AJP - Lung Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 294: L698-L704, 2008. First published February 29, 2008; doi:10.1152/ajplung.00529.2007
1040-0605/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/L698    most recent
00529.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Simeone-Penney, M. C.
Right arrow Articles by Simon, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Simeone-Penney, M. C.
Right arrow Articles by Simon, A. R.

PDGF-induced human airway smooth muscle cell proliferation requires STAT3 and the small GTPase Rac1

Marina C. Simeone-Penney,1 Mariano Severgnini,1,2 Lilliana Rozo,1,2 Satoe Takahashi,1,2 Brent H. Cochran,1 and Amy R. Simon1,2

1Department of Physiology, Tufts University School of Medicine, Boston; and 2Pulmonary and Critical Care Division, Tufts-New England Medical Center, Boston, Massachusetts

Submitted 20 December 2007 ; accepted in final form 22 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The signal transducers and activators of transcription (STAT) family of transcription factors regulates a variety of biological functions including cellular proliferation, transformation, apoptosis, and differentiation. We have previously determined that PDGF activates the STAT pathway in human airway smooth muscle cells (HASMC) and that the Jak and Src kinases are required for both PDGF-induced STAT activation and HASMC proliferation. As increased airway smooth muscle (ASM) volume is associated with airflow obstruction and disease severity in patients with asthma, it is important to elucidate the cellular and molecular pathways that regulate ASM accumulation. In this paper, we investigated the requirement of STAT3 for PDGF-induced HASMC proliferation. We demonstrate that knockdown of STAT3 expression in HASMC resulted in a significant decrease in mitogen-induced cellular proliferation. Additionally, PDGF-induced activation of STAT3 required the small GTP-binding protein Rac1, and Rac1 was also required for PDGF-induced HASMC proliferation. Furthermore, PDGF treatment induced STAT3 and Rac1 to physically associate and translocate to the nucleus, identifying one mechanism by which STAT3 is regulated in response to PDGF in HASMC. Finally, we determined that STAT3 expression is required for PDGF-mediated regulation of cell cycle targets cyclin D3 and p27. These data define a novel mitogenic signaling pathway in airway smooth muscle cells leading from PDGF to Rac1 and STAT3 and subsequent cell cycle gene regulation. Thus, targeting STAT3 may prove to be a novel therapeutic approach for patients with severe asthma and significant airway wall remodeling, as manifested by ASM accumulation.

asthma; airway remodeling; human airway smooth muscle cells

ALLERGIC ASTHMA is a Th2 cell-mediated disease that results in airway inflammation and variable airflow obstruction in genetically susceptible individuals (5). Airway inflammation and/or airway injury are thought to result in airway structural changes, referred to as airway remodeling. Airway remodeling is characterized by increased airway smooth muscle (ASM) accumulation along with mucus hypersecretion, subepithelial fibrosis, and angiogenesis (4). ASM volume can be increased three- to fourfold in severe asthmatics and is thought to contribute significantly to increased airway hyperreactivity and persistent airflow obstruction despite maximal anti-inflammatory treatment (16). Increased ASM cell proliferation is one mechanism by which changes in ASM mass occur. Thus, a better understanding of the cellular and molecular pathways that mediate ASM cell proliferation is important for the development of novel therapeutics for patients with severe asthma.

Airways of asthmatics contain increased levels of mitogens that are thought to result from serum leakage into the airways due to enhanced vascular permeability (29). In particular, mitogens such as PDGF and TGF-β have been shown to be elevated in the lungs of asthmatics and are thought to contribute to airway remodeling and ASM proliferation (30, 50). In vitro, airway myocytes are known to proliferate in response to multiple different signals including growth factors, cytokines, reactive oxygen species (ROS), and bronchoconstrictors such as histamines and endothelin (19, 34, 40, 53). These mitogens are known to signal through two types of receptors, tyrosine kinase receptors and G protein-coupled receptors (2). Previously, our laboratory determined that the signal transducers and activators of transcription (STAT) kinases, Jak and Src, are required for mitogen-induced signaling in human airway smooth muscle cells (HASMC) in vitro (43). Work done by others has demonstrated that the MAPK and PI3-kinase pathways, as well as Rac1, are also important in smooth muscle cell mitogenesis (31, 32, 35).

The STATs are a family of cytokine and growth factor-inducible transcription factors that are important in multiple cellular responses including proliferation, apoptosis, differentiation, and migration (9). Upon growth factor stimulation, the STATs become activated through tyrosine phosphorylation by growth factor receptor kinases, the Jak kinases, and Src kinases (43). This phosphorylation results in the STATs undergoing a conformational change, dimerizing, and translocating to the nucleus where they bind sequence-specific DNA-binding elements to regulate transcription (15). Additionally, we have previously demonstrated that PDGF-induced phosphorylation of STAT3 requires both ROS and the NADPH oxidase regulator Rac1 (42, 44).

STATs have also been shown to be important in the regulation of the allergic response in asthma. STAT1 is upregulated in the airway epithelium of patients with asthma, whereas STAT6 is known to be critical for Th2 differentiation and IgE production (20, 39). We have recently identified epithelial STAT3 as a critical regulator of allergen-induced allergic inflammation and airway hyperresponsiveness in a murine model of asthma (41). Additionally, we have previously shown that Jak and Src kinases are required for PDGF-induced STAT3 activation and proliferation in HASMC (43). Inhibition of STAT3 activation with kinase inhibitors of Jak and Src correlated with decreased HASMC proliferation in response to PDGF (43). Recently, others demonstrated that STAT3 single-nucleotide polymorphisms are associated with decreased lung function in patients with asthma (26). These combined data suggest that STAT3 may be an important mediator of ASM accumulation along with other features of airway remodeling.

