Spleen tyrosine kinase (Syk) is an immunoregulatory tyrosine kinase that was identified originally in leukocytes. It is a key regulator of innate immunity as well as hematopoietic cell differentiation and proliferation. A role for Syk in regulating normal cellular functions in nonhematopoietic cells is increasingly recognized. We have shown previously robust Syk expression in airway epithelium, where it regulates the early inflammatory response to human rhinovirus (HRV) infections, and HRV cell entry by clathrin-mediated endocytosis. To test the hypothesis that Syk plays a role in modulating airway epithelial cell proliferation, migration, and production of vascular endothelial growth factor and interleukin-8, we studied the BEAS-2B human bronchial epithelial cell line and primary human airway epithelia from normal and asthmatic donors using Syk-specific pharmacologic inhibitors and small interfering RNA. Using an in vitro “wounding” model, we demonstrated significant impairment of “wound” closure after treatment with the Syk inhibitors N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406) and 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]-nicotinamide dihydrochloride (BAY61-3606), overexpression of the kinase-inactive SykK396R mutant, and Syk knockdown by small interfering RNA. HRV infection also impaired wound healing, an effect that was partly Syk-dependent because wound healing was impaired further when HRV infection occurred in the presence of Syk inhibition. Further investigation of potential regulatory mechanisms revealed that inhibition of Syk suppressed HRV-induced vascular endothelial growth factor expression while promoting the activation of caspase-3, a mediator of epithelial cell apoptosis. Together, these results indicate that Syk plays a role in promoting epithelial cell proliferation and migration, while mitigating the effects of apoptosis.
Spleen tyrosine kinase (Syk) is an important immunoregulatory protein tyrosine kinase that has been well described in leukocytes to regulate multiple aspects of innate immunity. These include the regulation of myeloid differentiation and proliferation as well as the modulation of immunoreceptor signaling pathways that govern humoral, allergic, and cytotoxic immunity (Mócsai et al., 2010). A functional role for Syk is being recognized increasingly in nonhematopoietic cells. Our laboratory has shown Syk to be robustly expressed in airway epithelium, where it signals downstream of intercellular adhesion molecule-1 (ICAM-1), the major receptor for human rhinovirus (HRV), the most common cause of acute infections in humans. In the human bronchial epithelial cell line (BEAS-2B) and normal human bronchial epithelial cells, we have demonstrated previously the recruitment and activation of Syk within minutes of HRV-ICAM-1 binding (Wang et al., 2006). Syk then activates two signaling pathways: the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathway that leads to the expression of the neutrophil chemokine interleukin (IL)-8 (Wang et al., 2006) and the phosphatidylinositol 3-kinase (PI3K) pathway that mediates HRV cell entry by clathrin-mediated endocytosis (Lau et al., 2008).
Ulanova et al. (2005) also observed a role for Syk in mediating the airway epithelial cell inflammatory response: engagement of β1 integrin led to Syk activation, Syk-dependent induction of IL-6 expression, and enhanced surface expression of ICAM-1, whereas tumor necrosis factor-α stimulation resulted in Syk-dependent nitric oxide expression and induction of inducible nitric-oxide synthase (Ulanova et al., 2006). These observations, together with ours in the context of HRV infection, clearly indicate a role for Syk in the regulation of the airway epithelial cell inflammatory response.
HRV has been reported to induce cytotoxicity and impair wound healing in an in vitro model of tissue injury using BEAS-2B cells (Bossios et al., 2005). Other studies have shown HRV to induce airway epithelial cell apoptosis by activation of caspase-3 and caspase-9 (Deszcz et al., 2005; Wark et al., 2005). The role of Syk in these cellular events after HRV infection is not known, although Syk has been shown to be an important regulator of cell survival and proliferation in different cell types. For example, in addition to its critical role in the differentiation of hematopoietic cells to mature B and γδT lymphocytes, the normal and appropriate activity of Syk appears to be important for B cell homeostasis. Tonic activation of Syk has been found in several common B cell lymphoma subtypes (Gobessi et al., 2009) and is thought to be responsible for abnormal proliferation and survival of the lymphomatous cells.
