Fibronectin Stimulates TRPV1 Translocation in Primary Sensory Neurons

Summary

Extracellular matrix (ECM) molecules are highly variable in their composition and receptor recognition. Their ubiquitous expression profile has been linked to roles in cell growth, differentiation, and survival. Recent work has identified certain ECM molecules that serve as dynamic signal modulators, versus the more-recognized role of chronic modulation of signal transduction. In this study, we investigated the role that fibronectin plays in the dynamic modulation of TRPV1 translocation to the plasma membrane in trigeminal ganglia (TG) sensory neurons. Confocal immunofluorescence analyses identify co-expression of the TRPV1 receptor with integrin subunits that bind fibronectin. TG neurons cultured upon or treated with fibronectin experienced a leftward shift in the EC₅₀ of capsaicin-stimulated neuropeptide release. This fibronectin-induced increase in TRPV1 sensitivity to activation is coupled by an increase in plasma membrane expression of TRPV1, as well as an increase in tyrosine phosphorylation of TRPV1 in TG neurons. Furthermore, TG neurons cultured on fibronectin demonstrated an increase in capsaicin-mediated Ca⁺² accumulation relative to neurons cultured on poly-D-lysine. Data presented from these studies indicate that fibronectin stimulates tyrosine-phosphorylation-dependent translocation of the TRPV1 receptor to the plasma membrane, identifying fibronectin as a critical component of the ECM capable of sensory neuron sensitization.

Introduction

Fibronectin exists primarily as an extracellular matrix (ECM) molecule, mediating various cellular events such as cell growth, differentiation, and adhesion (Hedman et al. 1978; Wartiovaara et al. 1978). Since it’s initial characterization, fibronectin has been found to exist in many forms, due to the numerous alternative-splicing events that govern expression (Schwarzbauer et al. 1983). Although alternative splicing events can mediate fibronectin expression profiles, peptides contained with the fibronectin sequence serve as ligands for various members of the integrin receptor family, including integrin heterodimers α₃β₁, α₄β₁, α₅β₁, α_vβ₁, α_vβ₅, and α_IIbβ₃ (Smith et al. 1990; Vogel et al. 1990; Elices et al. 1991; Mould et al. 1991; Busk et al. 1992; Koivunen et al. 1993; Bowditch et al. 1994). The activation of integrin heterodimers, including those known to bind fibronectin, induce signaling cascades intracellularly that have been demonstrated to modulate certain receptors and pathways. Indeed, focal adhesion kinase and c-src are activated following fibronectin activation of integrin receptors in cell culture models (Hanks et al. 1992; Kaplan et al. 1995). The extent of signaling beyond these molecules and others are believed to modulate many intracellular systems, including some plasma membrane receptor systems that could possibly influence neuronal excitability.

The transient receptor potential family V type 1 receptor (TRPV1) is an ion channel that predominantly conducts Ca⁺² ions in response to stimuli including capsaicin, heat, protons, and certain cannabinoids (Caterina et al. 1997; Tominaga et al. 1998; Caterina et al. 2000; Smart et al. 2000). Originally identified in a dorsal root ganglia cDNA screen, TRPV1 is primarily expressed at the terminal endings of nociceptive, c-type fibers, where its activation in response to noxious stimuli can result in activation of central nervous system pain pathways.. Similar to other plasma membrane receptors, TRPV1 activity is sensitized upon phosphorylation by kinases including protein kinase A and protein kinase C (Premkumar and Ahern 2000; Vellani et al. 2001; Bhave et al. 2002). Interestingly,c-src also positively modulates TRPV1 activity, increasing both capsaicin-gated current and plasma membrane translocation of the channel (Jin et al. 2004; Zhang et al. 2005). The kinase activity of c-src, a non-receptor tyrosine kinase, is associated with the activation of receptors for growth factors including nerve growth factor and epidermal growth factor (Chinkers and Cohen 1981; Alema et al. 1985). Importantly, recent studies have characterized the importance of fibronectin-mediated integrin activation of c-src kinase activity as it pertains to cellular survival (Papp et al. 2007; Wu et al. 2008).