Given that the STAT pathway plays a critical role in the regulation of PDGF-mediated proliferation and that STAT3 has been shown to be activated in ASM cells during the allergic response, we sought to investigate whether STAT3 is directly required for HASMC proliferation. Here we report that STAT3 is required for PDGF-induced proliferation of HASMC. Moreover, the small GTP-binding protein, Rac1, is required for maximal PDGF-induced STAT3 activation and proliferation. Finally, we demonstrate that STAT3 is a regulator of the cell cycle target genes cyclin D3 and p27. Thus, STAT3 is a critical regulator of mitogen-induced ASM proliferation and may be a novel target for asthma patients with airway remodeling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
HASMC cell culture. HASMC were kindly provided by Dr. Reynold Panettieri (Univ. of Pennsylvania, Philadelphia, PA). These cells were derived by enzymatic dissociation from the trachea of lung transplant donors and characterized for the presence of smooth muscle actin and agonist-induced calcium changes as previously described (33). HASMCs were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml L-glutamine. Cells were growth arrested with DMEM without FBS for 48 h.

Small interfering RNA transfection protocol. HASMCs were transfected with control small interfering RNA (siRNA) (GFPi), STAT3 siRNA [STAT3i-1 from Dharmacon; STAT3i-2 generated by Konnikova L and Cochran BH (22)], or Rac1 siRNA (Rac1i-1 and Rac1i-2 from Dharmacon) using oligofectamine at 400 nM final concentration. Cells were transfected in Optimem on day 0 and again on day 2 to ensure sufficient knockdown. Following knockdown, HASMCs were growth arrested with 0.1% FBS/DMEM for 48 h and stimulated with PDGF-AB (25 ng/ml) for either 10 min, 4 h, or 36 h depending on the assay.

[3H]thymidine incorporation and cell proliferation assays. HASMC were plated at a density of 45,000 cells in 12-well plates in DMEM. Cells were transfected with either GFPi or STAT3i as outlined above. Cells were subsequently growth arrested for 48 h in 0.1% FBS-DMEM before being stimulated with PDGF-AB (25 ng/ml), serum (15% FCS), or thrombin (10 U/ml). After the addition of mitogen, cells were labeled with [methyl-3H]thymidine (1 µCi/well, 20 Ci/mmol) for 36 h. The experiment was terminated by washing the cells with ice-cold PBS and then with cold 6% TCA. Cells were then lysed in 0.2 N NaOH, and radioactivity was counted in a liquid scintillation counter. The results represent at least two separate experiments done in triplicate for each condition.

Cell number quantitation. Equivalent numbers or cells were plated and HASMC were transfected with STAT3i, Rac1i, or GFPi as a control on days 0 and 2 as stated above. Following knockdown of STAT3 or Rac1 with siRNAs, cells were treated on day 4 with either PDGF (25 ng/ml) or serum. Cell number was quantified using a Coulter counter as previously described (43).

Colocalization experiment. Quiescent HASMC were stimulated for 10 min with PDGF (25 ng/ml). Cells were washed, fixed in 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Cells were blocked with goat serum and incubated with anti-STAT3 antibody (Upstate) followed by goat anti-rabbit Alexa 488 antibody (Molecular Probes). Cells were washed and subsequently incubated with Rac1 MAb (Upstate) followed by goat anti-mouse Alexa 594 secondary antibody (Molecular Probes). Cells were fixed for analysis. Single stained negative controls were also performed to ensure each secondary antibody was specific. For both quiescent and PDGF-treated HASMC, 10–15 high-powered fields were evaluated, and representative images were shown. Colocalization experiments were repeated three times, and each treatment was done in duplicate.

Immunoprecipitation. Growth arrested HASMC were stimulated for 10 min with PDGF (25 ng/ml), washed with cold PBS containing protease and phosphatase inhibitors, and lysed in a high-salt lysis buffer. Proteins were quantified, and 750 µg of precleared protein lysates were spun and incubated with either anti-Rac1 MAb (Upstate; 1:100 dilution) or anti-STAT3 antibody (Cell Signaling; 1:100 dilution) overnight at 4°C. Lysates were subsequently incubated at 4°C with protein A/G beads (Sigma) for 2 h. Samples were spun, supernatant was drawn off, and remaining beads were washed, boiled, and resuspended in 1x SDS sample buffer. Lysates were run out on a gel and immunoblotted with anti-STAT3 antibody (Upstate) or anti-Rac1 (Upstate). Control lysates were immunoprecipitated with normal rabbit serum (NRS) or control IgG and immunoblotted with Rac1 and STAT3 as a control.

Western blot analysis. Growth-arrested HASMC were treated for 10 min with PDGF (25 ng/ml) and lysed in lysis buffer containing phosphatase inhibitors on ice. Forty micrograms of protein was run on a 10% SDS-PAGE gel. Antibodies against STAT3-pTyr705, cyclin D3, p27 (Cell Signaling), STAT3, and Rac1 (Upstate) were blotted, and membranes were processed via chemiluminescence.

Quantitative real-time PCR. Total RNA was isolated using the RNeasy kit (Qiagen) from HASMC transfected with STAT3 RNAi or Rac1 RNAi, as stated above. Genomic DNA was digested using DNase I for 30 min at 37°C. Subsequently, cDNAs were reverse-transcribed with SuperScriptIII (Invitrogen) for 60 min at 50°C and heat inactivated for 10 min at 70°C. cDNA samples were combined with sequence-specific primers and SYBR Green master mix for quantitative analysis (Qiagen). Sample differences ({Delta}CT) were analyzed and reported as the fold change following β-actin normalization.

