cGMP Inhibits TGF-β Signaling by Sequestering Smad3 with Cytosolic β2-Tubulin in Pulmonary Artery Smooth Muscle Cells (2024)

Two-dimensional differential proteomic and MS analyses of cytosolic Smad3-anchoring proteins in TGF-β1-treated PASMC with or without cGMP pretreatment

To test our novel hypothesis that cGMP treatment limits TGF-β-induced Smad3 nuclear translocation by enhancing Smad3 binding to cytosolic anchoring proteins, we carried out a differential proteomic analysis to identify candidate cytosolic proteins for Smad3 binding. Isolated PASMC were pretreated with cGMP or vehicle for 1 h followed by exposure to TGF-β1 or vehicle for 1 h, and the cytosolic and nuclear proteins were isolated. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone deacetylase 1 (HDAC1) as markers of the cytosolic and nuclear fractions (4), respectively, we demonstrated that the isolated cytosolic fraction was not contaminated with nuclear proteins (Fig. 1A). Cytosolic extracts were immunoprecipitated with anti-Smad3 to enrich for Smad3-interacting proteins, and the proteomes were profiled by 2D-DIGE. Differential proteomic expression among vehicle, TGF-β1, and cGMP + TGF-β1-treated cells was analyzed by DeCyder image analysis software.

TGF-β1 treatment significantly decreased Smad3 binding to 48 cytosolic proteins and increased Smad3 binding to four cytosolic proteins compared with vehicle-treated cells (Fig. 1B, left image, and Supplemental Table 1, published on The Endocrine Society's Journals Online web site at http://mend.endojournals.org). cGMP + TGF-β1 treatment increased Smad3 binding to 13 proteins and decreased Smad3 binding to 39 proteins compared with vehicle (Fig. 1B, middle image, and Supplemental Table 1). To test the effect of cGMP pretreatment on Smad3 interaction with cytosolic proteins in the presence TGF-β1, we focused on the comparison between the cGMP + TGF-β1 group and the TGF-β1 group. cGMP + TGF-β1 treatment increased Smad3 binding to 44 proteins and decreased Smad3 binding to eight proteins compared with TGF-β1 alone (Fig. 1B, right image, and Supplemental Table 1).

Among the 44 proteins that showed increased Smad3 binding with cGMP pretreatment, we selected six with fold increased more than 2 for further MS analysis (Fig. 1C). MS analysis identified the six proteins of interest as the cytoskeletal protein β2-tubulin, a lys-63-specific deubiquitinase BRCC36, vimentin and its three isoforms, and aldose reductase (Table 1). Because it has been reported previously that Smad3 binds to cytosolic microtubules in endothelial and epithelial cells (5, 6) and because cGMP treatment resulted in the highest fold (3.33) increase in Smad3 binding to β2-tubulin in our study, we focused on the Smad3-β2-tubulin interaction for further investigation.

TGF-β1 treatment decreases, but cGMP treatment increases, Smad3 binding to β2-tubulin in PASMC

We used a reciprocal Co-IP assay to further examine the Smad3-β2-tubulin interaction in PASMC. Quiescent PASMC were pretreated with cGMP or vehicle for 1 h and exposed to TGF-β1 or vehicle for 1 h. Whole cell protein extracts were subjected to IP with anti-Smad3 antibody followed by immunoblotting (IB) with anti-β2-tubulin antibody (Fig. 2A). β2-Tubulin was bound to Smad3 in vehicle-treated cells. TGF-β1 treatment led to a significant decrease in β2-tubulin binding to Smad3. cGMP treatment caused a marked increase in β2-tubulin binding to Smad3 compared with vehicle treatment and abolished the inhibitory effect of TGF-β1 on β2-tubulin binding to Smad3. In the reverse Co-IP, whole cell protein extracts were subjected to IP with selective anti-β2-tubulin antibody, followed by IB with anti-Smad3 antibody (Fig. 2B). We confirmed that Smad3 constitutively binds to β2-tubulin in the vehicle-treated cells. TGF-β1 treatment significantly decreased the binding of Smad3 to β2-tubulin, whereas cGMP treatment significantly increased Smad3 binding to β2-tubulin compared with vehicle treatment. Importantly, cGMP pretreatment abolished the TGF-β1-induced dissociation of Smad3 from β2-tubulin. ANP treatment mimicked the enhancing effects of cGMP on the interaction between Smad3 and β2-tubulin and inhibited TGF-β1-induced plasminogen activator inhibitor (PAI)-1 mRNA expression (Supplemental Fig. 1). These results suggest that the ANP-cGMP-PKG pathway and TGF-β signaling play counterregulatory roles in regulating the constitutive binding of Smad3 to β2-tubulin in PASMC.

