β-adrenergic receptor activation in immortalized human urothelial cells stimulates inflammatory responses by PKA-independent mechanisms
© Harmon et al; licensee BioMed Central Ltd. 2005
Received: 23 March 2005
Accepted: 09 August 2005
Published: 09 August 2005
Interstitial cystitis (IC) is a debilitating disease characterized by chronic inflammation of the urinary bladder, yet specific cellular mechanisms of inflammation in IC are largely unknown. Multiple lines of evidence suggest that β-adrenergic receptor (AR) signaling is increased in the inflamed urothelium, however the precise effects of these urothelial cell signals have not been studied. In order to better elucidate the AR signaling mechanisms of inflammation associated with IC, we have examined the effects of β-AR stimulation in an immortalized human urothelial cell line (UROtsa). For these studies, UROtsa cells were treated with effective concentrations of the selective β-AR agonist isoproterenol, in the absence or presence of selective inhibitors of protein kinase A (PKA). Cell lysates were analyzed by radioimmunoassay for generation of cAMP or by Western blotting for induction of protein products associated with inflammatory responses.
Radioligand binding demonstrated the presence of β-ARs on human urothelial UROtsa cell membranes. Stimulating UROtsa cells with isoproterenol led to concentration-dependent increases of cAMP production that could be inhibited by pretreatment with a blocking concentration of the selective β-AR antagonist propranolol. In addition, isoproterenol activation of these same cells led to significant increases in the amount of phosphorylated extracellular signal-regulated kinase (pERK), inducible nitric oxide synthase (iNOS) and the induced form of cyclooxygenase (COX-2) when compared to control. Moreover, preincubation of UROtsa cells with the selective PKA inhibitors H-89 or Rp-cAMPs did not diminish this isoproterenol mediated phosphorylation of ERK or production of iNOS and COX-2.
Functional β-ARs expressed on human urothelial UROtsa cell membranes increase the generation of cAMP and production of protein products associated with inflammation when activated by the selective β-AR agonist isoproterenol. However, the increased production of iNOS and COX-2 by isoproterenol is not blocked when UROtsa cells are preincubated with inhibitors of PKA. Therefore, UROtsa cell β-AR activation significantly increases the amount of iNOS and COX-2 produced by a PKA-independent mechanism. Consequently, this immortalized human urothelial cell line can be useful in characterizing potential AR signaling mechanisms associated with chronic inflammatory diseases of the bladder.
Interstitial cystitis (IC) is a debilitating disease characterized by chronic pain in the urinary bladder along with increased urinary frequency and urgency. IC is a complex disease with multiple etiologies, yet inflammatory pain is a common mechanism of all IC symptoms . Prostanoids, arachidonic acid metabolites of the cyclooxygenase (COX) pathway, and nitric oxide (NO), whose formation is catalyzed by nitric oxide synthase (NOS), both play major roles in regulating the inflammatory response. Increased levels of prostaglandins generated by the inducible form of cyclooxygenase (COX-2) mediate the vasodilatation and vascular permeability observed during the early events of inflammation . Moreover, animal models lacking the PGE2 prostaglandin receptor demonstrate a reduced algesic response indicating the importance of prostanoids in the signaling and perception of inflammatory pain . Finally, increased COX-2 expression documented for an in vivo model of cystitis supports the idea that increased prostaglandin signaling sensitizes bladder afferents that control micturition and pain .
The expression of the inducible form of NOS, iNOS, has been characterized in numerous cell types as a consequence of the inflammatory processes that follow tissue damage . Large amounts of NO generated by iNOS surpass homeostatic concentrations formed by endothelial eNOS or neuronal nNOS . This difference in kinetics of NO formation by iNOS leads to multiple inflammatory responses that include neutrophil activation, DNA damage, protein nitration and induction of apoptosis . Furthermore, animal models deficient in iNOS establish this enzyme's importance as a pathophysiological mediator of chronic inflammatory diseases . Moreover, increased levels of luminal NO, recognized as a causative agent for bladder excitability and micturition, has been documented in patients with IC, which could represent a mechanism of hyperexcitability documented for this disease .