Real-time PCR primers. The following primers were designed to anneal specifically to cDNA: STAT3, forward 5'-ACCTGCAGCAATACCATTGAC-3' and STAT3, reverse 5'-AAGGTGAGGGACTCAACCTGC-3'; Rac1, forward 5'-AGCTTTTGCGGAGATTTTGA-3' and Rac1, reverse 5'-CCCGTGACACTTTCATTCCT-3'; β-actin, forward 5'-CCTGGGCATGGAGTCCTGTGG-3' and β-actin, reverse 5'-CTGTGTTGGCGTACAGGTCTT-3'.

Statistical analysis. Data are expressed as means ± SE. A Student's t-test was performed on the means of two sets of sample data, and a difference was accepted as significant if the P value was <0.05. Experiments were performed at least three times, and, for proliferation and cell number assays, treatments were done in duplicate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transfection of STAT3 RNAi results in target-specific knockdown. To specifically determine the role of STAT3 in PDGF-induced proliferation in HASMC, we used an siRNA approach to knockdown STAT3 expression in vitro. Transfection of cells with two different STAT3i sequences resulted in knockdown of STAT3 protein expression, but not of another STAT family member, STAT1, as determined by Western blot (Fig. 1A). Transfection of a control siRNA (GFPi) had no effect on STAT3 expression levels. Additionally, STAT3i-transfected cells showed a reduction in STAT3 mRNA by 90% compared to GFPi-transfected cells as was determined by quantitative real-time PCR (Q-RT-PCR) (Fig. 1B).


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 1. STAT3 is specifically targeted by 2 STAT3 small interfering RNA (siRNA) sequences. Cells were transfected twice with 400 nM STAT3 siRNA (ST3i-1 and ST3i-2) on day 0 and day 2. Following transfection, cells were rescued with full serum media and lysed for Western blot analysis (A) or for RNA isolation and Q-RT-PCR analysis of mRNA levels (B).

 
STAT3 is required for PDGF-induced proliferation in HASMC. To determine whether STAT3 is also required for mitogen-induced HASMC proliferation, HASMC were transfected with STAT3i, growth arrested, and subsequently stimulated with various mitogens. As expected, treatment with PDGF, serum, or thrombin significantly increased thymidine uptake in HASMC (Fig. 2A). STAT3 knockdown significantly inhibited PDGF- and serum-induced thymidine uptake while having no effect on thrombin-induced thymidine uptake. In addition, STAT3 knockdown also resulted in a significant decrease in PDGF- and serum-induced cell number compared with that seen in the GFPi-treated cells (Fig. 2B). Importantly, cell viability was not compromised in STAT3i-treated cells as was measured by MTS assay (data not shown) (22). We also determined by Western blot analysis that STAT3 knockdown was still significantly reduced at the time point when cells were processed for thymidine uptake (data not shown). Thus STAT3 is required for some of the mitogen-induced cellular pathways that mediate proliferation in HASMC.


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 2. STAT3 is required for PDGF-induced human airway smooth muscle cell (HASMC) proliferation and cell number. Growth-arrested HASMC were transfected with STAT3i or GFPi as described above and treated with PDGF-AB (25 ng/ml), serum, or thrombin (10 U/ml) and [methyl-3H]thymidine (1 µCi/well, 20 Ci/mmol) for 36 h. Thymidine uptake was quantified for comparison (A). In addition to cellular proliferation, total cell number was evaluated in STAT3i- or GFPi-transfected HASMC in response to PDGF or serum (B). *P < 0.05 compared with untreated; **P < 0.05 vs. PDGF treatment.

 
Rac1 is required for PDGF-induced STAT3 activation. Previously, Rac1 has been shown to be required for PDGF-induced STAT3 activation in fibroblast cells (44). To determine if Rac1 is similarly required for PDGF-induced STAT3 activation in primary HASMC, we employed a knockdown approach using two Rac1 RNAi sequences. Transfection of cells with two different Rac1i sequences, but not GFPi, reduced Rac1 protein expression as was determined by Western blotting (Fig. 3A). Transfection with either of the two Rac1i sequences had no effect on the expression of a related Rac family member, Rac2 (Fig. 3A). Rac1i transfection also resulted in decreased Rac1 mRNA levels by 80% compared with GFPi-transfected cells (Fig. 3B). More importantly, knockdown of Rac1 by siRNA resulted in a decrease in PDGF-induced STAT3 phosphorylation (Fig. 3C). These data indicate that Rac1 is required for maximal PDGF-induced STAT3 activation in primary HASMC.


Figure 3
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 3. Rac1 is required for maximal PDGF-induced STAT3 activation. HASMC were transfected twice with 400 nM two different Rac1 siRNA sequences (Rac1i-1 and Rac1i-2) on day 0 and 2. Transfected HASMC were lysed for Western blot analysis of Rac1 and Rac2 levels (A) or for RNA isolation and Q-RT-PCR analysis of Rac1 mRNA levels (B). Levels were compared with loading controls in both A and B. Following transfection, cells were also growth arrested with 0.1% FBS/DMEM for 48 h. Subsequently, cells were stimulated with PDGF-AB (25 ng/ml) for 10 min, and levels of STAT3 activation were assessed via Western blot (C).