There also is evidence that Syk regulates proliferation and migration in nonhematopoietic cells. Indeed, the fatal intrauterine hemorrhage observed in homozygous Syk knockout mice is a result of aberrant development of the blood and lymphatic vessels due to abnormal endothelial cell proliferation and migration (Yanagi et al., 2001; Abtahian et al., 2003). Syk also regulates breast epithelial cell proliferation, migration, and differentiation: studies in human ductal cell carcinomas reveal that loss of Syk correlated with increased aggressiveness and metastases of the tumors (Coopman et al., 2000). Subsequent in vitro studies have shown that reconstitution of Syk expression abrogated the abnormal cell proliferation observed in a cancerous breast epithelial cell line (Moroni et al., 2004). Therefore, we tested the hypothesis that Syk regulates airway epithelial cell proliferation and migration and plays a role in mitigating the effects of HRV-induced epithelial cell damage and apoptosis.
Materials and Methods
Cell Lines and Primary Cells.
BEAS-2B cells, a human bronchial epithelial cell line (a generous gift from Dr. Curtis Harris, National Cancer Institute, Bethesda, MD), were cultured in Clonetics bronchial epithelial growth medium (BEGM; Lonza Walkersville, Inc., Walkersville, MD) at 37°C in 5% CO2 in a humidified environment as described previously (Lau et al., 2008).
Primary airway epithelial cells obtained from normal, nonsmoking donors and asthmatics were purchased from MatTek (Ashland, MA). The clinical history of the donors was provided by MatTek. The cells were equilibrated in Air 100-MM media (MatTek) for 16 to 18 h upon arrival, in an air-liquid interface in a humidified 5% CO2 incubator before experimentation.
Antibodies, Inhibitors, and HRV16.
Antibodies were purchased from the following sources: mouse monoclonal Syk 4D10 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit polyclonal phospho-Akt (Thr308), rabbit polyclonal Akt, rabbit polyclonal phospho-p38 and total p38, rabbit polyclonal full-length caspase-3, and rabbit polyclonal cleaved caspase-3 antibodies from Cell Signaling Technology (Danvers, MA). The horseradish peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). HRV16 was propagated in WI-38 cells (both from American Type Culture Collection, Manassas, VA), harvested by repeated freeze-thaw cycles at −80°C, purified, and stored at −80°C in BEGM in aliquots containing approximately 104.5 TCID50, as assessed using a microtiter plate assay (Lau et al., 2008).
The Syk-selective inhibitors N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4] oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406) and 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]-nicotinamide dihydrochloride (BAY61-3606) (Yamamoto et al., 2003; Matsubara et al., 2006a,b; Li et al., 2009; Ruzza et al., 2009; Riccaboni et al., 2010) were a gift of Boehringer Ingelheim AG (Biberach am Riss, Germany). N-[4-[6-(Cyclobutylamino)-9H-purin-2-ylamino]phenyl]-N-methylacetamide (NVP-QAB-205) was a gift from GSK Inc. (Mississauga, ON, Canada) (MacGlashan et al., 2008). Although the compounds have been evaluated extensively in vitro and in vivo (Matsubara et al., 2006a,b; Riccaboni et al., 2010), these studies all have been performed in the context of leukocyte signaling. Therefore, we evaluated the efficacy in airway epithelial cells by evaluating known Syk-mediated cellular events after HRV infection in BEAS-2B cells (Supplemental Fig. 1): R406, BAY61-3606, and NVP-QAB-205 impaired HRV-induced p38 mitogen-activated protein kinase phosphorylation and PI3K activation to similar degrees as had been reported previously by our group when using genetic means to knock down Syk activity (Wang et al., 2006; Lau et al., 2008).
Transfection, Plasmids, and Small Interfering RNA.
