HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 19/3/428 04-2869fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Krymskaya, V. P. Articles by Panettieri, R. A. Search for Related Content PubMed PubMed Citation Articles by Krymskaya, V. P. Articles by Panettieri, R. A., Jr

Src is necessary and sufficient for human airway smooth muscle cell proliferation and migration

Vera P. Krymskaya^*,1, Elena A. Goncharova^*, Alaina J. Ammit, Poay N. Lim^*, Dmitry A. Goncharov^*, Andrew Eszterhas^* and Reynold A. Panettieri, Jr^*

^* Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA; and
Faculty of Pharmacy, University of Sydney, NSW, Australia

¹Correspondence: Pulmonary, Allergy and Critical Care Division, University of Pennsylvania, 421 Curie Blvd., BRB II/III, Philadelphia, PA 19104-6160, USA. E-Mail: krymskay{at}mail.med.upenn.edu

SPECIFIC AIMS

The central aim of this study was to determine whether activation of Src regulates agonist- and growth factor-induced human airway smooth muscle (ASM) cell proliferation and migration and whether agonist-specific activation of Src underlies differential proliferative and migratory responses.

PRINCIPAL FINDINGS

1. Src is necessary and sufficient for proliferation of human ASM cells
Since Src regulation of growth factor-induced DNA synthesis is cell type specific, we investigated whether Src mediates ASM cell growth induced by PDGF, EGF, or thrombin using pharmacological and molecular approaches. In serum-deprived ASM cells, pretreatment with PP2, a Src inhibitor, followed by stimulation of cell monolayers with PDGF, EGF, or thrombin, PP2 abrogated PDGF-, thrombin-, and EGF-induced DNA synthesis measured by [³H]-thymidine incorporation in a concentration-dependent manner. In parallel experiments, PP2 inhibited mitogen-induced cell proliferation as demonstrated by cell counting. The IC₅₀ for PDGF-, EGF-, and thrombin-induced DNA synthesis was 0.3 µM, 5.5 µM, and 0.3 µM, respectively, and correlates with the IC₅₀ reported for inhibition of Src kinase by PP2 in studies using other cell types. Curiously, EGF-induced stimulation of DNA synthesis was somewhat less responsive to PP2 compared with that induced by PDGF or thrombin.

In earlier studies the specificity of pharmacological inhibitors prevented definitive characterization of the role of Src in modulating cell growth. To further address the role of Src in human ASM cell proliferation and determine whether Src is necessary and sufficient for ASM DNA synthesis, cells were microinjected with plasmids expressing wild-type (Src-WT), constitutively active (Src-CA), and dominant negative (Src-DN) Src. DNA synthesis was measured by BrdU incorporation and immunocytochemical staining. Src plasmids were coinjected with GFP-expressing plasmids for supravital analysis and immunostaining with anti-GFP antibodies to identify microinjected cells (Fig. 1 A). Overexpression of Src-WT or expression of Src-CA stimulated DNA synthesis in ASM cells (Fig. 1B ). Src-WT and Src-CA promoted BrdU incorporation to levels comparable with cells transfected with a GFP vector alone. These experiments were performed on serum-deprived cells without mitogenic stimuli. The data suggest that Src is sufficient to induced DNA synthesis in human ASM cells.

View larger version (19K): [in this window] [in a new window]   Figure 1. Src is necessary and sufficient for human ASM cell proliferation. ASM cells were comicroinjected with Src-WT or Src-CA and GFP to identify microinjected cells and compared with cells microinjected with GFP alone. Results are expressed as the mitotic index: % BrdU-positive/total number of cells. A) Representative images of immunostaining with anti-BrdU antibody to identify DNA synthesis and with anti-GFP antibody to identify cells microinjected with Src mutants. Cells were untreated (B) or stimulated with PDGF, EGF, or thrombin or diluent alone for 16 h (C). Statistical analysis was performed by 1-way ANOVA [Bonferonni Dunn]; 472 Src-CA-microinjected cells, 504 Src-WT-microinjected cells, and 226 Src-DN-microinjected cells were analyzed from 4 separate experiments.