Multiple lines of evidence suggest that increased signaling through the G protein-coupled β-adrenergic receptor (AR) may be linked to inflammation associated with IC. Patients with IC have been found to have increased nerve fiber innervation of the urinary bladder. Further study has shown these fibers to be solely sympathetic nerves, which would correspond to an increase in AR signaling . Moreover, elevated urinary levels of norepinephrine have been found in IC patients, which is also consistent with greater AR activity in the urinary bladder . Finally, genomic profiling found increased transcription of the β2-AR gene in a mouse bladder inflammation model . Together, these observations suggest that chronic β-AR stimulation may be linked to inflammatory bladder diseases like IC. Therefore, we hypothesize that urothelial β-AR activation mediates specific inflammatory responses that can be linked to bladder hyperexcitability and pain documented in chronic inflammatory bladder diseases like IC.
In order to test this hypothesis, we have studied the effects of β-AR activation in an immortalized cell line of human urothelium, UROtsa cells . UROtsa cells exhibit numerous properties of basal bladder epithelial cells, including the potential to differentiate into the stratified cell types found in the mammalian bladder lining. Our results using these UROtsa cells as an in vitro model of bladder urothelium, reveals a correlation between β-AR activation and the production of specific pro-inflammatory proteins via a PKA-independent mechanism.
Identification of specific β-AR binding sites
Isoproterenol Induces cAMP Accumulation
Selective Production of Inflammatory Mediators by β-AR Stimulation
β-AR Mediated Activation of MAPK Pathway
ERK Phosphorylation is Independent of cAMP Mediated Activation of PKA
PKA-Independent Production of Inflammatory Mediators
PKA-Dependent Phosphorylation of the Cyclic AMP-Responsive Element Binding Protein
This study characterizes a novel role of β-AR signaling in urothelial cells that leads to selective induction of protein products associated with inflammatory responses. Our results demonstrate that a previously described human urothelial cell line expressing functional β-ARs increases production of cAMP, phosphorylated ERK and heightened translation of COX-2 and iNOS in response to agonist activation. β-AR stimulation classically precedes cAMP accumulation, which regulates the activity of PKA leading to phosphorylation of PKA-sensitive substrates. However, phosphorylation of ERK and selective production of inflammatory mediators in UROtsa cells occurs independently of PKA activation, as similar results were observed in the presence of two analogous inhibitors specific for this cAMP-dependent kinase. Effective use of these compounds was confirmed by documenting the inhibition of PKA dependent protein phosphorylation in our same model system. Therefore, functional β-ARs present on these human urothelial cells elicit pro-inflammatory responses by a PKA-independent mechanism.
Previous studies by others have demonstrated the link between activation of MAPK pathways and the induction of inflammatory mediators . In these studies, receptor regulated expression of COX-2 and iNOS was dependent upon the intermediary phosphorylation of ERK. Moreover, β-AR activation, although classically linked to generation of cAMP, has been shown in other studies to influence MAPK activation in a PKA-independent manner . These PKA-independent mechanisms associated with β-AR mediated phosphorylation of ERK have been shown to involve β-arrestin scaffolding complexes . In our studies we show that β-AR mediated ERK phosphorylation in UROtsa cells is independent of active cAMP-dependent PKA. Whether other scaffolding complexes caused by β-AR stimulation in these cells are associated with ERK phosphorylation is currently under investigation by our laboratory.
Despite the fact that a specific etiology has yet to be identified, inflammatory pain is a common mechanism associated with the symptoms of IC . With reference to our human urothelial cell model, we demonstrate an induction of mediators associated with inflammatory pain and bladder hyperexcitability in response to β-AR activation. Clinical correlations have recognized an increased sympathetic innervation as well as elevated catecholamine levels in IC patients when compared to controls [9, 10]. Our studies suggest that chronic urothelial β-AR stimulation in these patients may induce COX-2 and iNOS leading to the increased progression of inflammatory pain and bladder hyperexcitability associated with this disease. Induction of COX-2 by bacterial lipopolysaccharide or endogenous cytokines has been shown to elevate prostanoid levels that are linked to the increased vasodilatation, vascular permeability and hyperalgesic responses of inflammation . In other models, induction of iNOS by these same agonists to generate nitric oxide contributes to the nociceptive processing of inflammatory pain . Therefore, we suggest that chronic urothelial β-AR stimulation leading to increased levels of prostaglandins and NO is one potential mechanism of inflammatory pain in IC. Moreover, higher levels of prostanoids and NO may also contribute to the symptomatic increases in urinary frequency and urgency diagnosed in patients with IC [4, 8].