BEAS-2B cells were transfected using the Amaxa Nucleofector system according to the manufacturer's instructions using 4 × 106 cells and 2 μg of plasmid DNA or 2 × 106 cells with 0.75 μg of control SMARTpool small interfering RNA (siRNA) reagent or Syk SMARTpool siRNA (Millipore, Billerica, MA). The cells were plated in normal culture medium after transfection and cultured at 37°C in 5% CO2 for 36 to 48 h before use for the experiments. Sham-transfected cells underwent the same procedure in the absence of siRNA.
The plasmids expressing human Syk mutants were generated from the pcDNA3 plasmid containing amino-terminal hemagglutinin-tagged wild-type (WT) human Syk using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The Syk cDNAs were excised from pcDNA3 as a 1.553-kb BamHI fragment and subcloned into the BglII and BamHI sites of pEGFP-N2 (Clontech, Mountain View, CA). All of the mutants were verified by sequencing and Western blot analysis to ensure expression of a protein of appropriate size and immunoreactivity before use for experimentation and have been validated already in BEAS-2B cells (Lau et al., 2008). We have shown previously that transfection of BEAS-2B cells with Syk SMARTpool siRNA (Millipore) specifically knocked down Syk expression and did not affect the expression of other HRV signaling pathway molecules, such as ICAM-1 or ezrin (Wang et al., 2006; Lau et al., 2008).
Scratch “Wound” Test and HRV 16 Stimulation Experiments.
BEAS-2B cells were grown to confluence on six-well plates in normal culture medium. “Wounding” was performed by scoring the cell monolayer using a sterile P100 pipette tip. Two “scratches” were made per well. Detached cells were removed by washing with prewarmed normal culture medium, and the cells were returned to normal culture conditions for 24 h. For the HRV stimulation experiments, purified HRV16 stock preparations were diluted 1:10 in BEGM and subsequently applied to the confluent BEAS-2B cells. Cells were incubated at 37°C for 1 h. Medium containing HRV16 was aspirated, and fresh normal culture medium was added back to the cells. Where indicated, “wounding” was performed at this time point. The cells were returned to incubation at 37°C for 24 h and harvested for immunoblotting experiments or fluorescence microscopy (described below). Nonstimulated cells were treated in the same manner in BEGM without HRV16. For the pharmacological studies, Syk inhibition was performed by preincubating the cells with 0.5 to 10 μM R406 or BAY61-3606 dissolved in 1 M dimethyl sulfoxide (DMSO) for 1 h before the HRV16 inoculation or “wounding.” DMSO alone (1 M) was used as the control. The presence of R406 and BAY61-3606 was maintained throughout the duration (24 h) of the experiment. In the experiments with the primary airway epithelial cells, HRV16 inoculation was performed on the apical surface using 200 μl of medium.
Transmission and Fluorescence Microscopy and Quantification of Wound Repair.
After “wounding” using a sterile P100 pipette, BEAS-2B cells were imaged immediately (0 h) and at 24 h later using an Eclipse TE 200 microscope (Nikon Canada, Mississauga, ON, Canada) and 10× objective using the Simple PCI software (Hamamatsu Corporation, Bridgewater, NJ). The images were saved as .tiff files and exported into Canvas X. We measured the size of the wound at 0 and 24 h at time of image acquisition using a size bar calibrated for the 10× objective. For each image, three measurements across the opposing edges of the wound were recorded. The mean of these measurements (counted as an n of 1) was expressed as a percentage of the initial measurement at 0 h. At least four separate experiments were performed for each of the experimental conditions studied.
SDS-Polyacrylamide Gel Electrophoresis, Western Blot Analysis, and Enzyme-Linked Immunosorbent Assay.