Expression of dominant negative Src significantly inhibited PDGF-, EGF-, and thrombin-induced DNA synthesis (Fig. 1C ). Microinjection of GFP plasmid without Src had no effect on growth-induced BrdU incorporation. Thrombin-induced DNA synthesis was markedly more sensitive to Src-DN transfection than that seen in PDGF- or EGF-stimulated cells. We found that microinjection of neutralizing antibody directed against the C terminus of Src inhibited thrombin-induced BrdU incorporation by 40.7 ± 21.7% (P=0.03), having little effect of EGF-induced DNA synthesis. These data show that constitutively active or wild-type Src is sufficient to induce DNA synthesis in human ASM cells; dominant negative Src appears to inhibit RTK- and GPCR-mediated DNA synthesis.

2. Src is necessary and sufficient for human ASM migration
In chronic severe asthma, ASM mass is increased and myofibroblast numbers are increased in the submucosa of the airways. An increased myofibroblast number likely represents increased migration of myofibroblasts into the airways after repetitive airway injury and repair. Since Src regulates motility in a cell type-specific manner and Src is activated by adhesion molecules, we addressed whether transient expression of Src mutants promote human ASM motility. Expression of Src-DN inhibited chemokinesis by 38.0 ± 4.0% (P<0.003) in serum-deprived cells in the absence of any stimuli (Fig. 2 A). Attenuation of cell migration was not associated with cell apoptosis or inhibition of cell attachment, since equal numbers of viable cells were observed in the upper well of the Boyden chamber after 4 h of the migration assay (data not shown).

View larger version (9K): [in this window] [in a new window]   Figure 2. Modulation of human ASM cell migration by Src. Growth-arrested ASM transfected with Src-DN or Src-CA were harvested and transferred to a Boyden chamber for analysis. Cells were untreated (A) or stimulated with 10 ng/mL PDGF (B, C), 10 ng/mL EGF, 1 U/mL thrombin, 1 µM bradykinin (C), or vehicle. Mean values ±SE from 3 observations by ANOVA (Bonferroni-Dunn).

Overexpression of Src-WT or expression of Src-CA is sufficient to promote cell migration in the absence of PDGF stimulation, suggesting that Src is a critical modulator of cell motility (Fig. 2A ). Whereas Src-DN attenuated PDGF-induced ASM cell motility, Src-WT or Src-CA synergistically enhanced cell migration in the presence of PDGF. PDGF alone induced a 4.84 ± 0.83-fold (P<0.001) increase, and Src-WT or Src-CA together with PDGF stimulated cell motility by 8.35 ± 0.52-fold (P<0.01) or 10.6 6± 0.1.62-fold (P<0.008), respectively. PDGF, EGF, and thrombin have differential effects on human ASM cell migration. PDGF markedly stimulated cell migration by 4.57 ± 1.30-fold; EGF increased cell motility by only 1.78 ± 0.19-fold (Fig. 2C ). Different contractile agonists such as thrombin and bradykinin had little effect on cell migration (Fig. 2C ). These data demonstrate that Src not only modulates cell proliferation but is necessary and sufficient to promote cell migration.

3. Mitogens activate Src in ASM cells
Parallel studies were performed to address whether mitogens directly activate Src. Confluent serum-deprived cells stimulated with PDGF, EGF, or thrombin promoted time-dependent phosphorylation of the Src activation site Tyr-418. EGF and thrombin, which have little effect on ASM cell migration, promoted a rapid Tyr-418 phosphorylation of Src after 1–2 min of stimulation, followed by an abrupt return to basal levels by 5 min. PDGF induced a rapid Tyr-418 Src phosphorylation at 2 min; however, there was a sustained phosphorylation at 416 that remained elevated at 30 min, then returned to basal levels only after 60 min. Our data demonstrate that EGF and thrombin have little effect on cell migration whereas PDGF markedly induces cell proliferation. A potential mechanism by which PDGF can promote migration while other mitogens lack this ability may relate to the sustained activation of Src.

