TGF-beta receptor (TbetaRI) (36). The activin receptor-like kinase (ALK5) has a ubiquitous distribution and represents the principle TbetaRI that mediates most of the cellular responses to TGF-beta1 (11). Once TGF-beta1-mediated ALK5 activation occurs, this receptor kinase subsequently phosphorylates Smad2 or Smad3, enabling association with the universal common Smad, Smad4, before nuclear translocation. These complexes translocate and accumulate in the nucleus, where they are directly involved in the transcriptional regulation of various target genes (10, 11). Therefore, to investigate whether ALK5 was involved in mediating the TGF-beta1-stimulated upregulation of hAT1R expression, a specific pharmacological inhibitor of ALK5 receptors was utilized (13, 31). hPFBs were treated with SB 431542 (1 µM, 1 h) followed by TGF-beta1 stimulation (4 ng/ml, 4 h), and Northern blot analyses and radioreceptor binding assays were subsequently performed. Pretreatment with SB 431542 completely abolished the TGF-beta1-mediated increases in hAT1R mRNA expression (Fig. 6A) and hAT1R protein levels (data not shown). To validate the specificity of SB 431542 in hPFB cells, Western blot experiments were performed utilizing phospho-Smad2 and -Smad3 antibodies. These experiments demonstrated that TGF-beta1 treatment led to the phosphorylation of Smad2 and Smad3 and that pretreatment of hPFBs with SB 431542 resulted in attenuated phosphorylation levels (Fig. 6B). To further confirm the involvement of ALK5 in specifically mediating the TGF-beta1 response, hPFBs were transduced with an adenovirus expressing a constitutively active ALK5 (caALK5-GFP) (29, 40), and Northern blot analyses and radioreceptor binding assays were again performed. Importantly, forced expression of caALK5 resulted in augmented expression of hAT1R mRNA (Fig. 6C) and hAT1R protein levels (data not shown), thus confirming the importance of ALK5 in this process. Control Western blot analyses were also performed to demonstrate that Smad2 and Smad3 were properly activated with the forced expression of caALK5 (Fig. 6D).


Figure 6
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  		Fig. 6. Inhibition of TGF-beta1 type 1 receptor (TbetaRI) activity abolishes TGF-beta1-stimulated increases in AT1R steady-state mRNA levels. A: hPFBs were serum-starved for 24 h, incubated with 1 µM SB 431542, a selective inhibitor of TGF-beta1 type 1 receptor kinase (ALK5), for 1 h, and subsequently stimulated with 4 ng/ml TGF-beta1 for 4 h. Total RNA was isolated, and Northern blot analysis was performed. Data are representative of 3 separate experiments. B: hPFBs were treated as described in A; however, cells were lysed in RIPA buffer and subjected to immunoblotting with phospho-specific or Smad2 and Smad3 antibodies. Data are representative of 3 separate experiments. C: hPFBs were grown to 30–60% confluence and subsequently transduced (100 multiplicity of infection) with adenovirus expressing constitutively active ALK5 (caALK5) or empty vector. Forty-eight hours after infection, total RNA was isolated and Northern analysis was performed. Data are representative of 3 separate experiments. D: hPFBs were treated as described in C; however, cells were lysed in RIPA buffer and subjected to immunoblotting with phospho-specific or Smad2 and Smad3 antibodies. Data are representative of 3 separate experiments.

 
To further investigate whether TGF-beta1 stimulation of hAT1R gene expression is mediated by a Smad-dependent pathway, hPFBs were transfected with control, ALK5-, Smad2-, Smad3-, or Smad4-specific siRNAs (25 nM final concentrations) and subsequently stimulated with TGF-beta1 (4 ng/ml, 8 h). Importantly, ANG II radioreceptor binding assays demonstrated that knockdown of ALK5, Smad2, Smad3, or Smad4 attenuated TGF-beta1 stimulation of hAT1R expression (Fig. 7A). In contrast, transfection of control siRNA had no effect on TGF-beta1-mediated changes in hAT1R density. Northern blot analyses of siRNA transfected cells treated with TGF-beta1 (4 ng/ml, 4 h) also demonstrated that each siRNA specifically reduced the hAT1R steady-state mRNA levels (data not shown). To confirm that the various siRNAs knocked down their appropriate target, Western blot analyses were performed. Immunoblotting results demonstrated that ALK5, Smad2, Smad3, and Smad4 protein levels were specifically reduced (Fig. 7B).