 
Rac1 mediates PDGF-induced proliferation. Since Rac1 is required for maximal STAT3 activation by PDGF, we evaluated whether Rac1 was also required for PDGF-induced proliferation in HASMC. To determine if Rac1 regulates PDGF-induced proliferation, thymidine uptake was evaluated in Rac1i-treated cells. As seen in Fig. 4A, Rac1 knockdown significantly attenuated both PDGF- and serum-induced HASMC proliferation compared with similarly treated GFPi-transfected cells. In addition, Rac1 knockdown resulted in a significant decrease in PDGF- and serum-induced cell number (Fig. 4B) but not in cell viability as determined by MTS assay (data not shown). Thus, these data demonstrate that endogenous Rac1 is required for maximal activation of endogenous STAT3 in response to PDGF as well as for PDGF-induced proliferation in HASMC. These data elucidate a novel pathway in HASMC from PDGF activation of Rac1 to STAT3 phosphorylation and HASMC proliferation.


Figure 4
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 4. Rac1 is required for PDGF-induced HASMC proliferation. HASMC were transfected twice with 400 nM Rac1i siRNA on day 0 and day 2. Transfected HASMC were growth arrested for 48 h and then treated with PDGF-AB or serum and [methyl-3H]thymidine (1 µCi/well, 20 Ci/mmol) for 36 h. Thymidine uptake in HASMC was measured to quantify proliferation in response to mitogenic signals (A). Additionally, total cell number was analyzed in Rac1i- or GFPi-transfected cells following PDGF or serum treatment. *P < 0.05 compared with untreated; **P < 0.05 vs. PDGF treatment.

 
STAT3 and Rac1 colocalize and physically interact upon PDGF stimulation. Since Rac1 is required for PDGF-induced STAT3 activation and proliferation in HASMC, and prior work in our laboratory has shown that Rac1 physically interacts with STAT3 (44), we sought to determine if this was the case in primary ASM. In quiescent HASMC, Rac1 and STAT3 had a diffuse staining pattern but colocalized at the membrane of the cell as determined by immunocytochemistry (Fig. 5A). However, upon PDGF stimulation of quiescent cells, STAT3 and a fraction of Rac1 colocalized to the nucleus (Fig. 5A). To confirm that STAT3 and Rac1 exist in a complex, we performed immunoprecipitation of HASMC treated with PDGF. Coimmunoprecipitation experiments in HASMC demonstrated that STAT3 was associated with Rac1 and that this association was enhanced by PDGF treatment, and to a lesser extent by oncostatin M (OSM) treatment (Fig. 5B). Immunoprecipitation with NRS or control IgG did not result in immunoprecipitation of either STAT3 or Rac1 (Fig. 5B). Thus, these combined data demonstrate that STAT3 and Rac1 physically interact and translocate to the nucleus following PDGF treatment.


Figure 5
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 5. STAT3 and Rac1 physically interact and colocalize to the nucleus in response to PDGF. HASMC were growth arrested with 0.1% FBS/DMEM for 48 h. Following growth arrest, cells were stimulated with PDGF-AB (25 ng/ml) for 10 min. The cellular localization of both STAT3 and Rac1 was analyzed using fluorescently conjugated STAT3 or Rac1 antibodies (A). Colocalization was compared both before and after PDGF stimulation (A). Growth-arrested HASMC were also stimulated with PDGF-AB or oncostatin M (OSM) and lysed for immunoprecipitation with a Rac1 antibody. Following Rac1 immunoprecipitation, lysates were run out on a 10% SDS-PAGE gel and immunoblotted for the presence of STAT3 (B). Values represent fold changes in the amount of immunoprecipitated STAT3 or Rac1 compared with quiescent lysates (B and C). NRS, normal rabbit serum.

 
STAT3 is required for PDGF-mediated induction of pro-proliferative targets. To identify a possible mechanism by which STAT3 regulates PDGF-induced HASMC proliferation, we evaluated the expression of cell cycle regulators that are also known to be STAT3-regulated (10, 45). We first analyzed cyclin D3 expression, given its requirement for G1 cell cycle progression. As seen in Fig. 6A, an increase in cyclin D3 expression was seen in HASMC cells stimulated with PDGF compared with quiescent cells as was determined by Western blotting (Fig. 6A). This PDGF-induced increase in cyclin D3 expression was inhibited by transfection of HASMC with STAT3i, indicating that STAT3 regulates the induction of cyclin D3 expression in response to PDGF (Fig. 6A). In addition, STAT3i-transfected quiescent cells also showed a significant decrease in cyclin D3 expression compared with quiescent GFPi-transfected cells. Thus, STAT3 also regulates basal cyclin D3 expression.


Figure 6
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 6. STAT3 expression is required for PDGF-induced regulation of cell cycle regulators cyclin D3 and p27. HASMC were transfected with 400 nM siSTAT3 on day 0 and day 2. Following knockdown of STAT3, HASMC were growth arrested for 48 h and stimulated with PDGF for 4 h. Cells were lysed and prepared for Western blotting. Levels of cyclin D3 were normalized against β-actin, and values represent the change in cyclin D3 expression compared with untreated cells (A). Cells were also lysed, and levels of p27 were also normalized to β-actin. Values at bottom represent the change in p27 expression compared with untreated cells.