TGF-β1 treatment decreases, but cGMP treatment increases, the colocalization of cytosolic Smad3 with β2-tubulin in PASMC

To test whether Smad3 colocalizes with β2-tubulin in the cells, we performed immunofluorescence staining with anti-Smad3 and anti-β2-tubulin antibodies. In vehicle-treated cells, Smad3 was distributed uniformly in the cytosol and nucleus, whereas β2-tubulin formed a typical filament-like structure around the nucleus (Fig. 3). Merging the images clearly demonstrated colocalization of Smad3 and β2-tubulin. Consistent with the results of the Co-IP studies, TGF-β1 treatment caused a significant reduction in colocalization of Smad3 and β2-tubulin and led to a significant nuclear accumulation of Smad3. In contrast, cGMP treatment significantly increased Smad3 colocalization with β2-tubulin compared with the vehicle control group. Further, cGMP pretreatment significantly blocked TGF-β1-induced Smad3 nuclear accumulation and increased Smad3 colocalization with β2-tubulin in the cytosol compared with the TGF-β1-treated group.

cGMP-induced hyperphosphorylation of Smad3 enhances the interaction between Smad3 and β2-tubulin

Our previous results suggested that PKG activation-induced hyperphosphorylation of Smad3 at Ser309 and Thr388 residues may play a critical role in the prevention of Smad3 nuclear translocation (7). To test the hypothesis that hyperphosphorylated Smad3 binds to β2-tubulin in the presence of cGMP, wild-type Smad3, S309G-Smad3, T388A-Smad3, or the empty vector were transiently transfected into HEK293 cells. Overexpression of Smad3 was confirmed by Western blot analysis. Compared with cells transfected with the empty vector, overexpression of Smad3 increased Smad3 binding to β2-tubulin in vehicle-treated cells. cGMP treatment potentiated Smad3 binding to β2-tubulin in cells transfected with wild-type Smad3 or S309G-Smad3 and significantly inhibited TGF-β1-induced PAI-1 mRNA expression (Supplemental Fig. 2). In contrast, mutation of Smad3 at the Thr388 significantly attenuated cGMP-induced Smad3 binding to β2-tubulin (Fig. 4A). Luciferase assay demonstrated that mutation of Smad3 at the S309 residue did not alter cGMP-induced inhibition of transcriptional activity of PAI-1 in the presence of TGF-β1 in PASMC. In contrast, mutation of Smad3 at Thr388 residue abolished the inhibitory effect of cGMP (Fig. 4B). These results suggest that the Thr388 residue of Smad3 plays an essential role in mediating cGMP-induced enhancement of Smad3 binding to β2-tubulin and inhibition of PAI-1 transcriptional activity in response to TGF-β1 treatment.