Figure 7
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  		Fig. 7. TGF-beta1-induced hAT1R expression is mediated by a Smad-dependent mechanism. A: hPFBs were grown to 30–60% confluence and transiently transfected with control, ALK5-, Smad2-, Smad3-, or Smad4-specific small interference RNAs (siRNAs; 25 nM final concentration). Forty-eight hours after transfection, hPFBs were serum-starved for an additional 24 h and subsequently stimulated with TGF-beta1 (4 ng/ml, 8 h), and AT1R radioreceptor binding assays were performed. Error bars represent SE of 3 independent experiments. *P < 0.01, TGF-beta1/ALK5, TGF-beta1/Smad2, TGF-beta1/Smad3, or TGF-beta1/Smad4 siRNA-treated vs. TGF-beta1-treated hPFBs. B: hPFBs were transfected and treated as described in A; however, cells were lysed and subjected to immunoblotting with the antibodies indicated. Tubulin immunoblots were performed as a protein loading control. Data are representative of 3 separate experiments.

 
TGF-beta1 stimulation of hPFB also enhances hAT1R mRNA expression by activating specific kinase signaling pathways. Although the Smad pathway is the main mediator of TGF-beta1 signaling, recent studies have implicated other pathways such as ERK1/2, p38 MAPK, PI3K, JNK, and PKC as either mediators or modulators of TGF-beta1-dependent biological effects (reviewed in Refs. 11, 28). To begin to investigate whether TGF-beta1 activation of these kinase signaling pathways also plays a role in the augmentation of hAT1R gene expression, hPFBs were pretreated with specific pharmacological inhibitors of these pathways. These cells were subsequently stimulated with TGF-beta1 (4 ng/ml, 4 h) and subjected to Northern blot analysis utilizing a radiolabeled probe specific for the hAT1R mRNA. These experiments demonstrated that PI3K (LY-294002), p38 MAPK (SB 203580), and JNK (SP6000125) inhibitors significantly attenuated TGF-beta1-induced hAT1R gene expression (i.e., ~6–7-fold, Fig. 8, A and B). In contrast, ERK1/2 (PD-98059, U0126) and PKC (R031-8425) inhibitors did not attenuate TGF-beta1-induced augmentation of hAT1R steady-state mRNA levels. Identical experiments measuring hAT1R density demonstrated that PI3K, p38 MAPK, and JNK inhibitors also prevented TGF-beta1-induced increases in hAT1R protein levels (data not shown). To validate the pharmacological inhibitor experiments, siRNA knockdown experiments were performed as described in Fig. 7. Importantly, PI3K, p38 MAPK, and JNK siRNAs attenuated TGF-beta1-induced hAT1R gene expression (Fig. 9A). Immunoblot experiments demonstrated that although MAPK1 siRNAs decreased MAPK1 protein expression (Fig. 9B), this siRNA did not decrease TGF-beta1-induced hAT1R gene expression (Fig. 9A), confirming that this signaling pathway is not involved. Together, these data suggest that TGF-beta1-mediated upregulation of hAT1R gene expression involves additional signaling pathways beyond the traditional Smad-mediated mechanisms.


Figure 8
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  		Fig. 8. TGF-beta1-induced hAT1R expression can be attenuated by PI3K, p38 MAPK (p38K), and JNK pharmacological inhibitors. A: hPFBs were grown to 70–80% confluence, washed twice, and serum-starved for 24 h. Cells were preincubated for 30 min with the following inhibitors at the indicated concentrations: PD-98059 (10 µM, MAPK inhibitor), LY-294002 [5 µM, phosphatidylinositol 3-kinase (PI3K) inhibitor], SB 203580 (1 µM, p38K inhibitor), SP6000125 (10 µM, JNK inhibitor), R031-8425 (10 µM, PKC inhibitor), and U0126 (10 µM, ERK inhibitor). After pretreatment with the various inhibitors, cells were stimulated with 4 ng/ml TGF-beta1 for 4 h. Total RNA was isolated, and Northern analysis was performed. B: hAT1R steady-state mRNA levels were quantitated by densitometric analysis. All values were normalized to GAPDH mRNA levels. Error bars represent SE of 3 independent experiments. *P < 0.01, TGF-beta1 + inhibitor vs. TGF-beta1.