 
For cell cycle progression to occur, cell cycle inhibitors, such as p27 and p16, must also be downregulated (24). To determine if STAT3 also regulates HASMC proliferation through suppression of cell cycle inhibitors, levels of p27 were analyzed in cells where STAT3 expression was knocked down. As expected, PDGF treatment resulted in a significant downregulation of p27 expression as was determined by Western blot analysis (Fig. 6B). However, this PDGF-induced decrease in p27 expression was significantly attenuated in cells transfected with STAT3i, indicating that STAT3 is also necessary for suppression of cell cycle inhibitors (Fig. 6B). These data indicate that STAT3 regulates HASMC proliferation through the regulation of gene expression of both cell cycle promoters and cell cycle inhibitors.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
While the tyrosine kinases Jak and Src have been shown to be important in HASMC proliferation, the role of the STAT transcription factors in mediating the response to these kinases has not been demonstrated (23, 43). We demonstrate here for the first time that STAT3 is specifically required for PDGF-induced proliferation of HASMC and the regulation of cell cycle regulators cyclin D3 and p27. In addition, we determined that PDGF-induced STAT3 activation and subsequent HASMC proliferation requires the small GTP binding protein Rac1. Furthermore, endogenous Rac1 and STAT3 proteins were shown to physically interact and colocalize to the nucleus upon PDGF stimulation, indicating that Rac1 is an important STAT3 regulator in the mitogenic response. Thus, we have identified a novel pathway in primary HASMC downstream of the growth factor PDGF that connects Rac1 to STAT3, leading to subsequent cellular proliferation.

While the STAT proteins often have tissue-specific functions, STAT3 has largely been found to function as a pro-proliferative transcription factor in multiple tissue types (9). STAT3 has been shown to be activated by a number of different growth factors (VEGF, PDGF, and EGF), cytokines (IL-6 and OSM), and chemokines (eotaxin) that are known to be present in the airways of asthmatics, and, thus, likely has pleiotropic functions in the regulation of airway inflammation and airway structural changes. Additionally, STAT3 has been shown to be required for the upregulation of growth factors and chemokines in ASM (11, 12). While the MAPK and PI3K signaling pathways in mitogen-induced smooth muscle cell proliferation have been well characterized (31, 35), the role of STAT3 was previously unknown. Here we show that knockdown of STAT3 by siRNA resulted in decreased PDGF-induced HASMC proliferation, indicating that STAT3 also plays a critical role in mitogen-induced ASM accumulation. STAT3 likely requires the cooperation of other signaling pathways since IL-11 activated STAT3 in HASMC, but did not result in increased cellular proliferation (data not shown). Current asthma therapeutics are poor at preventing and treating airway remodeling, thus identifying novel regulators of airway structural changes may have important therapeutic implications. Our data suggest that targeting STAT3 in vivo may be a novel way to modulate airway remodeling by altering ASM proliferation.

Prior work in our laboratory has shown that STATs are regulated by both Jak and Src kinases in HASMCs as well as in vivo, in the lung, in a house dust mite model of allergic asthma (41, 43). Additionally, we have previously determined that STAT3 activation is directly regulated by the small GTPase Rac1 in fibroblasts (44). Recent studies by us and others have shown that Rac1 contributes to maximal STAT3 activation, transcriptional activity, and nuclear translocation and that the STAT3/Rac1 interaction is important in STAT3 function (21, 36, 37, 44, 46, 47). However, the results regarding the direct regulation of STAT3 by Rac1 have been somewhat contradictory, likely due to the fact that the studies have used different cell types and stimuli, and have often relied on overexpression studies using dominant negative forms of Rac1 (DnRac1). Given that DnRac1 has been shown to also bind multiple guanine nucleotide exchange factors that activate other small GTPases, results from these prior experiments may relate to altering the activity of other GTPases, such as ras, and not Rac1 (38, 44). Our data using RNAi show that Rac1 specifically is needed for the rapid activation of STAT3 by PDGF (within minutes), and thus, this STAT3 activation is not likely dependent on an autocrine signaling pathway as has been previously suggested by others in another cell type (13).

In addition to its role in regulating STAT3, Rac1 is well known to be a component of the multi-subunit NADPH oxidase in non-phagocytic cells, including ASM, and thus plays a role in growth factor-induced ROS production (18, 32). Thus, another possible mechanism by which Rac1 may regulate STAT3 activation is through the production of ROS by PDGF, as we have previously determined that STAT3 activation by PDGF requires ROS in HASMC (42). Finally, it has also been suggested that one mechanism by which Rac1 activates STAT3 is through the rapid activation of Jak kinases (37, 44). Jak kinases are likely required for STAT3 activation in response to PDGF. Furthermore, both the Jak and Src kinases have been shown to be redox-regulated, and so it is possible that Rac-mediated ROS production facilitates activation of the STAT kinases as well (7, 8, 28, 42, 48, 49).

In this study, we provide further evidence that endogenous Rac1 and STAT3 physically interact and colocalize to the nucleus in HASMC. Interestingly, work by others has identified a nuclear localization signal in Rac1 (51) and has shown by fluorescence resonance energy transfer that activated Rac1 can localize to the nucleus (52). The function of the nuclear Rac1, however, is still not understood. While STAT3 requires facilitated import into the nucleus via importin {alpha}5 (27), molecules other than importins, such as Rac1, are specifically required for the nuclear translocation of STAT3 (46). Kawashima et al. (21) show that activated Rac1 is specifically required for the interaction of phospho-STAT5A with importins. In our studies, PDGF treatment results in Rac1 and STAT3 nuclear colocalization, suggesting that Rac1 not only facilitates nuclear import of STAT3 but also enters the nucleus with it. These combined data suggest that Rac1 may also regulate STAT3 activity in the nucleus.