We have described previously the methods used for harvesting cell lysates and measuring protein concentrations (Wang et al., 2006; Lau et al., 2008). For Western blot analysis of whole-cell lysates, 30 μg of protein were loaded per lane and separated by SDS-polyacrylamide gel electrophoresis using a 7.5 to 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoblotted with primary and secondary antibodies as described previously (Wang et al., 2006). Quantification of IL-8, vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and epidermal growth factor (EGF) expression was performed by enzyme-linked immunosorbent assay according to the manufacturer's instructions. The kits for IL-8 (Hu IL-8/NAP-1), VEGF (Hu VEGF), EGF (Hu EGF), and TGF-β (multispecies TGF-β1) were purchased from Medicorp Inc. (Montreal, PQ, Canada).
Analysis of variance was used for factorial analysis. Post hoc tests were conducted when analysis of variance reached p < 0.05, using Tukey's adjustment method. The statistical analysis program Prism 4.0c was used for analysis (GraphPad Software Inc., San Diego, CA).
Syk Mediates Cell Proliferation and Wound Closure after Injury.
The role of Syk in airway epithelial cell proliferation was assessed using the human BEAS-2B cell line after transfection with Syk-siRNA to knock down Syk expression. Equal numbers of cells were plated immediately after transfection with Syk- or control-siRNA, and the cells were returned to normal culture conditions for an additional 72 h. At this time, total cell counts were determined after trypsinization in an automated cell counter (Beckman Coulter, Fullerton, CA). As shown in Fig. 1, A and B, total cell numbers were consistently lower in BEAS-2B cells that were transfected with Syk-siRNA compared with those of cells transfected with control-siRNA (p < 0.05, n = 4/group). Western blot analysis of the whole-cell lysates revealed effective Syk knockdown at 72 h after transfection (Fig. 1B, lower right corner). Proliferation of sham-transfected cells was similar to that of control-siRNA-transfected BEAS-2B cells (data not shown).
Next, we used an in vitro model of tissue injury to evaluate the role of Syk in wound repair. Confluent monolayers of BEAS-2B cells grown on six-well plates were wounded as described under Materials and Methods. Wound repair/cell migration then was assessed at 24 h postinjury. To evaluate the role of Syk, we used three different approaches: 1) inhibition with the Syk-selective inhibitors R406 and BAY61-3606, 2) Syk knockdown with siRNA, and 3) overexpression of the dominant-negative kinase-inactive SykK396R mutant. As shown in Fig. 1C, control BEAS-2B cells and those treated with DMSO (vehicle control) exhibit almost complete closure of the wound at 24 h after wound induction. In contrast, wound closure was impaired in cells treated with the Syk-selective inhibitors R406 (0.5–1 μM) and BAY61-3606 (1–5 μM). Both R406 and BAY61-3606 significantly attenuated wound repair at 24 h (Table 1, no HRV, *, p < 0.05, n = 4/group). Although the higher concentrations of the inhibitors appeared to impair wound healing to a greater degree, this effect was not statistically significant.
We also used complementary techniques to assess the role of Syk activity in this in vitro model of wound repair to minimize any ambiguity about the role of Syk in this cellular function. As shown in Table 1 (no HRV), knockdown of Syk using siRNA also impaired wound healing compared with those of sham-transfected and control-siRNA-transfected cells (**, p < 0.05, n = 4/group); no differences were noted between the sham- and control-siRNA-transfected cells. Expression of the kinase-inactive SykK396R mutant and the dual SH2 domain SykR46,201A mutant also impaired wound healing compared with those of sham- and WT-Syk-transfected cells (***, p < 0.05, n = 4/group). Because integrity of the SH2 domains is critical for Syk activation and function (Lau et al., 2011), impaired wound healing in cells overexpressing SykR46,201A provides additional evidence in support of a role for Syk in wound healing after injury.
HRV Infection Impairs Wound Repair and Is Impaired Further by Syk Inhibition.