 

Figure 9
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  		Fig. 9. TGF-beta1-induced hAT1R expression can be attenuated by PI3K, p38K, and JNK siRNAs. A: hPFBs were grown to 30–60% confluence and transiently transfected with control, MAPK1-, PI3K-, p38K-, or JNK-specific siRNAs (25 nM final concentration). Forty-eight hours after transfection, hPFBs were serum-starved for an additional 24 h and subsequently stimulated with TGF-beta1 (4 ng/ml, 8 h), and AT1R radioreceptor binding assays were performed. Error bars represent SE of 3 independent experiments. *P < 0.01, TGF-beta1/PI3K, TGF-beta1/p38K, or TGF-beta1/JNK siRNA-treated vs. TGF-beta1-treated hPFBs. B: hPFBs were transfected and treated as described in A; however, cells were lysed and subjected to immunoblotting with the antibodies indicated. Tubulin immunoblots were performed as a protein loading control. Data are representative of 3 separate experiments.

 

   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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In this study, we have analyzed the mechanisms by which TGF-beta1 regulates hAT1R gene expression in hPFBs. For the first time, we have demonstrated that TGF-beta1 stimulation of hPFBs activates hAT1R gene transcription, which, in turn, leads to a robust upregulation of hAT1R steady-state mRNA levels (~6- to 8-fold). Importantly, the increased hAT1R mRNA levels resulted in augmented hAT1R protein densities (~5-fold increase 8 h after TGF-beta1 treatment), which correlated with enhanced ANG II-induced signaling via the hAT1R. We also present evidence that the ubiquitously expressed TGF-beta1 type I receptor, ALK5, mediates TGF-beta1 activation of the hAT1R gene. This conclusion was based on the observations that forced expression of caALK5 mimicked TGF-beta1 stimulation of the hAT1R gene and that TGF-beta1 augmentation of hAT1R gene expression was attenuated by an ALK5-specific pharmacological inhibitor or by transfection of an ALK5-specific siRNA.

It is well established that once ALK5 is activated by TGF-beta1, the downstream receptor-regulated Smads (R-Smads) are phosphorylated at two distal serine residues located in the COOH terminus. Subsequently, the phosphorylated R-Smads (Smad2 and Smad3) associate with a co-Smad, Smad4, and enter the nucleus to modulate the transcription of TGF-beta1-responsive genes (11, 36). Consensus DNA-binding sequences for R-Smad/Smad4 complexes have been identified that contain the palindrome GTCTAGAC, half-sites of this sequence, or CAGA motifs (10, 14, 45–47). R-Smad/Smad4 complexes are capable of binding to DNA alone, but they do so with low affinity, and their interaction with additional transcription factors is required for target gene regulation (12, 42, 46, 47).

We present siRNA knockdown evidence that Smad2/3 and Smad4 are involved in the TGF-beta1 induction of the hAT1R gene. Computer analysis (MatInspector; http://www.genomatix.com) of several thousand base pairs of the hAT1R promoter sequence demonstrated that several putative Smad binding elements are harbored in this region, including one binding site at –1355 bp and another at +50 bp (with respect to the transcription initiation start site; Ref. 48). We were unable to activate hAT1R promoter luciferase constructs that encompassed both of the sites described above with TGF-beta1 (data not shown). Since we have clearly shown that TGF-beta1 transcriptionally activates the hAT1R gene, we hypothesize that the appropriate Smad binding element(s) needed for TGF-beta1 activation is(are) missing in our reporter constructs. Therefore, we are currently generating new reporter constructs that harbor additional putative Smad binding sites further upstream within the hAT1R promoter region.

Many studies have demonstrated that in addition to Smads, other signaling pathways such as ERK1/2, p38 MAPK, JNK, PI3K, Rho/ROCK, PKC, and CaMKII have also been shown to mediate TGF-beta1 function (reviewed in Refs. 11, 28). We present evidence that PI3K, p38 MAPK, and JNK signaling pathways are critical to TGF-beta1-mediated induction of hAT1R expression in hPFBs. This conclusion was based on the observation that blockade of PI3K, p38 MAPK, and JNK signaling with any of the pharmacological inhibitors LY-294002, SB 203580, or SP6000125 or kinase-specific siRNAs significantly attenuated expression of the hAT1R gene. In contrast, PD-98059 or U0126, two well-known inhibitors of the ERK1/2 signaling pathways, and blockade of PKC signaling with the inhibitor R031-8425 demonstrated that these pathways were not essential for the TGF-beta1-mediated upregulation of the hAT1R gene, since pretreatment with these inhibitors did not block the TGF-beta1 response. The utilization of a MAPK1-specific siRNA confirmed that this signaling pathway was not involved. Although PKC-specific siRNAs were not utilized in this study, other investigators (11, 28) have utilized 10 µM R031-8425 to successfully inhibit the PKC signaling pathways. Together, our studies suggest that TGF-beta1-mediated induction of hAT1R expression results from the ability of TGF-beta1 to activate Smads, PI3K, p38 MAPK, and JNK since the elimination of any of these signaling pathways attenuates the potential for TGF-beta1 to stimulate hAT1R expression. Therefore, we conclude that the Smad and kinase signaling pathways do not act independently but involve some level of intracellular cross talk or scaffolding.