Nocodazole pretreatment abolishes the inhibitory effect of cGMP on TGF-β1-induced PAI-1 mRNA expression

To test the functional significance of β2-tubulin binding in preventing nuclear translocation of Smad3, we used the microtubule depolymerizer nocodazole to disrupt the β2-tubulin (5). Incubation of vehicle-treated cells with nocodazole led to the formation of punctuate-like structures, in which cytosolic β2-tubulin was localized, in addition to increased Smad3 accumulation in the nucleus (Fig. 5A). TGF-β1 treatment further enhanced nocodazole-induced Smad3 nuclear translocation. Importantly, nocodazole pretreatment abolished cGMP-induced Smad3 binding to β2-tubulin. cGMP pretreatment had no effect on TGF-β1-induced Smad3 nuclear accumulation in nocodazole-treated cells. These results provide pivotal evidence that the structural integrity of β2-tubulin has an essential role in mediating its interaction with Smad3.

Additionally, to test the functional significance of β2-tubulin-mediated Smad3 sequestration in the cytosol in modulating TGF-β-induced stimulation of target gene expression, quiescent PASMC were pretreated with nocodazole or vehicle for 1 h, followed by cGMP treatment for 1 h and then exposed to TGF-β1 for 12 h. PAI-1 mRNA expression was the indicator of activated TGF-β-Smad3 signaling (8). In the absence of nocodazole (control), cGMP pretreatment significantly decreased basal PAI-1 mRNA expression and attenuated the stimulatory effect of TGF-β1 (Fig. 5B), without affecting the viability of the cells (data not shown). However, nocodazole treatment significantly increased PAI-1 mRNA expression in all treatment groups. Further, nocodazole pretreatment significantly enhanced the stimulatory effect of TGF-β1 and abolished the inhibitory effect of cGMP on TGF-β1-induced PAI-1 expression. Similar results were also observed in cells treated with the microtubulin depolymerizer colchicine (Supplemental Fig. 3). Thus, disruption of microtubules abolishes the inhibitory effect of cGMP on TGF-β1-Smad3 signaling in PASMC.

Stabilizing microtubules with pacilitaxel enhances the inhibitory effect of cGMP on TGF-β signaling

We next tested whether stabilizing microtubules with pacl*taxel enhances the inhibitory effect of cGMP on TGF-β signaling. Quiescent PASMC were pretreated with pacl*taxel or vehicle for 1 h, followed by cGMP treatment for 1 h and then exposed to TGF-β1 for 1 h to detect Smad3 distribution by immunostaining or for 12 h to determine PAI-1 mRNA expression. Immunostaining analysis showed that pacl*taxel pretreatment significantly inhibited TGF-β1-induced Smad3 nuclear translocation compared with TGF-β1 alone. Pacl*taxel pretreatment potentiated colocalization of Smad3 and β2-tubulin and enhanced the inhibitory effect of cGMP on TGF-β1-induced Smad3 nuclear translocation (Fig. 6A). Quantitative real-time RT-PCR analysis showed that stabilization of microtubulin with either pacl*taxel or epothilone B caused a decrease in PAI-1 mRNA expression compared with vehicle-treated control cells. Further, both microtubulin stabilizers inhibited TGF-β1-induced PAI-1 mRNA expression (Fig. 6B and Supplemental Fig. 3). This inhibitory effect was also present in cGMP-pretreated cells, indicating that stabilizing β2-tubulin enhances Smad3 sequestration in the cytosol and thus interferes with TGF-β1 stimulation of target gene expression.

PASMC isolation and culture

PASMC were isolated from distal segments of 10- to 12-wk-old male Sprague Dawley rat pulmonary arteries (second to third branches, 0.1–0.2 mm external diameter) using the explant method as described previously (3). PASMC were used for experiments at passage 3 or 4. Before each study, PASMC were subjected to serum starvation for 24 h. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham and were consistent with the Guide for Care and Use of Laboratory Animals published by the United States National Institutes of Health (Department of Health, Education, and Welfare publication no. 96-01, revised in 2002).