In addition to providing insight into the regulation of STAT3 by Rac1, our work provides a better understanding of the mechanism by which STAT3 regulates ASM mass. We have shown here that STAT3 is required for PDGF-induced expression of cyclin D3 and PDGF-regulated suppression of p27 in HASMC. These findings correlate with work by others indicating that STAT3 is a critical regulator of cyclin D expression in eosinophils and that STAT3 binds directly to the cyclin D1 promoter to enhance transcriptional activity (3, 45). Additionally, STAT3 has been shown to regulate p27 expression in keratinocytes (14). However, the exact mechanism by which STAT3 regulates p27 expression remains to be elucidated. One possibility is that p27 is regulated translationally in response to PDGF, as has been previously suggested (1). Thus one mechanism by which HASMC undergo enhanced cellular proliferation and accumulation in asthmatics may be through STAT3-mediated regulation of both cell cycle promoters and cell cycle inhibitors. Finally, we and others have demonstrated that STAT3 is antiapoptotic in other cell types, thus it is possible that STAT3 regulates ASM accumulation by antiapoptotic mechanisms as well in HASMC (6, 22).

An increasing number of studies have identified STAT3 as a potentially important target gene in asthma pathogenesis and in allergic diseases. Recently, STAT3 SNPs have been shown to be associated with decreased lung function in asthmatics (26). Our laboratory has characterized a role for epithelial STAT3 in the regulation of allergic inflammation and AHR in a murine model of asthma (41). Additionally, mutations in the STAT3 gene have recently been shown to underlie hyper-IgE syndrome, further characterizing the increasing importance of this transcription factor in the regulation of immune and allergic inflammatory diseases (17, 25). We have now specifically demonstrated the importance of STAT3 in the regulation of ASM mitogenesis. Moreover, we have identified endogenous STAT3 and Rac1 as functional binding partners and critical regulators of HASMC proliferation downstream of growth factor stimulation. Our data further broaden the roles of STAT3 in asthma and suggest that targeting STAT3 in patients may be a novel therapeutic approach for people with severe asthma and airway remodeling.


   ACKNOWLEDGMENTS
 
We thank Dr. Reynold Panettieri for kindly providing the human airway smooth muscle cells used in this paper. Without his generous donation to our laboratory, the work above would not have been possible.