HRV has been reported to induce cytotoxicity and impair wound healing (Bossios et al., 2005), although a role for Syk in this process has not been described. Therefore, we evaluated the role of Syk in wound healing in BEAS-2B cells after HRV infection (Fig. 2). Although noninfected control and DMSO-treated cells exhibited almost complete wound closure at 24 h, the presence of HRV infection significantly impaired closure in both the control and DMSO-treated cells (Table 1, p < 0.05, n = 4/group). The same phenomenon was observed in cells undergoing sham, control-siRNA, and WT-Syk transfection (i.e., wound closure was almost 100% at 24 h in the noninfected cells but decreased by one third in the HRV-infected cells; Table 1, p < 0.05 compared with no HRV cells, n = 4).
Syk inhibition in the setting of HRV infection further impaired wound healing regardless of the method employed to knock down Syk activity (Fig. 2). In HRV-infected cells, treatment with R406 and BAY61-3606 significantly reduced wound healing to a greater extent than noninfected cells (Table 1, p < 0.05, n = 4/group). Likewise, Syk knockdown by siRNA and overexpression of the kinase-inactive SykK396R or SykR46,201A mutants had a greater effect on delaying wound healing in the presence of HRV infection compared with cells treated with comparable conditions but not infected with HRV (Table 1, p < 0.05, n = 4/group).
Syk Appears to Protect from Caspase-3-Induced Apoptosis after HRV Infection.
Although not as cytotoxic as other respiratory viruses such as respiratory syncytial virus, HRV has been reported to induce airway epithelial cytotoxicity and apoptosis by activation of caspase-3 (Deszcz et al., 2005; Wark et al., 2005). To evaluate the role of Syk in HRV-induced activation of caspase-3, we used Western blot analysis to detect the presence of the 17- and 19-kDa caspase-3 cleavage products. As shown in Fig. 3, HRV16 infection alone resulted in the activation of caspase-3, with the emergence of the 19-kDa cleavage product being most apparent at 3 h after HRV inoculation in sham- and control-siRNA-transfected cells. However, transfection with Syk-siRNA (Fig. 3A, far right five panels) enhanced caspase-3 activation at all of the time points evaluated. Note that Syk expression was decreased only in cells transfected with Syk-siRNA. Similar observations were made in BEAS-2B cells treated with 1 and 5 μM BAY61-3606 (Fig. 3B).
HRV-Induced Expression of VEGF and IL-8 Is Mediated by Syk.
VEGF is known to be induced by HRV infections and has been implicated as a mediator of airway remodeling in asthma (Leigh et al., 2008). Therefore, we assessed the role of Syk in the regulation of HRV-induced VEGF expression. We observed significantly increased VEGF expression at 3 and 9 h after HRV infection in control and DMSO-treated BEAS-2B cells (Fig. 4A, p < 0.001, n = 3/group). Inhibition of Syk with 10 μM R406 and BAY61-3606 significantly decreased VEGF expression at both time points (p < 0.05, n = 3/group). We had previously shown HRV-induced IL-8 expression to be attenuated when Syk expression was knocked down by siRNA (Wang et al., 2006). Therefore, we evaluated the effects of R406 and BAY61-3606 on IL-8 expression and observed that both inhibitors significantly decreased IL-8 expression at 3 and 9 h after HRV infection (Fig. 4B, p < 0.05). Subsequent studies using 1 and 2 μM R406 and BAY61-3606 yielded similar observations with no obvious dose dependence (data not shown). Taken together, it appears that Syk mediates the induction of VEGF expression and is also responsible for maintaining IL-8 expression after HRV infection.
Syk Mediates VEGF and IL-8 Expression in Primary Normal and Asthmatic Airway Epithelia.
To further validate the role of Syk in airway epithelial cell function, we assessed the effects of R406 and BAY61-3606 on VEGF and IL-8 secretion by primary airway epithelia obtained from donors with and without asthma. As shown in Fig. 5A, we observed high basal VEGF expression in both nonasthmatic and asthmatic primary airway epithelial cells (p = N.S., n = 3/group). R406 and BAY61-3606 exhibited no significant effect on basal VEGF expression in either nonasthmatic or asthmatic epithelium (n = 3/group). Increased VEGF expression was observed after HRV inoculation, which was decreased to varying degrees in cells treated with inhibitors, with R406-treated cells showing significant differences (*, p < 0.05, n = 3).