The hypothesis of this study is that integrin heterodimers that recognize fibronectin positively modulate TRPV1 activity via src-dependent phosphorylation of the receptor channel following fibronectin exposure. Indeed, exposure to both soluble and adherent fibronectin has been demonstrated to drive src kinase activity in various cell models (Meerschaert et al. 1999b; Ren et al. 2005; Tvorogov et al. 2005). In our studies utilizing native trigeminal ganglia (TG), integrin subunits known to heterodimerize to form fibronectin receptors are expressed in TRPV1-positive neurons. Furthermore, fibronectin sensitizes capsaicin-stimulated neuropeptide release from cultured TG neurons, in a src-dependent manner. Fibronectin-dependent increases in capsaicin-responsive neurons, tyrosine phosphorylation, and surface expression suggest that ECM molecules such as fibronectin both chronically and dynamically modulate TRPV1 activity.

Materials and Methods

Tissue Culture

All procedures utilizing animals were approved by the Institutional Animal Care and Use Committee of The University of Texas Health Science Center at San Antonio and were conducted in accordance with policies for the ethical treatment of animals established by the National Institutes of Health. Trigeminal ganglia (TG) were removed bilaterally from male Sprague-Dawley rats (200-250g, Charles River, Wilmington, MA), and dissociated by collagenase treatment (30 min, Worthington, Lakewood, NJ, Lot # S5K8219), followed by trypsin treatment (15 min, Sigma, St.Louis, MO). Cells were centrifuged and re-suspended between each treatment with Pasteur pipettes. Cells were centrifuged, aspirated, and re-suspended in DMEM (Gibco, Grand Island, NY) with 10% FBS (Gibco, Lot # 1389H39), 250 ng/ml NGF (Harlan, Indianapolis, IN, Lot # 05-J1), 1% 5-fluoro deoxyuridine (Sigma), 1% penicillin/streptomycin (Gibco), and 1% L-glutamine (Sigma), and then plated onto plates coated with either poly-D-lysine (PDL), or fibronectin (FN, BD Biocoat, San Diego, CA). Cultures were maintained at 37°C, 5% CO₂, and grown for 5 - 7 days for all experiments. 24 hours before the experiment, cultures were switched to and maintained in serum-free, NGF-free media up to the time of treatment/manipulation. Soluble rat fibronectin and PP2 were purchased from Calbiochem, San Deigo, CA. All other reagents, unless otherwise noted, were purchased from Sigma-Aldrich, St. Louis, MO.

Immunohistochemistry

TG were dissected from 3 rats, sectioned (30 sections/ganglion) with a cryostat and fixed with 4% paraformaldehyde in 0.1M phosphate buffer for 30 min at 25°C. Following fixation, 3 representative tissue sections from each ganglion were rinsed three times in 0.1M phosphate buffered saline (PBS), incubated with 2% normal goat serum (NGS, Sigma-Aldrich, St. Louis, MO), 0.3% Triton X-100 (Fisher Scientific, Pittsburgh, PA), and 20mg/ml bovine-γ-globulin (Sigma-Aldrich) in PBS (blocking solution) for 90 min at 25°C. Tissue sections were then incubated with antisera directed specifically towards rat TRPV1 (1:1,000, Guinea pig, Chemicon/Millipore, Bedford, MA) (Woodbury et al. 2004; Miranda et al. 2007), in combination with either β1 (1:100, Santa Cruz Biotechnology, Santa Cruz Biotechnology, CA) (Cang et al. 2007; Zucchi et al. 2007), β3 (1:100, Chemicon) (Trikha et al. 2002; Rosenberg et al. 2003), β5 (1:100, Santa Cruz Biotechnology) (Wu et al. 2005; Nandrot et al. 2007), β6 (1:100, Chemicon) (Nettles et al. 2004; Trevillian et al. 2004), β7 (1:100, Santa Cruz) (Park et al. 2007), β8 (1:100, Santa Cruz) (McCarty et al. 2005), α4 (1:100, Santa Cruz Biotechnology) (Kumar and Ponnazhagan 2007), α5 (1:100, Santa Cruz Biotechnology) (Usatyuk et al. 2006; Tran et al. 2007), αIIb (1:100, Chemicon) (Trikha et al. 2002), or αv (1:100, Chemicon) (Murase and Horwitz 2002) integrin subunits in blocking solution overnight at 4°C. Tissue sections were then rinsed three times and incubated with species-specific Alexa Fluor secondary antibodies (Molecular Probes, Eugene, OR) in blocking solution (1:100) for 90 min at 25°C. Following three rinses with PBS, tissue sections were air-dried, coverslipped with Vectashield (Vector Labs, Burlingame, CA) and images obtained with a Nikon C1si confocal microscope.