Support of this hypothesis has been reported using a mouse model of bladder inflammation in which genes encoding for iNOS and the β2-AR subtype were upregulated when compared to control . Interestingly, a significant increase in genomic expression of the β2-AR subtype was only observed in a chronic and not an acute bladder inflammation model. Conversely, other investigators have shown using transient application of β-AR agonists that an increase in cAMP is sufficient to generate maintenance levels of NO in primary rat urothelial cells . However, our studies using a human urothelial cell model, demonstrates that cAMP-dependent PKA activation is not necessary to induce inflammatory mechanisms for generating NO. Moreover, generation of homeostatic levels of NO in the rat model was sensitive to Ca2+ indicating that the responsible enzyme was eNOS, although transcriptional message (mRNA) for iNOS was well documented in this same report . This suggests that chronic β-AR stimulation may induce expression of iNOS, which would generate higher levels of NO contributing to the production of inflammatory pain and increased micturition associated with IC.
In our human urothelial cell model we document a β-AR stimulated, PKA-independent signaling pathway that simultaneously increases the expression of two mediators of inflammation, COX-2 and iNOS. In addition, the pathophysiology linked to increased prostaglandin and NO production correlate well with the clinical manifestations associated with chronic inflammatory diseases like IC. Consequently, we believe that UROtsa cells serve as a readily accessible model for studying the β-AR-effector system associated with inflammation in IC. Nonsteroidal anti-inflammatory drugs (NSAIDs), which block the synthesis of prostaglandins by inhibiting COX, are commonly prescribed to relieve discomforts associated with IC. Moreover, NSIADs have been shown to decrease the amount of NO in vivo indicating the importance of COX-2 activity in regulating NO production during inflammation . Furthermore, combined pharmacological inhibition of COX-2 and iNOS in a rat model of tonic pain, produces a synergistic antinociceptive effect . This data suggests a common mechanism of action between these two drug classes, however, the associations between COX-2 and iNOS effector systems are currently unknown. Therefore, UROtsa cells represent a unique cell model whereby signal-transduction pathways common to the induction of both COX-2 and iNOS can be investigated. These studies not only may reveal novel targets of inflammatory pain that could be exploited therapeutically, but would increase our understanding of the etiology for general bladder inflammation and hyperexcitability in IC.
Stimulation of β-ARs expressed on cultured human urothelial cells leads to ERK phosphorylation and production of the pro-inflammatory enzymes. While cAMP levels rise in these cells after β-AR activation, production of COX-2 and iNOS are not dependent upon an increased cAMP regulated PKA activity. Continual initiation of AR function documented for patients diagnosed with IC would likely stimulate urothelial cell inflammatory responses thereby contributing to the etiology of this disease. Our results suggest that by focusing on common urothelial β-AR mediated inflammatory signaling pathways, reasonable pathophysiological mechanisms and potential therapeutic strategies could be developed for chronic inflammatory diseases like IC.
The immortalized human urothelial (UROtsa) cell line was a gift from Donald Sens (University of North Dakota) and was propagated as previously reported . Briefly, undifferentiated UROtsa cells were grown to confluence in serum-containing Dulbecco's Modified Eagle's Medium (DMEM) under standard cell culture conditions. Confluent UROtsa cells were washed in serum-free DMEM and pre-incubated with or without inhibitors protein kinase A (PKA) inhibitors 1 hr before addition of the selective β-AR agonist, isoproterenol. The PKA inhibitors H-89 (Sigma, St. Louis, MO) was used at a final concentration of 100 nM, while Rp-cAMPS (BioLog, Bremen, Germany) was used at 10 μM. Unless noted otherwise, isoproterenol (Sigma, St. Louis, MO) was added to cells at a final concentration of 100 nM. As a nonspecific initiator of inflammation control, cells were incubated with 300 nM lipopolysaccharide (CalBioChem, La Jolla, CA).