We also measured IL-8 expression in the primary airway epithelia (Fig. 5B). Basal IL-8 expression was significantly higher in the nonasthmatic epithelial cells compared with that in the asthmatic epithelial cells (p < 0.05, n = 3/group). Treatment with the Syk inhibitors did not affect basal IL-8 expression in either asthmatic or nonasthmatic epithelium. HRV induced IL-8 expression in both nonasthmatic and asthmatic epithelial cells to similar levels at 9 h after inoculation, despite differences in basal expression. In both asthmatic and nonasthmatic epithelia, treatment with R406 and BAY61-3606 attenuated the expression of IL-8 compared with those in control and DMSO-treated cells (*, p < 0.05, respectively, n = 3/group). This was most apparent at 9 h after HRV inoculation.
We also evaluated a third-generation Syk inhibitor, NVP-QAB-205, a purine derivative with an IC50 value of 10 nM that is similar to BAY61-3606, an imidazopyrimidine analog, but with greater potency than R406, an imidazopyrimidine analog that has an IC50 value of 40 to 160 nM (Li et al., 2009; Ruzza et al., 2009; Riccaboni et al., 2010). NVP-QAB-205 significantly attenuated HRV-induced expression of VEGF and IL-8 (Fig. 5, C and D, *, p < 0.05 compared with control and DMSO-treated cells, same time point, n = 3).
Our observations in an in vitro model using BEAS-2B cells clearly reveal a role for Syk in wound repair after injury and HRV infection. The effect of HRV is, in part, independent of Syk, because infection alone was sufficient to significantly reduce wound repair, with evidence of an additive effect when infection occurred in the presence of Syk inhibition. In primary cells, treatment with R406 and NVP-QAB-205 significantly attenuated VEGF expression at 3 and 9 h after HRV infection, reducing expression levels below that at baseline, suggesting a role for Syk in mediating basal VEGF expression. In the BEAS-2B cell line, basal VEGF expression was negligible, and treatment with Syk inhibitors only partially reduced VEGF expression at 3 and 9 h after HRV infection. This disparity is most likely due to inherent differences between cell lines and primary cells. Indeed, although the BEAS-2B cell line is used extensively as a surrogate for airway epithelial cells, they were derived by transforming human bronchial epithelial cells with an adenovirus 12-simian virus 40 construct (Reddel et al., 1988). In the process of immortalization, they have lost some of the characteristics of airway epithelium, such as the formation of tight junctions. Moreover, the primary cells were grown at an air-liquid interface to allow for epithelial cell differentiation and maturation, whereas BEAS-2B cells were grown in submerged culture, another difference that contributes to the observed difference in the BEAS-2B and primary airway epithelial cell responses (Figs. 4 and 5).
Differences between the BEAS-2B and the primary cells were most notable in the EGF response to HRV infection (Supplemental Fig. 2). EGF and EGF receptor signaling have been shown to promote chemotaxis and wound repair in scrape-wounded airway epithelial cell monolayers (Puddicombe et al., 2000). Whereas HRV infection suppressed EGF expression in BEAS-2B cells, it induced expression in normal primary airway epithelia; in asthmatic primary airway epithelia, basal levels were already high, and HRV infection did not induce further augmentation of EGF production. Under no condition was EGF expression Syk-dependent, because treatment with R406, BAY61-3606, and NVP-QAB-205 had no effect compared with control and DMSO-treated cells.
Although previous studies have reported Syk to regulate cell proliferation and migration during malignant transformation of breast ductal epithelial cells (Coopman et al., 2000; Moroni et al., 2004), our observations suggest that Syk is important in maintaining the homeostatic integrity of the epithelium in response to mechanical injury and after infection with a common virus, such as HRV.