Cultured rat TG cells were grown on poly-D lysine coated coverslips, rinsed with PBS, and fixed with 4% paraformaldehyde in 0.1M PB for 20 min at 25°C. Following fixation, coverslips were rinsed twice with PBS, and incubated with 5% NGS and 0.5% Triton X-100 in PBS for 30 min at 25°C. Coverslips were then incubated with antisera directed specifically towards rat TRPV1 in combination with antisera against either β1, β3, β5, β6, β7, β8, α4, α5, αIIb, or αv integrin subunits overnight at 4°C. Coverslips were then rinsed three times and incubated with appropriate Alexa Fluor antibodies for one hour at 25°C. Following three rinses with PBS, coverslips were mounted to microscope slides with Vectashield and dried overnight. Double-label images were acquired using a 40X objective lens mated to a Nikon E600 microscope (Melville, NY, USA) equipped with a Photometrics SenSys digital CCD camera (Roper Scientific, Tucson, AZ, USA) using Metamorph V4.1 image analysis software (Universal Image Corporation, Downingtown, PA, USA). Results are representative of 4-5 individual coverslips. Quantification of single- and double-labeled neurons was conducted by taking 10 random images of 3 independently generated coverslips, and counting integrin-positive, TRPV1-positive, and integrin- and TRPV1-positive neurons per image. Counts were averaged across 30 total images, and represented as percentage of total integrin-positive or TRPV1-positive.

Calcium Imaging

Cultured TG neurons were plated on fibronectin or poly-D-lysine coated coverslips (BD Biocoat, Bedford, MA) and grown for 5 - 7 days. To measure intracellular [Ca⁺²] levels following capsaicin (50nM) exposure, the dye Fura-2 AM (2 μM; Molecular Probes) was loaded for 30 min at 37°C into cells in the presence of 0.02% Pluronic (Invitrogen). Fluorescence was detected with a Nikon Eclipse TE 2000-U microscope fitted with a 20×/0.8 NA Fluor objective. Fluorescence images from 340 nm and 380 nm excitation wavelengths were collected and analyzed with the MetaFluor Software (MetaMorph, Web Universal Imaging Corporation, Downingtown, PA). The net change in Ca⁺² (ΔF340/380) was calculated by subtracting the basal F340/380 Ca⁺² level (mean value collected for 60 s prior to agonist addition) from the peak F340/380 Ca⁺² level achieved after exposure to the agonists. A minimum ΔF340/380 of 0.03 was designated as the peak-baseline for determination for CAP-responding cells. For each transfection/treatment group, 40-60 cells were imaged, statistical significance determined by one-way ANOVA analysis, *p<0.05.

Western Blot and Biotinylation

The lysis of TG cells, quantification of protein concentration, immunoprecipitation, and Western blotting were conducted as previously described (Jeske et al. 2006). Biotinylation was also performed as previously described (Jeske et al. 2004), using biotin and streptavidin-bound agarose available from Pierce. Antibodies used for Western blotting were TRPV1 (1:1000, Calbiochem), β1 (1:1000, Santa Cruz), α5 (1:1000, Santa Cruz), β-actin (1:2000, Sigma-Aldrich), and c-src (1:1000, Santa Cruz). Antibodies utilized for immunoprecipitation were phospho-tyrosine platinum (1 μg/IP sample, Millipore). Densitometry data analyzed by one-way ANOVA, *p<0.05, **p<0.01, NS=not significant, results are representative of 3-5 independent trials.

CGRP neuropeptide release

TGG were plated onto 24-well plates coated with poly-D lysine, laminin, collagen type IV or fibronectin (BD Biosciences, Franklin Lakes, NJ). Following maintenance at 37°C, 5% CO₂ for 5 days, TG cultures were washed twice with release buffer (HBSS supplemented with 10.9 mM HEPES, 4.2 mM sodium bicarbonate, 10 mM dextrose and 0.1% bovine serum albumin). Following washes, cultures were treated with increasing concentrations of capsaicin (Sigma, St. Louis, MO) for 10 min, after which release buffer and released CGRP were collected and quantified by radioimmunoassay (RIA, (Patwardhan et al. 2008)). Results are represented as means ± SEM of peak values taken from 3 - 5 quadruplicate trials.