Isolation of cytosolic Smad3 complexes from PASMC

To obtain cytosolic proteins for the differential protomic study, 60 dishes (15-cm dish) of PASMC were treated with vehicle, TGF-β1 (2 ng/ml, 1 h, catalog no. T1654; Sigma-Aldrich, St. Louis, MO) alone, or cGMP (0.5 mm, catalog no. B1381; Sigma-Aldrich) pretreatment for 1 h followed by TGF-β1 (2 ng/ml for 1 h), respectively. Cells were harvested and fractionated using NE-PER nuclear and cytoplasmic extraction reagent (Pierce, Rockford, IL) according to the manufacturer's instructions. The quality of the separation was assessed by Western blot analysis using GAPDH and HDAC1 as markers of the cytosolic and nuclear fractions, respectively. To enrich the cytosolic Smad3 complex, a Crosslink IP kit was used (catalog no. 26147; Pierce) according to the manufacturer's instructions. Briefly, a selective antibody against Smad3 (sc-8332; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was precoupled to proteinA/G-plus resin and covalently immobilized to the support by cross-linking with disuccinmidyl suberate. The purified cytosolic protein was precleaned with normal rat IgG and Pierce control proteinA/G-plus agarose resin and incubated with the cross-linked anti-Smad3 resin in a 10-ml Pierce centrifuge columns (part no. 89898; Pierce) at 4 C overnight. After washing to remove nonbound proteins from the sample, the Smad3-bound proteins were recovered by dissociation from anti-Smad3 with the supplied elution buffer. Coelution of anti-Smad3 antibody with the Smad3-bound cytosolic proteins was minimized and thus reduced the influence of IgG from anti-Smad3 on the proteomic study. Protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 5 mg/ml leupeptin, and 20 μg/ml aprotinin) and phosphotase inhibitors co*cktail (Sigma-Aldrich) were added to all buffers except the elution buffer.

2D-DIGE and protein identification by MS

2D-DIGE was performed at Applied Biomics (Hayward, CA), as previously described (22). Briefly, samples were thawed and 2D lysis buffer [7 m urea, 2 m thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 30 mm Tris-HCl (pH 8.8)] was added (10% final concentration). Samples were concentrated using Amicon 3K MWCO spin columns (Millipore, Bedford, MA), and buffer exchange was performed into 2D lysis buffer. Protein concentration was measured using the Bradford assay (Bio-Rad, Hercules, CA). Samples from vehicle, TGF-β1, and cGMP + TGF-β1-treated cells were labeled with Cy2, Cy3, and Cy5, respectively. Labeled samples were mixed and subjected to isoelectric focusing on a 13-cm precast linear immobilized pH gradient strip (pH 3–10; Amersham, Piscataway, NJ). Samples were then separated by 12% SDS-PAGE in the second dimension. Gel images were scanned immediately using Typhoon TRIO (Amersham BioSciences, Piscataway, NJ). The scanned images were analyzed by Image Quant software (version 6.0; Amersham BioSciences), followed by in-gel analysis using DeCyder software (version 6.0; Amersham BioSciences). The fold change of protein expression levels was obtained from in-gel DeCyder analysis.

Based on 2D-DIGE assessment, six protein spots of interest that were expressed differentially in response to cGMP treatment and had the maximal change (increased >2.0-fold in the comparison between cGMP + TGF-β1 and TGF-β1) were picked up using an Ettan spot picker (Amersham BioSciences), digested with trypsin, extracted with 2% trifluoroacetic acid and 40 μl of acetonitrile, and desalted with a ZipTip C18 column (Millipore). Peptides were eluted from the ZipTip and spotted on the matrix-associated laser desorption/ionization plate (model ABI 01-192-6-AB). Matrix-associated laser desorption/ionization-time-of-flight (TOF) MS and TOF/TOF tandem MS/MS were performed on an ABI 4700 mass spectrometer (AB Sciex, Framingham, MA). Candidates with either protein score confidence interval (C.I.)% or Ion C.I.% greater than 95 were considered significant.