A crude cell membrane preparation was prepared as previously described . Briefly, UROtsa membranes were prepared by transferring suspended cells to a 50 mL conical tube and twice washing by centrifugation at 1000 × g using cold Hank's balance salt solution (HBSS). The intact cell pellet was resuspended in 10 mL of 0.25 M sucrose containing 10 μg/mL bacitracin, 10 μg/mL benzamidine, 10 μg/mL leupeptin, and 20 μg/mL phenylmethysulfonylfluoride. The cells were disrupted by freezing followed by Dounce homogenization of the thawed suspension using 20 strokes from a loose fitting (B) pestle. This mixture was then centrifuged at 1260 × g for 5 min at 4°C. Buffer A (20 mM HEPES, pH 7.5, 1.4 mM EGTA, 12.5 mM MgCl2) was added to the supernatant and centrifuged again at 30,000 × g for 15 min at 4°C. The resultant pellet was kept, resuspended in buffer A then centrifuged once more at 30,000 × g for 15 min at 4°C. The final crude membrane pellet was resuspended in buffer A containing 10% glycerol and stored in aliquots at -70°C until used for radioligand binding. Protein concentrations were measured using the method of Bradford .
The radioligand binding protocol used for this study was performed as previously described . Briefly, the density of expressed β-ARs on UROtsa cells was determined by saturation binding experiments using the nonselective β-AR antagonist 125I-CYP as the radiolabel (NEN Life Sciences, Boston, MA). Crude UROtsa cell membranes were allowed to equilibrate at 37°C with increasing concentrations of 125I-CYP (5–600 pM) in a 0.25 mL total volume of buffer A using 10-5 M propranolol to determine non-specific binding. Binding was stopped by filtering the membranes though Whatman GF/C glass fiber filters, followed by 5 – 5 mL washes with cold buffer A to remove any unbound drug. Amounts of total and non-specific radiolabel bound to cell membranes were calculated from radioactive counts remaining on the glass fiber filters. From the plotted saturation hyperbola, β-AR density (Bmax) and the equilibrium dissociation constant (Kd) of 125I-CYP for specific UROtsa cell binding sites were calculated using iterative non-linear regression analysis .
Confluent UROtsa cells used for the quantification of cAMP were treated in serum-free DMEM containing 1 mM 1-methyl-3-isobutylxanthine (IBMX) to inhibit phosphodiesterase. After 30 min of isoproterenol treatment, cells were lysed using 0.1 M HCl and collected for determination of cAMP production according to the Biotrak Assay System protocol (Amersham, Buckinghamshire, UK). Briefly, 3H-cAMP added to cell lysates was used to compete with endogenous cAMP for binding to a specific cAMP-binding protein. 3H-cAMP levels were then counted by liquid scintillation and related to endogenously generated cAMP by comparison with known standards. The concentration of isoproterenol that caused a half-maximal generation of cAMP (EC50) was calculated from non-linear regression analysis using Prism 4 (Graphpad Software, San Diego, CA).
After an appropriate period of time, treated cells were lysed using a modified RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% sodium dodecylsulphate, 1.0% Triton X-100, 1.0% sodium deoxycholate, 5 mM EDTA, 1.0% protease inhibitor cocktail; Sigma, St. Louis, MO). Total cell lysate protein concentrations were estimated using Bradford protein assay reagent (Bio-Rad, Hercules, CA) before lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Protein expression was measured by 4°C overnight immunoblotting with diluted antibodies: extracellular signal-regulated kinase 2 (mouse monoclonal ERK2, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated ERK1/2 (mouse monoclonal pERK, 1:500; Santa Cruz Biotechnology), COX-2 (goat polyclonal, 1:500; Santa Cruz Biotechnology) iNOS (rabbit polyclonal, 1:500; Santa Cruz Biotechnology) and phosphorylated CREB (rabbit polyclonal, 1:1000; AbCam, Cambridge, MA). After washing, membranes were incubated at 25°C for 90 min with diluted horseradish peroxidase-linked secondary antibody (1:1000–5000). Bound antibody was visualized by the Supersignal West Pico chemiluminescent system (Pierce, Rockford, IL) and exposed to radiographic film. Developed films were subsequently photographed and protein band intensity was estimated by semi-quantitative densitometric methods using LabWorks v.4.5 (UVP, Upland, CA). Protein levels are presented as the mean fold increase in pixel intensity over control, plus or minus the standard error for n experiments. Differences between control and drug treated groups was determined using a paired one-tailed Student's t test with a p < .05 level of probability accepted as significant. Equal protein loading was confirmed by Ponceau-S staining of nitrocellulose membranes.
This work was supported in part by grants from the National Science Foundation 0235146 and the National Institutes of Health R21DK062865 (JEP).
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