Results

Fibronectin serves as an extracellular ligand for many heteromeric integrin receptors throughout the mammalian system. In the trigeminal system, immunocytochemistry and immunohistochemistry were performed to determine which integrin receptor systems co-expressed with TRPV1. In cultured trigeminal ganglia from the rat, strong co-localization (yellow) was observed between TRPV1 (red) and various integrin receptor subunits, all of which form heteromeric receptor systems documented to recognize and become activated by fibronectin (Figure 1A-J). In particular, co-localization was noted for TRPV1 with β1, β3, β5, β6, β7, β8, α4, α5, αIIb, and αv integrin subunits. Further characterization of TRPV1/integrin subunit co-localization by means of randomized cell counting from coverslips revealed distinct differences between the TRPV1-positive TG cells that co-expressed one of the integrin subunits, and integrin subunit-positive TG cells that co-express TRPV1 (Figure 1K). In intact TG sections, TRPV1 co-localization with specific integrin subunits was also observed (Figure 2A-J). These data indicate that many of the integrin subunits that form functional heteromeric receptor systems that bind fibronectin are co-expressed with TRPV1 in TG.

Figure 1.

Fibronectin integrin receptor subunits co-express with TRPV1 in cultured trigeminal ganglia. Primary cultures of trigeminal ganglia probed for TRPV1 expression (red) with integrin subunits known to heterodimerize and recognize fibronectin (green): A. β1, B. β3, C. β5, D. β6, E. β7, F. β8, G. α4, H. α5, I. α11b, and J. αv. Yellow bars indicate 100 μm. K. 30 randomly chosen coverslips from each immunocytochemical pairing were counted for total TRPV1-positive cells, total integrin subunit-positive cells, and cells that co-express both TRPV1 and the targeted integrin subunit. Results are displayed as number of co-expressing cells per total of each singular protein, dark grey bars indicate percentage of integrin-subunit-positive cells that co-express TRPV1, light grey bars indicate percentage of TRPV1-positve cells that co-express the integrin subunit.

Figure 2.

Fibronectin integrin receptor subunits co-express with TRPV1 in intact trigeminal ganglia. Fixed slices of intact trigeminal ganglia probed for TRPV1 expression (red) with integrin subunits known to heterodimerize and recognize fibronectin (green): A. β1, B. β3, C. β5, D. β6, E. β7, F. β8, G. α4, H. α5, I. αv, and J. α11b. Yellow bars indicate 100 μm.

In order to evaluate the effect of fibronectin on TRPV1 activity, capsaicin- (CAP) mediated neuropeptide release measurements were taken from TG cultures plated onto either poly-D-lysine- (PDL), laminin (Lam), collagen type IV (Coll IV) or fibronectin- (FN) coated plates. As depicted in Figure 3A, TG cells cultured and grown on FN-coated plates revealed a greater sensitivity to CAP-mediated calcitonin gene-related protein (CGRP) release compared to those cultured and grown on PDL, Lam or Coll IV. Indeed, the dose response curve observed for CAP-stimulated CGRP release from TG cells grown on FN had an EC₅₀ of 1.78 × 10^-8 M and an E_max of 106.1 fmol, compared to an EC₅₀ of 3.39 × 10^-8 M and E_max of 64.1 fmol for cells grown on PDL, an EC₅₀ of 3.91 × 10^-8 M and E_max of 64.6 fmol for cells grown on Lam, and an EC₅₀ of 4.18 × 10^-8 M and E_max of 70.5 fmol for cells grown on Coll IV. Control experiments designed to determine whether growth of cultured TG cells on FN, PDL, Lam, or Coll IV affected total CGRP content revealed no statistical difference between FN- and PDL- or Coll IV-coated cultures. Indeed, both the 0.1N HCl and 50 mM KCl challenges resulted in similar amounts of released CGRP from FN- and PDL- or Coll IV-coated cultures. (Figure 3B). In contrast, 0.1N HCl and 50 mM KCl stimulated CGRP release from TG cells cultured on Lam-coated vessels was significantly less than that seen from TG cells cultured on PDL-coated vessels (*p < 0.05, as determined by one-way ANOVA). Immunohistochemical counts of TG cells grown on FN, PDL, Lam, or Coll IV using antibodies specific to CGRP and TRPV1 yielded little difference in the numbers of TG cells co-expressing both proteins (Figure 3C). Taken together, Figure 3 results indicate that fibronectin modulation of TRPV1 activity does not occur via a transcriptional process, but possibly by a post-translational process.