   FOOTNOTES
 

Addressfor reprint requests and other correspondence: A. R. Simon, Pulmonary and Critical Care Division (Box 369), 750 Washington St., Boston, MA 02111 (e-mail: amy.simon{at}tufts.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Agrawal D, Hauser P, McPherson F, Dong F, Garcia A, Pledger WJ. Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells. Mol Cell Biol 16: 4327–4336, 1996.[Abstract]
  2. Ammit AJ, Panettieri RA Jr. Invited review: the circle of life: cell cycle regulation in airway smooth muscle. J Appl Physiol 91: 1431–1437, 2001.[Abstract/Free Full Text]
  3. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, Darnell JE Jr. Stat3 as an oncogene. Cell 98: 295–303, 1999.[CrossRef][Web of Science][Medline]
  4. Busse W, Elias J, Sheppard D, Banks-Schlegel S. Airway remodeling and repair. Am J Respir Crit Care Med 160: 1035–1042, 1999.[Free Full Text]
  5. Busse WW, Lemanske RF Jr. Asthma. N Engl J Med 344: 350–362, 2001.[Free Full Text]
  6. Chen CL, Hsieh FC, Lieblein JC, Brown J, Chan C, Wallace JA, Cheng G, Hall BM, Lin J. Stat3 activation in human endometrial and cervical cancers. Br J Cancer 96: 591–599, 2007.[CrossRef][Web of Science][Medline]
  7. Chen KC, Zhou Y, Zhang W, Lou MF. Control of PDGF-induced reactive oxygen species (ROS) generation and signal transduction in human lens epithelial cells. Mol Vis 13: 374–387, 2007.[Web of Science][Medline]
  8. Choudhury GG, Marra F, Kiyomoto H, Abboud HE. PDGF stimulates tyrosine phosphorylation of JAK 1 protein tyrosine kinase in human mesangial cells. Kidney Int 49: 19–25, 1996.[Web of Science][Medline]
  9. Darnell JE Jr. STATs and gene regulation. Science 277: 1630–1635, 1997.[Abstract/Free Full Text]
  10. de Koning JP, Soede-Bobok AA, Ward AC, Schelen AM, Antonissen C, van Leeuwen D, Lowenberg B, Touw IP. STAT3-mediated differentiation and survival and of myeloid cells in response to granulocyte colony-stimulating factor: role for the cyclin-dependent kinase inhibitor p27(Kip1). Oncogene 19: 3290–3298, 2000.[CrossRef][Web of Science][Medline]
  11. Faffe DS, Flynt L, Mellema M, Moore PE, Silverman ES, Subramaniam V, Jones MR, Mizgerd JP, Whitehead T, Imrich A, Panettieri RA Jr, Shore SA. Oncostatin M causes eotaxin-1 release from airway smooth muscle: synergy with IL-4 and IL-13. J Allergy Clin Immunol 115: 514–520, 2005.[CrossRef][Web of Science][Medline]
  12. Faffe DS, Flynt L, Mellema M, Whitehead TR, Bourgeois K, Panettieri RA Jr, Silverman ES, Shore SA. Oncostatin M causes VEGF release from human airway smooth muscle: synergy with IL-1β. Am J Physiol Lung Cell Mol Physiol 288: L1040–L1048, 2005.[Abstract/Free Full Text]
  13. Faruqi TR, Gomez D, Bustelo XR, Bar-Sagi D, Reich NC. Rac1 mediates STAT3 activation by autocrine IL-6. Proc Natl Acad Sci USA 98: 9014–9019, 2001.[Abstract/Free Full Text]
  14. Hauser PJ, Agrawal D, Hackney J, Pledger WJ. STAT3 activation accompanies keratinocyte differentiation. Cell Growth Diff 9: 847–855, 1998.[Abstract]
  15. Hayes TE, Kitchen AM, Cochran BH. A rapidly inducible DNA-binding activity which binds upstream of the c-fos proto-oncogene. J Cell Physiol Suppl 5: 63–68, 1987.[Medline]
  16. Hogg JC. Pathology of asthma. J Allergy Clin Immunol 92: 1–5, 1993.[CrossRef][Web of Science][Medline]
  17. Holland SM, DeLeo FR, Elloumi HZ, Hsu AP, Uzel G, Brodsky N, Freeman AF, Demidowich A, Davis J, Turner ML, Anderson VL, Darnell DN, Welch PA, Kuhns DB, Frucht DM, Malech HL, Gallin JI, Kobayashi SD, Whitney AR, Voyich JM, Musser JM, Woellner C, Schaffer AA, Puck JM, Grimbacher B. STAT3 mutations in the hyper-IgE syndrome. N Engl J Med 357: 1608–1619, 2007.[Abstract/Free Full Text]
  18. Hordijk PL. Regulation of NADPH oxidases: the role of Rac proteins. Circ Res 98: 453–462, 2006.[Abstract/Free Full Text]
  19. Janat MF, Liau G. Transforming growth factor beta 1 is a powerful modulator of platelet-derived growth factor action in vascular smooth muscle cells. J Cell Physiol 150: 232–242, 1992.[CrossRef][Web of Science][Medline]
  20. Kaplan MH, Schindler U, Smiley ST, Grusby MJ. Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4: 313–319, 1996.[CrossRef][Web of Science][Medline]
  21. Kawashima T, Bao YC, Nomura Y, Moon Y, Tonozuka Y, Minoshima Y, Hatori T, Tsuchiya A, Kiyono M, Nosaka T, Nakajima H, Williams DA, Kitamura T. Rac1 and a GTPase-activating protein, MgcRacGAP, are required for nuclear translocation of STAT transcription factors. J Cell Biol 175: 937–946, 2006.[Abstract/Free Full Text]
  22. Konnikova L, Kotecki M, Kruger MM, Cochran BH. Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells. BMC Cancer 3: 23, 2003.[CrossRef][Medline]
  23. Krymskaya VP, Goncharova EA, Ammit AJ, Lim PN, Goncharov DA, Eszterhas A, Panettieri RA Jr. Src is necessary and sufficient for human airway smooth muscle cell proliferation and migration. FASEB J 19: 428–430, 2005.[Abstract/Free Full Text]
  24. Kudo Y, Kitajima S, Ogawa I, Miyauchi M, Takata T. Down-regulation of Cdk inhibitor p27 in oral squamous cell carcinoma. Oral Oncol 41: 105–116, 2005.[CrossRef][Web of Science][Medline]
  25. Levy DE, Loomis CA. STAT3 signaling and the hyper-IgE syndrome. N Engl J Med 357: 1655–1658, 2007.[Free Full Text]
  26. Litonjua AA, Tantisira KG, Lake S, Lazarus R, Richter BG, Gabriel S, Silverman ES, Weiss ST. Polymorphisms in signal transducer and activator of transcription 3 and lung function in asthma. Respir Res 6: 52, 2005.[CrossRef][Medline]
  27. Ma J, Cao X. Regulation of Stat3 nuclear import by importin alpha5 and importin alpha7 via two different functional sequence elements. Cell Signal 18: 1117–1126, 2006.[CrossRef][Web of Science][Medline]
  28. Nakamura K, Hori T, Sato N, Sugie K, Kawakami T, Yodoi J. Redox regulation of a src family protein tyrosine kinase p56lck in T cells. Oncogene 8: 3133–3139, 1993.[Web of Science][Medline]
  29. Naureckas ET, Ndukwu IM, Halayko AJ, Maxwell C, Hershenson MB, Solway J. Bronchoalveolar lavage fluid from asthmatic subjects is mitogenic for human airway smooth muscle. Am J Respir Crit Care Med 160: 2062–2066, 1999.[Abstract/Free Full Text]