4. PI3K is activated by Src stimulation
Recent studies from our laboratories show that human ASM cell proliferation and migration are dependent on mitogen-induced PI3K activation. Although all mitogens require PI3K activation, not all mitogens (e.g., thrombin) promote cell migration. We examined whether there is cross-talk between Src and PI3K activation in ASM cells. PP2, a Src inhibitor, attenuated PI3K activity associated with tyrosine phosphorylated proteins, suggesting a potential role of Src in PI3K activation. To address whether Src and PI3K may physically associate to regulate their activation state, studies were performed in which confluent serum-deprived monolayers of ASM were stimulated with PDGF, EGF, thrombin, or bradykinin for 2, 5, 15, 30, and 60 min; lysed and Src immunoprecipitates were assayed for PI3K activation. PDGF and thrombin, but not EGF or bradykinin, induced PI3K activity associated with Src that reached a maximum level at 15 or 30 min for PDGF and thrombin, respectively, then declined to basal levels. Bradykinin had little effect on DNA synthesis or cell migration but robustly induced increased calcium responses and force generation of ASM cells. Src-associated PI3K activity in EGF-stimulated cells was not detected, suggesting PI3K activation may be important for PDGF and thrombin-induced Src signaling in human ASM cells. These data suggest a differential regulation of Src and its associated PI3K activity in GPCR- and RTK-activated signaling pathways that potentially modulates cell proliferation and motility.

CONCLUSIONS AND SIGNIFICANCE

Src protein tyrosine kinase integrates GPCR- and RTK-induced signaling, which ultimately regulates cell proliferation, migration, differentiation, and gene transcription. Little information is available on whether contractile agonists or growth factors activate Src and whether Src modulates human ASM cell proliferation and migration. Here, we show that Src activation is required for human ASM mitogenesis and motility. PDGF, EGF, and thrombin induce rapid activation of Src, and inhibition of Src induces a concentration-dependent abrogation of PDGF-, EGF-, and thrombin-induced ASM cell DNA synthesis. Src immunoprecipitates had associated PI3K activation in response to PDGF and thrombin, but not EGF. Microinjection of Src-DN in thrombin-stimulated cells completely inhibited BrdU incorporation, suggesting a critical role of Src for thrombin-induced DNA synthesis that correlates with the well-established role of Src in GPCR-induced mitogenesis. Thrombin-induced mitogenesis involves activation of PI3K and Erk, suggesting a potential cross-talk between Src and PI3K, Src and Erk. By expressing Src-DN or Src-CA, we found that Src is necessary and sufficient for human ASM cell migration. Our data demonstrate that not all ASM mitogens are promigratory factors. Only PDGF markedly induced human ASM cell migration; EGF and thrombin had little effect on cell motility. These differences in cell motility may be associated with differences in the kinetics of Src activation. Thus, stimulation of human ASM cells with PDGF induced a sustained Src activation; in contrast, EGF and thrombin stimulated transient Src activation.

Our current findings provide new evidence that Src plays an important role in regulating human ASM cell proliferation and migration. Src is deferentially regulated by growth factors and contractile agonists that may be associated with the relative contribution of Src signaling in human ASM cell proliferation and migration (Fig. 3 ). In asthma and chronic obstructive pulmonary disease (COPD), Src may serve as a molecule of potential importance in integrating complex signaling events and as a potential therapeutic target in airway remodeling. Molecular mechanisms of airway remodeling associated with pathobiology of asthma and COPD are not well understood. Our study identified the function of Src as a critical regulator of human ASM cell proliferation and migration, which fills a gap in understanding agonist-induced smooth muscle cell proliferation and migration.

View larger version (33K): [in this window] [in a new window]   Figure 3. Src integrates signaling from receptor protein tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCRs), which ultimately regulate human ASM cell proliferation and migration. Our study demonstrates that Src is differentially regulated by PDGF, EGF, and thrombin, but not bradykinin, which signals by activation of RTKs, GPCRs.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2869fje;

This article has been cited by other articles:

				M. Sharma, J. Batra, U. Mabalirajan, S. Sharma, R. Nagarkatti, J. Aich, S. K. Sharma, P. V. Niphadkar, and B. Ghosh A Genetic Variation in Inositol Polyphosphate 4 Phosphatase A Enhances Susceptibility to Asthma Am. J. Respir. Crit. Care Med., April 1, 2008; 177(7): 712 - 719. [Abstract] [Full Text] [PDF]

				M. C. Simeone-Penney, M. Severgnini, L. Rozo, S. Takahashi, B. H. Cochran, and A. R. Simon PDGF-induced human airway smooth muscle cell proliferation requires STAT3 and the small GTPase Rac1 Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L698 - L704. [Abstract] [Full Text] [PDF]