Figure 3.

Chronic exposure to fibronectin increases capsaicin-mediated neuropeptide release from trigeminal ganglia. A. Primary cultures of trigeminal ganglia were grown on poly-D-lysine (PDL), laminin (Lam), collagen type IV (Coll IV) or fibronectin (FN), and treated with increasing concentrations of capsaicin (CAP) to stimulate calcitonin gene-related peptide (CGRP) release, as determined by RIA. B. Primary cultures of trigeminal ganglia grown on PDL, Lam, Coll IV or FN subjected to 0.01N HCL- or 50 mM KCl-stimulated CGRP release. C. Primary cultures of trigeminal ganglia grown on PDL, Lam, Coll IV or FN revealed similar staining patterns of CGRP in TRPV1-positve cells (CGRP/TRPV1), and of TRPV1 in CGRP-positive cells (TRPV1/CGRP). *p < 0.05, determined by one-way ANOVA.

To further characterize fibronectin sensitization of CAP-mediated activation of TRPV1, we conducted Ca⁺² imaging of TG cells cultured on either PDL- or FN-coated coverslips. As shown in Figure 4A, a significant difference in CAP-stimulated Ca⁺² accumulation was observed in TG cells grown on the two types of coatings. One potential explanation for this observation is that TG cells grown on FN expressed more TRPV1 on the plasma membrane available for CAP-mediated activation. Indeed, there were almost twice as many CAP-responsive TG cells on FN-coated coverslips than PDL-coated coverslips (Figure 4B), even though the number of TRPV1-positive cells from the total NeuN-positive population on each type of coverslip were not significantly different (Figure 4C). Total populations chosen for Figure 4B (identifiable neurons in an observable field) were differently chosen from those in Figure 4C (total NeuN-positive: all neurons). Furthermore, baseline calcium accumulation levels from TG cells cultured on either PDL or FN prior to capsaicin were indistinguishable (data not shown). These data suggest that TRPV1 sensitivity to CAP is increased in TG cells grow on FN versus PDL, possibly through post-translational modification of TRPV1.

Figure 4.

Chronic exposure to fibronectin increases the number of capsaicin-sensitive trigeminal ganglia cells. A. Quantification of total calcium accumulation in primary cultured trigeminal ganglia cells grown on PDL (light grey) or FN (dark grey) in response to 50 nM CAP. B. Percentage of CAP (50nM)-responsive neurons observed of total pre-determined population of primary cultured trigeminal ganglia, grown on either PDL or FN (actual number shown in parentheses). C. Percentage of TRPV1-positive neurons of total NeuN-positive neurons from primary cultured trigeminal ganglia grown on PDL or FN. *p<0.05, NS = no significance.

Src-mediated phosphorylation of TRPV1 drives translocation of the receptor to the plasma membrane, potentially decreasing threshold activity (Zhang et al. 2005). Firstly, to determine whether TG cellular growth on fibronectin increases tyrosine phosphorylation of TRPV1, we immunoprecipitated from lysates of TG cells grown on either PDL or FN using an antibody directed against phospho-tyrosine, and then probed for TRPV1 and src. As shown in Figure 5A, TRPV1 and src both revealed greater tyrosine phosphorylation from TG cells grown on FN versus PDL. In Figure 5B, we determined that a 20 min treatment of TG cultures with FN also led to an increase in tyrosine phosphorylation in a PP2 sensitive manner. These results then prompted analysis of whether the FN-mediated tyrosine phosphorylation of TRPV1 stimulated translocation of the receptor to the plasma membrane.

Figure 5.