Co-IP analysis

To test the effect of TGF-β1 and ANP/cGMP on the interaction between Smad3 and β2-tubulin, quiescent PASMC were treated with ANP (1 μm, catalog no.A8208; Sigma-Aldrich), cGMP (0.5 mm), or vehicle for 1 h, followed by TGF-β1 (2 ng/ml) or vehicle for 1 h. The cells were lysed in Co-IP buffer [120 mm NaCl, 20 mm Tris (pH 7.5), 2 mm EDTA, 1% Triton-X100, 1 mm sodium vanadate, and 10% glycerol] containing 0.5 mm phenylmethylsulfonylfluoride and 20 μg/ml apotinin, and then centrifuged at 12,000 × g for 15 min at 4 C. Protein concentration was determined by a Bradford-based method (Bio-Rad). After cell lysis, 600 μg of total protein per sample were precleaned with normal rat/mouse IgG and proteinA/G-plus beads (sc-2003; Santa Cruz Biotechnology, Inc.) and then immunoprecipitated with anti-Smad3 (sc-8332; Santa Cruz Biotechnology, Inc.) or anti-β2-tubulin (T8453; Sigma-Aldrich), respectively, at 4 C for overnight. The bound proteins on proteinA/G-plus beads were washed using Co-IP buffer, centrifuged, eluted with 2× sample loading buffer, boiled at 95 C for 5 min, and stored at −80 C. Each Co-IP experiment was repeated for at least three times.

Western blot analysis

Proteins samples were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane, as described previously (3). Blots were probed with anti-Smad3, anti-GAPDH (sc-32233; Santa Cruz Biotechnology, Inc.), anti-HDAC1 (sc-7872; Santa Cruz Biotechnology, Inc.), anti-β-actin (sc-47778; Santa Cruz Biotechnology, Inc.) and anti-β2-tubulin primary antibodies, and a horseradish peroxidase-conjugated secondary antibody, respectively. Bands were visualized by use of a Super Western Sensitivity Chemiluminescence Detection System (Pierce). Autoradiographs were quantitated by densitometry (ImageJ; NIH, Bethesda, MD).

Colocalization analysis by immunofluorescence staining

To detect whether Smad3 colocalize with β2-tubulin in PASMC, cells were seeded on glass coverslips. Indirect immunofluorescence staining was carried out, as described previously (23). Quiescent PASMC were pretreated with microtubule depolymerizer nocodazole (5 μg/ml, catalog no. M1404; Sigma-Aldrich), microtubule stabilizer pacl*taxel (1 μm, catalog no. T1972; Sigma-Aldrich), or vehicle for 1 h, followed by cGMP (0.5 mm) for 1 h and then exposed to TGF-β1 (2 ng/ml) for additional 1 h. The treated cells were washed, fixed with 4% formaldehyde, and permeabilized with 0.5% Triton X-100. After washing with PBS, cells were blocked with 10% normal goat serum and then incubated with anti-Smad3, anti-β2-tubulin, or normal rabbit/mouse IgG at 4 C for overnight. The slides were incubated with a Texas-red-conjugated antirabbit secondary antibody (1:100, catalog no. TI-1000; Vector Laboratories, Inc., Burlingame, CA) and a fluorescein-conjugated antimouse secondary antibody (1:100, catalog no. FI-2000; Vector Laboratories, Inc.) for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (50 ng/ml) in PBS for 15 min. Coverslips were washed, mounted with 90% glycerol, and visualized by fluorescence microscopy (×400). To provide a valid comparison, identical acquisition parameters were used for all observations. Images were randomly acquired from different fields.