Syk as a regulator of cell proliferation and differentiation is well described in hematopoietic cells. Studies in chimeric mice that were Syk-deficient in the hematopoietic progenitor lineages revealed Syk to be critical for the expansion and differentiation of B and γδT lymphocytes. Normal Syk function is critical for maintaining B cell homeostasis; dysregulated Syk activation results in aberrant proliferation and the development of several common types of B cell lymphomas and chronic lymphocytic leukemia (Gobessi et al., 2009; Young et al., 2009). Increased B cell survival has been attributed to tonic Syk activation, leading to sustained Akt activation, up-regulation of antiapoptotic proteins such as myeloid cell leukemia-1, and increased resistance to apoptosis (Longo et al., 2008; Gobessi et al., 2009). Conversely, inhibition of Syk with R406 induced apoptosis, correlating with decreased extracellular signal-regulated kinase and Akt signaling and myeloid cell leukemia-1 expression (Gobessi et al., 2009). In addition to B cells, Syk promotes the survival of natural killer cells in response to osmotic stress and UV irradiation (Jiang et al., 2002) and eosinophils in response to cytokine-induced apoptosis (Yousefi et al., 1996).
Our observations indicate Syk to have a positive role in promoting airway epithelial cell migration, proliferation, and survival: wound closure requires both cell migration and proliferation. A similar role for Syk had been shown in vascular endothelium. The fatal bleeding diathesis of the Syk-deficient mouse in utero results from abnormal vascular endothelial cell development, with reduced cell numbers and aberrant morphogenesis (Yanagi et al., 2001; Abtahian et al., 2003). Studies with human umbilical vein endothelial cells revealed that Syk was required for cell proliferation and migration: overexpression of a dominant-negative Syk decreased cell growth and impaired wound healing (using the in vitro wound assay) compared with that of WT-Syk (Inatome et al., 2001). Similar observations have been made in rat aortic smooth muscle cells, where the inhibition of Syk pharmacologically or with Syk-siRNA decreased cell proliferation and migration in response to mitogens such as platelet-derived growth factor-BB (Lee et al., 2007), soluble vascular cell adhesion molecule-1 (Lee et al., 2008), and angiotensin II (Mugabe et al., 2010). Syk also regulates cell migration in myeloid cells: in the neutrophilic HL-60 cell line, Syk mediates cell migration by regulating the formation of the leading edge after β2 integrin engagement and regulates activation and recruitment of PI3K-δ to the leading edge (Schymeinsky et al., 2005, 2007). CD95 ligand-mediated recruitment of myeloid cells to sites of injury also is mediated by Syk recruitment and activation (Letellier et al., 2010).
Previously reports of Syk as a promoter of cell survival, proliferation, and migration in epithelial cells are controversial. Studies in breast ductal cell carcinomas revealed that progressive loss of Syk expression in breast epithelial cells was associated with increasing malignancy and worse prognosis (Coopman et al., 2000). Studies to date have suggested that Syk suppresses abnormal proliferation of the cancerous cells by regulating centrosome function and transcription as well as the urokinase-type plasminogen activator via the PI3K/nuclear factor κB pathway rather than by the regulation of apoptosis (Coopman et al., 2000; Mahabeleshwar and Kundu, 2003; Zyss et al., 2005). However, a report in breast epithelial cell lines of various cancerous origins found the presence of Syk, rather than its absence, to have a protective role in apoptosis (Zhou and Geahlen, 2009). Adding to the controversy is a report in melanoma cells identifying Syk as a tumor suppressor that induces senescence-like growth arrest (Bailet et al., 2009). It is possible that the role of Syk in cell survival and apoptosis is cell-type- and/or stimulus-specific. Our observations using siRNA indicating that Syk mitigates the cytotoxicity of HRV infections by attenuating caspase-3 activation are supported further by similar observations using BAY61-3606 to inhibit Syk activity.