Chronic and acute exposure to fibronectin increases tyrosine phosphorylation of src tyrosine kinase and TRPV1 in trigeminal ganglia cells. A. Immunoprecipitation of primary cultured trigeminal ganglia grown on fibronectin (FN) or poly-D-lysine (PDL) with antibodies directed against phospho-tyrosine residues (P-Tyr), and blotted for TRPV1 and src. B. Immunoprecipitation of primary cultured trigeminal ganglia treated with FN (10 μg/ml, 20 min) following pre-treatment with PP2 (src tyrosine kinase inhibitor, 1mM, 5 min) with antibodies directed against phospho-tyrosine residues (P-Tyr), and blotted for TRPV1 and src expression.

As previously shown by Zhang and colleagues, src-mediated tyrosine phosphorylation of TRPV1 following NGF treatment results in a dramatic translocation of TRPV1 to the plasma membrane in stably transfected HEK293 cells (Zhang et al. 2005). In our TG culture model, we employed surface membrane biotinylation to determine whether FN exposure had similar effects on TRPV1. In Figure 6A, biotintylation of TG cultures demonstrated a significantly greater surface expression of TRPV1 in those cultures grown on FN versus PDL. Furthermore, no difference was seen in the surface expression of integrin receptor subunits β1 or α5 (Figure 6A), which heterodimerize to form a receptor that recognizes and is activated by FN (Birkenmeier et al. 1991). Quantitative analysis of densitometric measurements showed a significant 60% increase (p<0.05, t-test) in TRPV1 surface biotinylation when normalized to β1 and α5 biotinylation, and a significant 75% increase (p<0.01, t-test) is revealed when normalized to cell lysate TRPV1 (Figure 6B). Importantly, there is no difference in TRPV1 cell lysate expression depending on FN or PDL growth (Figure 6A).

Figure 6.

Chronic exposure to fibronectin increases plasma membrane surface expression of TRPV1 in trigeminal ganglia cells. A. Primary cultures of trigeminal ganglia were grown on poly-D-lysine (PDL) or fibronectin (FN), and biotinylated to identify surface proteins. Streptavidin-agarose precipitates (Strept) and 50 μg total cell lysate (CL) aliquots were probed for TRPV1, β1 integrin, α5 integrin, and β-actin expression. Densitometry measurements of streptavidin-precipitated TRPV1 normalized to those for streptavidin-precipitated β1 integrin (B), streptavidin-precipitated α5 integrin (C), and CL TRPV1 (D). *p<0.05, **p<0.01.

TG cultures grown on PDL and treated for 20 min with FN reveal similar results to those generated by TG cultures grown on FN, as shown in Figure 6. Transient FN treatment resulted in a significant increase in surface biotinylation of TRPV1 (Figure 7A), with no apparent differences observed for b1 or a5 integrin subunits. Furthermore, quantitative analysis of densitometric measurements demonstrate significant TRPV1 surface biotinylation when normalized to biotinylated β1, biotinylated α5, and TRPV1 cell lysate expression (p<0.01, t-test) (Figure 7B-D). Interestingly, transient FN treatment appears to increase the difference in surface membrane TRPV1 over that seen from TG cultures grown on FN, although the difference is not significant. Additionally, we determined that TRPV1 biotinylation following 20 min FN treatment was sensitive to the src inhibitor PP2 (Figure 7E), which is congruent with tyrosine phosphorylation results depicted in Figure 5.

Figure 7.

Acute exposure to fibronectin increases plasma membrane surface expression of TRPV1 in trigeminal ganglia cells. A. Primary cultures of trigeminal ganglia were treated with vehicle or fibronectin (FN, 10 μg/ml, 20 min), and biotinylated to identify surface proteins. Streptavidin-agarose precipitates (Strept) and 50 μg total cell lysate (CL) aliquots were probed for TRPV1, β1 integrin, and α5 integrin expression. Densitometry measurements of streptavidin-precipitated TRPV1 normalized to those for streptavidin-precipitated β1 integrin (B), streptavidin-precipitated α5 integrin (C), and CL TRPV1 (D). E. Primary cultures of trigeminal ganglia were pre-treated with the src tyrosine kinase inhibitor PP2 (1 μM, 5 min), and then with FN (10 μg/ml, 20 min), and biotinylated to identify surface proteins. Streptavidin-agarose precipitates (Strept) and 50 μg total cell lysate (CL) aliquots were probed for TRPV1, and β1 integrin. *p<0.05, **p<0.01.