				Y. Ohta, M. Hayashi, T. Kanemaru, K. Abe, Y. Ito, and M. Oike Dual Modulation of Airway Smooth Muscle Contraction by Th2 Cytokines via Matrix Metalloproteinase-1 Production J. Immunol., March 15, 2008; 180(6): 4191 - 4199. [Abstract] [Full Text] [PDF]

				S. Zhuang, G. R. Kinsey, K. Rasbach, and R. G. Schnellmann Heparin-binding epidermal growth factor and Src family kinases in proliferation of renal epithelial cells Am J Physiol Renal Physiol, March 1, 2008; 294(3): F459 - F468. [Abstract] [Full Text] [PDF]

				R. A. Panettieri Jr., M. I. Kotlikoff, W. T. Gerthoffer, M. B. Hershenson, P. G. Woodruff, I. P. Hall, and S. Banks-Schlegel Airway Smooth Muscle in Bronchial Tone, Inflammation, and Remodeling: Basic Knowledge to Clinical Relevance Am. J. Respir. Crit. Care Med., February 1, 2008; 177(3): 248 - 252. [Abstract] [Full Text] [PDF]

				J. K. Bentley and M. B. Hershenson Airway Smooth Muscle Growth in Asthma: Proliferation, Hypertrophy, and Migration Proceedings of the ATS, January 1, 2008; 5(1): 89 - 96. [Abstract] [Full Text] [PDF]

				W. T. Gerthoffer Migration of Airway Smooth Muscle Cells Proceedings of the ATS, January 1, 2008; 5(1): 97 - 105. [Abstract] [Full Text] [PDF]

				K. Parameswaran, K. Radford, A. Fanat, J. Stephen, C. Bonnans, B. D. Levy, L. J. Janssen, and P. G. Cox Modulation of Human Airway Smooth Muscle Migration by Lipid Mediators and Th-2 Cytokines Am. J. Respir. Cell Mol. Biol., August 1, 2007; 37(2): 240 - 247. [Abstract] [Full Text] [PDF]

				C. J. Delvecchio, P. Bilan, K. Radford, J. Stephen, B. L. Trigatti, G. Cox, K. Parameswaran, and J. P. Capone Liver X Receptor Stimulates Cholesterol Efflux and Inhibits Expression of Proinflammatory Mediators in Human Airway Smooth Muscle Cells Mol. Endocrinol., June 1, 2007; 21(6): 1324 - 1334. [Abstract] [Full Text] [PDF]

				M. Hirakawa, Y. Karashima, M. Watanabe, C. Kimura, Y. Ito, and M. Oike Protein Kinase A Inhibits Lysophosphatidic Acid-Induced Migration of Airway Smooth Muscle Cells J. Pharmacol. Exp. Ther., June 1, 2007; 321(3): 1102 - 1108. [Abstract] [Full Text] [PDF]

				E. D. Fixman, A. Stewart, and J. G. Martin Basic mechanisms of development of airway structural changes in asthma Eur. Respir. J., February 1, 2007; 29(2): 379 - 389. [Abstract] [Full Text] [PDF]

				S. Qiu, L. F. Zhao, K. M. Korwek, and E. J. Weeber Differential Reelin-Induced Enhancement of NMDA and AMPA Receptor Activity in the Adult Hippocampus J. Neurosci., December 13, 2006; 26(50): 12943 - 12955. [Abstract] [Full Text] [PDF]

				K. C. Kong, C. K. Billington, U. Gandhi, R. A. Panettieri Jr., and R. B. Penn Cooperative mitogenic signaling by G protein-coupled receptors and growth factors is dependent on Gq/11 FASEB J, July 1, 2006; 20(9): 1558 - 1560. [Abstract] [Full Text] [PDF]

				T. Pertel, D. Zhu, R. A. Panettieri, N. Yamaguchi, C. W. Emala, and C. A. Hirshman Expression and muscarinic receptor coupling of Lyn kinase in cultured human airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, March 1, 2006; 290(3): L492 - L500. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/3/428 04-2869fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Krymskaya, V. P. Articles by Panettieri, R. A. Search for Related Content PubMed PubMed Citation Articles by Krymskaya, V. P. Articles by Panettieri, R. A., Jr