  30. Ohno I, Ohtani H, Nitta Y, Suzuki J, Hoshi H, Honma M, Isoyama S, Tanno Y, Tamura G, Yamauchi K, Nagura H, Shirato K. Eosinophils as a potential source of platelet-derived growth factor B-chain (PDGF-B) in nasal polyposis and bronchial asthma. Am J Respir Cell Mol Biol 13: 639–647, 1995.[Abstract]
  31. Orsini MJ, Krymskaya VP, Eszterhas AJ, Benovic JL, Panettieri RA Jr, Penn RB. MAPK superfamily activation in human airway smooth muscle: mitogenesis requires prolonged p42/p44 activation. Am J Physiol Lung Cell Mol Physiol 277: L479–L488, 1999.[Abstract/Free Full Text]
  32. Page K, Li J, Hodge JA, Liu PT, Vanden Hoek TL, Becker LB, Pestell RG, Rosner MR, Hershenson MB. Characterization of a Rac1 signaling pathway to cyclin D(1) expression in airway smooth muscle cells. J Biol Chem 274: 22065–22071, 1999.[Abstract/Free Full Text]
  33. Panettieri RA, Murray RK, DePalo LR, Yadvish PA, Kotlikoff MI. A human airway smooth muscle cell line that retains physiological responsiveness. Am J Physiol Cell Physiol 256: C329–C335, 1989.[Abstract/Free Full Text]
  34. Panettieri RA, Yadvish PA, Kelly AM, Rubinstein NA, Kotlikoff MI. Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression. Am J Physiol Lung Cell Mol Physiol 259: L365–L371, 1990.[Abstract/Free Full Text]
  35. Papagianni M, Hatziefthimiou A, Chachami G, Gourgoulianis K, Molyvdas PA, Paraskeva E. Insulin causes a transient induction of proliferation via activation of the PI3-kinase pathway in airway smooth muscle cells. Exp Clin Endocrinol Diabetes 115: 118–123, 2007.[CrossRef][Web of Science][Medline]
  36. Park EJ, Ji KA, Jeon SB, Choi WH, Han IO, You HJ, Kim JH, Jou I, Joe EH. Rac1 contributes to maximal activation of STAT1 and STAT3 in IFN-gamma-stimulated rat astrocytes. J Immunol 173: 5697–5703, 2004.[Abstract/Free Full Text]
  37. Pelletier S, Duhamel F, Coulombe P, Popoff MR, Meloche S. Rho family GTPases are required for activation of Jak/STAT signaling by G protein-coupled receptors. Mol Cell Biol 23: 1316–1333, 2003.[Abstract/Free Full Text]
  38. Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6: 167–180, 2005.[CrossRef][Web of Science][Medline]
  39. Sampath D, Castro M, Look DC, Holtzman MJ. Constitutive activation of an epithelial signal transducer and activator of transcription (STAT) pathway in asthma. J Clin Invest 103: 1353–1361, 1999.[Web of Science][Medline]
  40. Shapiro PS, Evans JN, Davis RJ, Posada JA. The seven-transmembrane-spanning receptors for endothelin and thrombin cause proliferation of airway smooth muscle cells and activation of the extracellular regulated kinase and c-Jun NH2-terminal kinase groups of mitogen-activated protein kinases. J Biol Chem 271: 5750–5754, 1996.[Abstract/Free Full Text]
  41. Simeone-Penney MC, Severgnini M, Tu P, Homer RJ, Mariani TJ, Cohn L, Simon AR. Airway epithelial STAT3 is required for allergic inflammation in a murine model of asthma. J Immunol 178: 6191–6199, 2007.[Abstract/Free Full Text]
  42. Simon AR, Rai U, Fanburg BL, Cochran BH. Activation of the JAK-STAT pathway by reactive oxygen species. Am J Physiol Cell Physiol 275: C1640–C1652, 1998.[Abstract/Free Full Text]
  43. Simon AR, Takahashi S, Severgnini M, Fanburg BL, Cochran BH. Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 282: L1296–L1304, 2002.[Abstract/Free Full Text]
  44. Simon AR, Vikis HG, Stewart S, Fanburg BL, Cochran BH, Guan KL. Regulation of STAT3 by direct binding to the Rac1 GTPase. Science 290: 144–147, 2000.[Abstract/Free Full Text]
  45. Stout BA, Bates ME, Liu LY, Farrington NN, Bertics PJ. IL-5 and granulocyte-macrophage colony-stimulating factor activate STAT3 and STAT5 and promote Pim-1 and cyclin D3 protein expression in human eosinophils. J Immunol 173: 6409–6417, 2004.[Abstract/Free Full Text]
  46. Tonozuka Y, Minoshima Y, Bao YC, Moon Y, Tsubono Y, Hatori T, Nakajima H, Nosaka T, Kawashima T, Kitamura T. A GTPase-activating protein binds STAT3 and is required for IL-6-induced STAT3 activation and for differentiation of a leukemic cell line. Blood 104: 3550–3557, 2004.[Abstract/Free Full Text]
  47. Tsai CT, Lai LP, Kuo KT, Hwang JJ, Hsieh CS, Hsu KL, Tseng CD, Tseng YZ, Chiang FT, Lin JL. Angiotensin II activates signal transducer and activators of transcription 3 via Rac1 in atrial myocytes and fibroblasts: implication for the therapeutic effect of statin in atrial structural remodeling. Circulation 117: 344–355, 2008.[Abstract/Free Full Text]
  48. Vignais ML, Gilman M. Distinct mechanisms of activation of Stat1 and Stat3 by platelet-derived growth factor receptor in a cell-free system. Mol Cell Biol 19: 3727–3735, 1999.[Abstract/Free Full Text]
  49. Vignais ML, Sadowski HB, Watling D, Rogers NC, Gilman M. Platelet-derived growth factor induces phosphorylation of multiple JAK family kinases and STAT proteins. Mol Cell Biol 16: 1759–1769, 1996.[Abstract]
  50. Vignola AM, Chanez P, Chiappara G, Merendino A, Pace E, Rizzo A, la Rocca AM, Bellia V, Bonsignore G, Bousquet J. Transforming growth factor-beta expression in mucosal biopsies in asthma and chronic bronchitis. Am J Respir Crit Care Med 156: 591–599, 1997.[Abstract/Free Full Text]
  51. Williams CL. The polybasic region of Ras and Rho family small GTPases: a regulator of protein interactions and membrane association and a site of nuclear localization signal sequences. Cell Signal 15: 1071–1080, 2003.[CrossRef][Web of Science][Medline]
  52. Wong KW, Isberg RR. Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1. PLoS Pathog 1: e16, 2005.
  53. Zhou L, Hershenson MB. Mitogenic signaling pathways in airway smooth muscle. Respir Physiol Neurobiol 137: 295–308, 2003.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 IOVSHome page
E. Syriani, G. Cuesto, E. Abad, T. Pelaez, A. Gual, J. Pintor, M. Morales, and X. Gasull
Effects of Platelet-Derived Growth Factor on Aqueous Humor Dynamics
Invest. Ophthalmol. Vis. Sci., August 1, 2009; 50(8): 3833 - 3839.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/L698    most recent
00529.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Simeone-Penney, M. C.
Right arrow Articles by Simon, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Simeone-Penney, M. C.
Right arrow Articles by Simon, A. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.