In order to determine whether FN-mediated tyrosine phosphorylation by src is responsible for the significant differences observed in Figure 3, we measured CAP-mediated CGRP release from TG cultures pre-treated with vehicle, FN, or PP2 and FN. As shown in Figure 8, 20 min FN pre-treatment, did not produce identical CGRP release results as in Figure 3, although the E_max for FN-treated cultures was higher than vehicle-treated or PP2-co-treated cultures. The measured differences in E_max are supportive of the hypothesis that greater TRPV1 expression on the plasma membrane of TG neurons, delays desensitization of the receptor to CAP stimulation.

Figure 8.

Acute exposure to fibronectin increases CAP-responsive neuropeptide release in trigeminal ganglia cells in a PP2-sensitive manner. Primary cultures of trigeminal ganglia were prr-treated with vehicle, fibronectin (FN, 10 μg/ml, 20 min), or PP2 (1 μM, 5 min) and FN (10 μg/ml, 20 min), and then treated with increasing concentrations of capsaicin (CAP) to stimulate calcitonin gene-related peptide (CGRP) release, as determined by RIA.

Discussion

Few studies have characterized the dynamic effects of ECM molecules on the biological chemistry of nociceptive neurons. In this group of studies, fibronectin was found to have a significant effect on TRPV1 responsiveness and plasma membrane localization in cultured TG neurons. Furthermore, the sensitization of TRPV1 activity and translocation of TRPV1 to the plasma membrane was sensitive to src kinase inhibition. Hence, results presented here suggest that fibronectin activates fibronectin-specific integrin receptors expressed on TG neurons, increasing src phosphorylation/activity, driving TRPV1 translocation to the plasma membrane, increasing its availability for activation by capsaicin.

These results are in agreement with previously published data, illustrating the effects of src tyrosine kinase activity on TRPV1 translocation to the plasma membrane in nociceptive neurons (Zhang et al. 2005). Although the effect of NGF reported by Zhang et al. was significantly greater than that seen in our TG culture system, they did perform the majority of their studies in transfected cell lines, while our work was conducted in primary neuronal cultures. Additionally, it has been shown that NGF drives PKC activation in TrkA-expressing cells (Plo et al. 2004), which could also sensitize TRPV1 responses, while fibronectin-mediated translocation of TRPV1 to the plasma membrane is not considered dependent upon PKC. Indeed, TrkA is considered a direct activator of src tyrosine kinase, while integrin heterodimeric receptor-stimulated activation of src is more divergent in signal strength (Lipfert et al. 1992; Ohmichi et al. 1992; Bouchard et al. 2008). However, this should not be considered a concession that FN-mediated translocation of TRPV1 is a weaker regulator of TRPV1 translocation, as integrin-regulation of nociceptive physiology is reported in cases of peripheral hyperalgesia, including those within the orofacial region(Dina et al. 2005; Bereiter et al. 2006; Alessandri-Haber et al. 2008).

Hyperalgesia, more importantly inflammatory hyperalgesia, is related to dynamic changes in fibronectin content and expression. In contrast to the static nature of fibronectin exposure that studies have demonstrated to be important for cellular attachment and development, tissue injury and/or inflammatory conditions can spur increases in available fibronectin in a dynamic manner. Various tissues, including epithelial, cardiac, and respiratory, demonstrate dynamic increases in fibronectin production following an inflammatory challenge (Richards and Saba 1983; Stecher et al. 1986; Fernandez and Mosquera 2002; Kolachala et al. 2007). Although fibronectin is generated by multiple cell types, it is unlikely that the increased content occurs throughout the organism, since significant increases in fibronectin mRNA are observed at localized areas of inflammation (Meerschaert et al. 1999a; Kolachala et al. 2007). Therefore, similar to other inflammatory mediators whose protein levels are increased following inflammation (nerve growth factor (Bienenstock et al. 1987), serotonin (Spector and Willoughby 1957), histamine (Ogle and Lydic 1951)), fibronectin may also dynamically increase.

The identification of the ECM protein fibronectin as a dynamic modulator of TRPV1 translocation to the plasma membrane emphasizes the role played by certain integrins in nociceptor activation. Although the focus of this study was on fibronectin, it is possible that other ECM molecules activate similar integrin heterodimers and consequently increase src activity in neuronal tissues. Further studies will evaluate whether collagens, laminin, and/or vitronectin can also direct TRPV1 translocation to the plasma membrane, thereby sensitizing receptor function.