Site-directed mutagenesis and transfection

The full-length (wild type) human Smad3 were subcloned into pMSCV-neo vector (Xu Cao, University of Alabama at Birmingham). Smad3 mutants at the Ser309 (S309G) and Thr388 (T388A) sites were constructed using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's protocol, and their sequences confirmed by DNA sequencing. For overexpression of wild-type Smad3 and mutant Smad3 in HEK293 cells, cells were grown in 100-mm dishes to approximately 70% confluence and were transfected 15 μg of plasmids of Smad3, mutant Smad3, or pMSCV-neo empty vector using Lipofectamine LTX with Plus (Invitrogen, Carlsbad, CA). After 72 h of transfection, serum-starved cells were treated with vehicle or cGMP (0.5 mm) for 1 h and then harvested. Whole cell lysates were used for Co-IP analysis using a selective antibody against Smad3.

Luciferase study

To test the functional effects of the mutation of Smad3 at Ser309 and Thr388 on cGMP-induced inhibition of TGF-β/Smad3 signaling, quiescent PASMC were transiently cotransfected with a TGF-β-responsive p3TP-Lux plasmid that contains the −740/−636 region of the PAI-1 promoter bearing the −730 CAGA box, a pRL-TK plasmid (as a control for transfection efficiency) and wild-type Smad3, S309G-Smad3, T388A-Smad3, or empty vector using the Lipofectamine LTX with Plus Transfection Reagent (Invitrogen). At 48 h after transfection, PASMC were treated with TGF-β1 (2 ng/ml) or vehicle for 24 h. PASMC were harvested, and luminescence from transfected PASMC was quantified by measuring firefly/Renilla luciferase activity using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI).

Real-time quantitative RT-PCR analysis

To determine the effects of cGMP, nocodazole/colchicine (catalog no. C9754; Sigma-Aldrich) and pacl*taxel/epothilone B (catalog no.E2656; Sigma-Aldrich) on TGF-β1-induced PAI-1 mRNA expression, quiescent PASMC were pretreated with nocodazole (5 μg/ml)/colchicine (1 μm), pacl*taxel (1 μm)/epothilone B (50 ng/ml), or vehicle for 1 h, followed by cGMP (0.5 mm) for 1 h and then exposed to TGF-β1 (2 ng/ml) for additional 12 h. Total RNA was extracted using TRIzol Reagent (catalog no. 15596-018; Invitrogen). The purified RNA was reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis System (catalog no. 18080-051; Invitrogen). cDNA was amplified by real-time quantitative PCR using the SYBR Green RT-PCR kit (part no. 4309155; Applied Biosystems, Foster City, CA) in a Bio-Rad iCycler with specific primers of rat PAI-1 (forward, 5′-GCC CTA CCA CGG CGA AAC C-3′ and reverse, 5′-AGG ATG AGG AGG CGG GGC AG-3′) and rat GAPDH (forward, 5′-ATT CTT CCA CCT TTG ATG C-3′ and 5′-TGG TCC AGG GTT TCT TAC T-3′) (Invitrogen). In addition, the expression of PAI-1 mRNA in HEK293 cells was measured using specific primers of human PAI-1 (forward, 5′-TCC AGC CCT CAC CTG CCT AGT C-3′ and reverse, 5′-ACC TGC TGA AAC ACC CTC ACC CC-3′) and human 18S (forward, 5′-GAG AAA CGG CTA CCA CAT CC-3′ and 5′-CAC CAG ACT TGC CCT CCA-3′) (Invitrogen). Relative RNA levels were calculated using the iCycler software (Bio-Rad) and normalized using GAPDH or 18S RNA.

Statistical analysis

Results were expressed as mean ± sem. Analyses were carried out using the SigmaStat statistical package (Jandel Scientific softwave). Our primary statistical test was one-way ANOVA. If ANOVA results were significant, a post hoc comparison among groups was performed with the Newman-Keuls test. A P value of less than 0.05 was considered statistically significant.

This work was supported, in part, by National Heart, Lung, and Blood Institute Grants HL080017;, HL044195; (to Y.-F.C.), and HL07457 (to S.O.) and by American Heart Association Grants 10POST3180007; (to K.G.) and 09BGIA2250367 (to D.X.).

Disclosure Summary: The authors have nothing to disclose.

Abbreviations:

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