In support of our hypothesis, several studies from other investigators have suggested that the PI3K signaling pathway can be modulated by TGF-beta1 (1, 17, 34, 43). The activation of PI3K signaling by TGF-beta1 may be direct, since coimmunoprecipitation between the p85 subunit of PI3K and both TbetaRI and TbetaRII has been demonstrated in airway smooth muscle cells (17). In addition, it has been shown that the activated TbetaRI serine-threonine kinase can potently induce PI3K activity (43). Interestingly, Bakin et al. (1) showed that the PI3K inhibitor LY-294002 blocked TGF-beta1-induced Smad2 phosphorylation in breast cancer cells, suggesting that Smad proteins may be potential targets of the PI3K pathway. Furthermore, Runyan et al. (34) demonstrated that TGF-beta1 stimulated the PI3K/Akt pathway, which, in turn, augmented the ability of Smad3 to transcriptionally activate collagen I expression in human mesangial cells. These investigators also demonstrated that TGF-beta1 activation of PI3K resulted in the phosphorylation of Smad3 at serine residues other than the direct TbetaRI/ALK5 target site located in the COOH terminus.

In further support of our observations, it has been shown that TGF-beta1 can activate the p38 kinase and JNK signaling pathways, possibly through the activation of TGF-beta-activated kinase (TAK1/Map3K7) (15, 44). Recent studies by Kamaraju and Roberts (15) demonstrated that the p38 MAPK pathway is activated by TGF-beta1, and this activation results in the phosphorylation of Smad2/3 in the linker region. Since COOH-terminal phosphorylation of Smad2 and 3 was not affected by p38 MAPK inhibitors, these investigators surmised that these pathways were activated in parallel and independently of one another. In addition, they concluded that hierarchically, the p38 MAPK pathway is likely upstream to Smad signaling and modulates Smad function through phosphorylation in the linker regions of Smad2 and Smad3. Finally, Yoshida et al. (44) demonstrated that in hepatic stellate cells treated with TGF-beta1, JNK and p38 MAPK were activated. Importantly, they demonstrated that the activated kinases could directly phosphorylate Smad2 and Smad3 in their linker regions, which in turn resulted in augmented PAI-1 transcription rates. Together, these studies demonstrate that there is interdependence between Smad signaling and specific kinase pathways that may involve the phosphorylation of specific amino acids localized in the linker region of Smad2 and Smad3. On the basis of these studies, we hypothesize that in hPFBs, TGF-beta1 stimulation of hAT1R expression arises from the synergy of the activation of Smads and the PI3K, p38 MAPK, and JNK signaling pathways, which, in turn, results in the phosphorylation of Smad2/3 at multiple sites (Fig. 10). Once the R-Smad/Smad4 complex is formed and translocated into the nucleus, we speculate that hyperphosphorylation of Smad2/3 in the linker and COOH-terminal regions allows for the recruitment of specific transcriptional coactivators that are required to stimulate the expression of a novel subset of TGF-beta1-inducible genes (i.e., the hAT1R gene) (Fig. 10).


Figure 10
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  		Fig. 10. Schematic model of the proposed synergistic interaction among TGF-beta1 activation of Smads, PI3K, p38K, and JNK signaling pathways, and augmented hAT1R gene expression. Traditionally, TGF-beta1 signaling is initiated by ligand binding to the transmembrane receptors TbetaRI (i.e., ALK5) and TbetaRII. The activated TbetaRI subsequently phosphorylates Smad2 and Smad3 within their conserved COOH-terminal SSXS motif (11, 36). These activated Smad proteins, together with Smad4, translocate to the nucleus and regulate the transcription of target genes. Our study demonstrates that there is intracellular cross talk between the described Smad pathway and the PI3K, p38K, and JNK signaling pathways. We propose that activation of PI3K, p38K, and JNK by TbetaRI/TbetaRII leads to phosphorylation (P) of Smad2/3 at additional serine/threonine sites located in the linker region of these proteins. The hyperphosphorylated Smad2/3, together with Smad4, are translocated to the nucleus, specific coactivators are recruited to the transcriptional complex, and hAT1R gene expression is stimulated. Alternatively, TGF-beta1 activation of PI3K, p38K, and JNK may result in the direct or indirect phosphorylation of distinct transcription factors, which translocate to the nucleus and merge their signals with the activated R-Smad/Smad4 complex, and hAT1R gene expression is subsequently activated.

 
An alternative explanation for intracellular cross talk among the Smad, PI3K, p38 MAPK, and JNK pathways for TGF-beta1-stimulated hAT1R expression is that each kinase pathway phosphorylates specific transcriptional coactivators that are necessary for the activation of the hAT1R gene (Fig. 10). In support of this model, it has been demonstrated that the downstream targets of JNK include the transcription factors c-Jun, ATF-2, ELK-1, and p53 (27). Targets of the p38 MAPK include multiple transcription factors such as MEF2, ATF-2, ELK-1, Ets-1, and p53 (27). Together, these studies clearly demonstrate that the kinase pathways that are activated by TGF-beta1 can stimulate the phosphorylation of many transcription factors; therefore, we speculate that the Smad, PI3K, p38 MAPK, and JNK pathways merge their signals within the nucleus to mediate TGF-beta1-stimulated hAT1R gene expression (Fig. 10). Therefore, if one kinase pathway is inhibited, the transcriptional complex would be missing a key component and hAT1R gene expression would not be activated by TGF-beta1. Our current studies cannot distinguish between the two proposed models and must be the focus of future research.

Convincing evidence indicates that ANG II, via the AT1R, activates the TGF-beta1 axis in the lung by both direct and indirect mechanisms (2, 18, 21–23, 32, 39). For example, ANG II is mitogenic for human fetal and adult lung fibroblasts in vitro, and this response is attenuated by anti-TGF-beta1 antibodies, suggesting that the mitogenic effect is mediated by autocrine production of TGF-beta1 (23, 24). TGF-beta1 is a potent profibrotic cytokine: it enhances fibroblast chemotaxis and proliferation and induces extracellular matrix synthesis; therefore, TGF-beta1 has a decisive role in pulmonary fibrotic diseases (2, 41). In a variety of forms of pulmonary pathology, including chronic lung disease of prematurity as well as several forms of acute and chronic adult lung disease, the expression of TGF-beta1 is increased (2). Interestingly, TGF-beta1 has now been shown to activate specific components of the RAS axis (18, 41). Specifically, TGF-beta1 stimulates angiotensinogen gene expression in proximal tubular cells (3). Furthermore, TGF-beta1 was shown to enhance hAT1R gene expression in lung fibroblasts (33), adrenal cells (19), and trophoblasts (38). Our current findings have extended these observations by demonstrating that TGF-beta1 augmentation of hAT1R protein levels results from the interdependence of ALK5 activation of Smad and specific kinase signaling pathways. These studies support the existence of a self-potentiating loop between the RAS and TGF-beta1 system. The clinical implications of these studies are highly significant, since the resulting amplification of the profibrotic effects of both systems may play a role in mediating the pathogenesis of pulmonary fibrosis. Although ACE inhibitors and ARBs can block experimental models of lung fibrosis (18), there are currently no published prospective or retrospective studies regarding the use of these drugs in humans with pulmonary fibrosis. Therefore, it is still unclear whether inhibition of the RAS would indeed have beneficial effects on lung fibrosis. Together, these studies may suggest the need to pharmacologically inhibit both systems to treat fibrotic diseases.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL48848 (to T. S. Elton), American Heart Association Award GRT00001380 (to T. S. Elton), and NHLBI Grant HL084498-01A2 (to D. S. Feldman).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. S. Elton, Davis Heart and Lung Research Institute, The Ohio State Univ., DHLRI 515, 473 West 12th Ave., Columbus, OH 43210 (e-mail: terry.elton{at}osumc.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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 DISCUSSION
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