It should be noted, however, that the effects of HRV infection on airway epithelia also remain controversial. Some investigators reported no appreciable airway epithelial cytotoxicity or apoptosis after HRV infection, despite evidence of the disruption of tight junctions and zonula occludens-1 localization leading to increased paracellular permeability, bacterial adhesion, and transmigration (Sajjan et al., 2008). Others demonstrated HRV serotypes to have differential cytotoxicity, with HRV7 being the most and HRV16 the least cytotoxic (Bossios et al., 2005; Wark et al., 2009). We observed peak caspase-3 activation at 3 h after inoculation; induction of apoptosis at this early time point after HRV infection may be a protective mechanism to limit the extent of infection, because propagation of the virus depends on intracellular replication, which typically occurs 8 to 12 h after inoculation. Recent studies have suggested intrinsic differences between the airway epithelia of asthmatic and nonasthmatic individuals (Freishtat et al., 2011; Hackett et al., 2011); HRV infections in asthmatic patients are prolonged and are more severe compared with those in nonasthmatics (Corne et al., 2002; Message et al., 2008). A recent study found the asthmatic airway epithelia to be more susceptible to HRV-induced apoptosis and cell lysis and to have a blunted antiviral interferon, β and λ, response (Contoli et al., 2005; Wark et al., 2005), mechanisms that favor enhanced infections in asthmatics. However, these and our own studies comparing normal and asthmatic airway epithelial responses are not adequately powered, or in fact designed, to address the question of whether intrinsic differences in susceptibility underlie the differences observed. As such, these limitations must be acknowledged.
We had previously reported Syk to be an important early modulator of HRV signaling by regulating the replication-independent activation of the p38 mitogen-activated protein kinase and PI3K signaling pathways leading to IL-8 expression and mediating viral cell entry by clathrin-mediated endocytosis (Wang et al., 2006; Lau et al., 2008). In the current article, we have expanded on these observations in primary airway epithelia using three novel Syk-specific inhibitors: our observations reveal that HRV-induced expression of VEGF and IL-8 are attenuated in the presence of Syk inhibitors at 3 and 9 h postinfection. These time points precede viral replication and corroborate earlier studies made in BEAS-2B and human bronchial epithelial cells, where VEGF expression was first detected at 8 h after inoculation (Leigh et al., 2008). Our studies using the pharmacological inhibitors also indicate Syk to be important for promoting airway epithelial cell growth and proliferation and for modulating activation of caspase-3 in response to HRV infection, observations that were confirmed in parallel studies in which Syk activity was down-regulated by Syk-siRNA and by overexpression of dominant-negative Syk mutants. Taken together, our studies suggest that novel Syk inhibitors may be potential therapeutic agents that can modulate some of the inflammatory sequelae of HRV infections.
Participated in research design: Wang, Lau, Scott, and Chow.
Conducted experiments: Wang, Mychajlowycz, and Lau.
Performed data analysis: Wang, Mychajlowycz, Gutierrez, and Chow.
Wrote or contributed to the writing of the manuscript: Lau, Scott, and Chow.
We acknowledge the generous support of Boehringer Ingelheim A.G. (Biberach am Riss, Germany) for providing the Syk inhibitors, R406 and BAY61-3606. We also thank Dr. Andreas Schnapp (Boehringer Ingelheim A.G.) for critical analysis and reading of the manuscript.
This work was supported by grants from the Canadian Institutes for Health [MOP 83388], Ontario Thoracic Society Grants in Aid, and the Canadian Foundation for Innovation.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- spleen tyrosine kinase
- bronchial epithelial growth medium
- dimethyl sulfoxide
- epidermal growth factor
- human rhinovirus
- intercellular adhesion molecule-1
- phosphatidylinositol 3-kinase
- small interfering RNA
- transforming growth factor-β
- vascular endothelial growth factor
- 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]-nicotinamide dihydrochloride
- tissue culture infective dose.
- Received July 27, 2011.
- Accepted October 21, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics