MIF-Modulated Spinal Proteins Associated with Persistent Bladder Pain: A Proteomics Study.

Bladder pain is a prominent symptom in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). We studied spinal mechanisms of bladder pain in mice using a model where repeated activation of intravesical Protease Activated Receptor-4 (PAR4) results in persistent bladder hyperalgesia (BHA) with little or no bladder inflammation. Persistent BHA is mediated by spinal macrophage migration inhibitory factor (MIF), and is associated with changes in lumbosacral proteomics. We investigated the contribution of individual spinal MIF receptors to persistent bladder pain as well as the spinal proteomics changes associated with relief of persistent BHA by spinal MIF antagonism. Female mice with persistent BHA received either intrathecal (i.t.) MIF monoclonal antibodies (mAb) or mouse IgG1 (isotype control antibody). MIF antagonism temporarily reversed persistent BHA (peak effect: 2 h), while control IgG1 had no effect. Moreover, i.t. antagonism of the MIF receptors CD74 and C-X-C chemokine receptor type 4 (CXCR4) partially reversed persistent BHA. For proteomics experiments, four separate groups of mice received either repeated intravesical scrambled peptide and sham i.t. injection (control, no pain group) or repeated intravesical PAR4 and: sham i.t.; isotype IgG1 i.t. (15 µg); or MIF mAb (15 µg). L6-S1 spinal segments were excised 2 h post-injection and examined for proteomics changes using LC-MS/MS. Unbiased proteomics analysis identified and relatively quantified 6739 proteins. We selected proteins that showed significant changes compared to control (no pain group) after intravesical PAR4 (sham or IgG i.t. treatment) and showed no significant change after i.t. MIF antagonism. Six proteins decreased during persistent BHA (V-set transmembrane domain-containing protein 2-like confirmed by immunohistochemistry), while two proteins increased. Spinal MIF antagonism reversed protein changes. Therefore, spinal MIF and MIF receptors mediate persistent BHA and changes in specific spinal proteins. These novel MIF-modulated spinal proteins represent possible new targets to disrupt spinal mechanisms that mediate persistent bladder pain.

Lumbosacral spinal proteomic changes during PAR4-induced persistent bladder pain.

Repeated intravesical activation of protease-activated receptor-4 (PAR4) in mice results in persistent bladder hyperalgesia (BHA). We investigated spinal proteomic changes associated with persistent BHA. Persistent BHA was induced in female mice by repeated (3x; days 0,2,4; n = 9) intravesical instillation of PAR4 activating peptide (PAR4-AP) while scrambled peptide served as the control (no pain; n = 9) group. The threshold to lower abdominal von Frey stimulation was recorded prior to and during treatment. On day 7, L6-S1 spinal segments were excised and examined for proteomic changes using LC-MS/MS. In-depth, unbiased proteomic tandem-mass tag (TMT) analysis identified and relatively quantified 6739 proteins. We identified significant changes with 29 decreasing and 51 increasing proteins in the persistent BHA group and they were associated with neuroprotection, redox modulation, mitochondrial factors, and neuronal-related proteins. In an additional experiment, decreases in protein levels were confirmed by immunohistochemistry for metallothionein 1/2. Our results show that persistent bladder pain is associated with central (spinal) protein changes. Previous work showed that PAR4-induced bladder pain is mediated, at least in part by spinal MIF. Further functional studies of these top changing proteins may lead to the discovery of novel potential therapeutic targets at the spinal level to modulate persistent bladder pain. Future studies will examine the effect of spinal MIF antagonism on PAR4-induced spinal proteomics associated with persistent bladder pain.

Urothelial Oxidative Stress and ERK Activation Mediate HMGB1-Induced Bladder Pain.

Activation of intravesical protease activated receptors-4 (PAR4) results in bladder pain through the release of urothelial macrophage migration inhibitory factor (MIF) and high mobility group box-1 (HMGB1). We aimed to identify HMGB1 downstream signaling events at the bladder that mediate HMGB1-induced bladder pain in MIF-deficient mice to exclude any MIF-related effects. We studied whether oxidative stress and ERK activation are involved by examining bladder tissue in mice treated with intravesical disulfide HMGB1 for 1 h and analyzed with Western blot and immunohistochemistry. HMGB1 intravesical treatment increased urothelium 4HNE and phospho-ERK1/2 staining, suggesting that HMGB1 increased urothelial oxidative stress and ERK activation. Furthermore, we examined the functional roles of these events. We evaluated lower abdominal mechanical thresholds (an index of bladder pain) before and 24 h after intravesical PAR4 or disulfide HMGB1. Intravesical pre-treatments (10 min prior) included: N-acetylcysteine amide (NACA, reactive oxygen species scavenger) and FR180204 (FR, selective ERK1/2 inhibitor). Awake micturition parameters (voided volume; frequency) were assessed at 24 h after treatment. Bladders were collected for histology at the end of the experiment. Pre-treatment with NACA or FR significantly prevented HMGB1-induced bladder pain. No significant effects were noted on micturition volume, frequency, inflammation, or edema. Thus, HMGB1 activates downstream urothelial oxidative stress production and ERK1/2 activation to mediate bladder pain. Further dissection of HMGB1 downstream signaling pathway may lead to novel potential therapeutic strategies to treat bladder pain.

Bladder Oxidative Stress and HMGB1 Release Contribute to PAR4-Mediated Bladder Pain in Mice.

Activation of intravesical PAR4 receptors leads to bladder hyperalgesia (BHA) through release of urothelial macrophage migration inhibitory factor (MIF) and urothelial high mobility group box-1 (HMGB1). MIF deficiency and/or MIF antagonism at the bladder block BHA in mice yet the mechanisms are not clear. Since oxidative stress and ERK phosphorylation are involved in MIF signaling we hypothesized that oxidative stress and/or ERK signaling, activated by MIF release, promote intravesical HMGB1 release to induce BHA. We induced BHA by intravesical PAR4 infusion in female C57BL/6 mice. Mechanical sensitivity was evaluated by measuring abdominal von Frey (VF) 50% thresholds before (baseline) and 24 h post-infusion. Intravesical pre-treatment (10 min infusion prior to PAR4) with N-acetylcysteine amide (NACA; reactive-oxygen species scavenger; 3 mg in 50 µl), FR180204 (selective ERK1/2 inhibitor; 200 µg in 50 µl), ethyl pyruvate (EP; HMGB1 release inhibitor; 600 µg in 50 µl), or diluent controls (50 µl) tested the effects of pre-treatment on PAR4-induced BHA. Intravesical fluid was collected after each treatment and HMGB1 concentration was measured using ELISA. Awake micturition parameters (volume and frequency) were assessed at the end of the experiments. Bladders were collected and examined for histological signs of edema and inflammation. Pre-treatment with PBS followed by PAR4 induced BHA in mice but PBS followed by scrambled peptide did not. Pre-treatment with NACA or EP partially blocked PAR4-induced BHA while FR180204 had no effect. A significant correlation between intravesical HMGB1 levels and 50% VF thresholds was observed. All PAR4 treated groups had increased levels of HMGB1 in the intravesical fluid compared to PBS-Scrambled group although not statistically significant. No significant effects were noted on awake micturition volume, micturition frequency or histological evidence of bladder edema or inflammation. Our results show that intravesical antagonism of bladder reactive-oxygen species accumulation was effective in reducing PAR4-induced bladder pain. The correlation between intravesical levels of HMGB1 and bladder pain indicates that released HMGB1 is pivotal to bladder pain. Thus, modulating events in the MIF signaling cascade triggered by PAR4 activation (including bladder oxidative stress and HMGB1 release) warrant further investigation as possible therapeutic strategies.

Intravesical CD74 and CXCR4, macrophage migration inhibitory factor (MIF) receptors, mediate bladder pain.

BACKGROUND: Activation of intravesical protease activated receptor 4 (PAR4) leads to release of urothelial macrophage migration inhibitory factor (MIF). MIF then binds to urothelial MIF receptors to release urothelial high mobility group box-1 (HMGB1) and elicit bladder hyperalgesia. Since MIF binds to multiple receptors, we investigated the contribution of individual urothelial MIF receptors to PAR4-induced HMGB1 release in vivo and in vitro and bladder pain in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We tested the effect of intravesical pre-treatment with individual MIF or MIF receptor (CD74, CXCR4, CXCR2) antagonists on PAR4-induced HMGB1 release in vivo (female C57/BL6 mice) and in vitro (primary human urothelial cells) and on PAR4-induced bladder hyperalgesia in vivo (mice). In mice, PAR4 induced HMGB1 release and bladder hyperalgesia through activation of intravesical MIF receptors, CD74 and CXCR4. CXCR2 was not involved in these effects. In primary urothelial cells, PAR4-induced HMGB1 release through activation of CD74 receptors. Micturition parameters in mice were not changed by any of the treatments. CONCLUSIONS/SIGNIFICANCE: Urothelial MIF receptors CD74 and CXCR4 mediate bladder pain through release of urothelial HMGB1. This mechanism may set up persistent pain loops in the bladder and warrants further investigation. Urothelial CD74 and CXCR4 may provide novel targets for interrupting bladder pain.

MIF mediates bladder pain, not inflammation, in cyclophosphamide cystitis.

Macrophage migration inhibitory factor (MIF), a proinflammatory mediator, is recognized as a player in inflammatory and neuropathic pain. Cyclophosphamide (CYP) results in bladder inflammation and pain and it's a frequently used animal model of interstitial cystitis/bladder pain syndrome (IC/BPS). Because pretreatment with a MIF inhibitor (ISO-1) prevented both CYP-induced bladder pain and inflammation we used genetic MIF knockout (KO) mice to further investigate MIF's role in CYP-induced bladder pain and inflammation. Abdominal mechanical threshold measured bladder pain induced by CYP in wild type (WT) and MIF KO mice at several time points (0-48 hours). End-point (48 hours) changes in micturition parameters and histological signs of bladder inflammation were also evaluated. Abdominal mechanical hypersensitivity developed within 4 hours after CYP injection (and lasted for the entire observation period: 48 hours) in WT mice. MIF KO mice, on the other hand, did not develop abdominal mechanical hypersensitivity suggesting that MIF is a pivotal molecule in mediating CYP-induced bladder pain. Both WT and MIF KO mice treated with CYP showed histological signs of marked bladder inflammation and showed a significant decrease in micturition volume and increase in frequency. Since both changes were blocked in MIF KO mice by pretreatment with a MIF inhibitor (ISO-1) it is likely these are non-specific effects of ISO-1. MIF mediates CYP-induced bladder pain but not CYP-induced bladder inflammation. The locus of effect (bladder) or central (spinal) for MIF mediation of bladder pain remains to be determined.

Erectile Dysfunction Medications and Skin Cancer: An Analysis in US Veterans.

OBJECTIVE: To examine the relationship between phosphodiesterase 5 inhibitor drugs (PDE5i) and skin cancers in a large-scale study of Veterans. METHODS: This was a retrospective database review using the Department of Veterans Affairs Informatics and Computing Infrastructure database. Veterans Affairs Informatics and Computing Infrastructure was searched 19 years for Veterans who received PDE5i treatment of erectile dysfunction. A non-PDE5i group of Veterans was selected for comparison analysis. Follow-up time, outpatient clinic visits and incidence of malignant melanoma (MM), and basal cell carcinoma (BCC) were measured in both groups. RESULTS: A total of 2.55 million Veterans were included in this study (1.27 million in each group). Mean age of the PDE5i group and non-PDE5i group was 59.2 years (standard deviation [SD] ± 10.8) and 58.7 (SD ± 10.8), respectively. Mean follow-up time for the PDE5i group was 8.9 years (SD ± 4.2) and 8.5 years (SD ± 4.3) for non-PDE5i group. Odds ratio for malignant melanoma and BCC in the PDE5i group was 1.25 (confidence interval 95%, 1.22-1.28, P <.0001) and 1.49 (confidence interval 95%, 1.46-1.51, P <.0001), respectively. PDE5i users showed more mean outpatient visits/year (8.9 SD ± 9.50) compared to non-PDE5i users (5.9 SD ± 10.0; P <.0001). CONCLUSION: Veterans prescribed PDE5is to treat erectile show a minimal increased risk of MM and a greater risk of BCC compared to non-PDE5i users. PDE5i users visited outpatient VA clinics at a higher rate than non-PDE5i users in this study. These findings suggest confounding variables are likely involved in the relationship between skin cancers and PDE5i use. PDE5i drugs remain a safe treatment for erectile dysfunction.

Spinal macrophage migration inhibitory factor and high mobility group box 1 mediate persistent bladder pain.

Repeated intravesical PAR4 (protease activated receptor 4) activation elicits persistent bladder pain lasting 5 days after the last treatment. Persistent bladder pain was fully reversed by a systemic HMGB1 (high mobility group box 1) inhibitor while a MIF (macrophage migration inhibitory factor) antagonist partly reversed it. Since there is growing evidence that spinal MIF and HMGB1 mediate inflammatory and neuropathic pain we examined whether there were spinal changes occurring during persistent bladder pain that may be responsible for maintaining bladder pain. In addition, we tested whether we could modulate persistent bladder pain with spinal MIF or HMGB1 antagonists. Persistent bladder pain was elicited in female C57 mice by repeated (3x) intravesical instillation of PAR4-activating peptide while control animals received scramble peptide treatment. On day 4, spinal cord (L6-S1) changes in c-fos (non-specific marker of spinal activation) was assessed with immunofluorescence while MIF and HMGB1 were assessed with immunofluorescence, western blotting and real-time PCR. On day 7, mice received an intrathecal injection of a neutralizing MIF monoclonal antibody (15 µg in 5 µl PBS) or a HMGB1 inhibitor glycyrrhizin (25 µg in 5 µl of 5% alcohol in PBS) and abdominal mechanical threshold was tested. On day 9, mice were treated with vehicle or control and abdominal mechanical threshold was tested. Immunofluorescence showed that c-fos and MIF in the dorsal horn, dorsal grey commissure and intermediolateral areas significantly increased in PAR4-treated mice while HMGB1 was decreased. In addition, intrathecal treatment with MIF neutralizing mAb or glycyrrhizin significantly alleviated abdominal mechanical hypersensitivity at 1 and 2 h and the analgesic effect diminished at 6 h. Vehicle or control treatment had no effect. Persistent bladder pain is associated with spinal changes in MIF and HMGB1 levels. Furthermore, spinal treatment with MIF monoclonal antibody and HMGB1 inhibitor temporarily reversed bladder pain. Our findings suggest that spinal MIF and HMGB1 participate in persistent bladder pain induced by repeated intravesical PAR4 and may be potential therapeutic targets in chronic bladder pain conditions.

Protease activated-receptor 4 activation as a model of persistent bladder pain: Essential role of macrophage migration inhibitory factor and high mobility group box 1.

OBJECTIVES: To develop a rodent model of persistent non-inflammatory bladder pain and to test macrophage migration inhibitory factor and high mobility box group 1 as mediators of bladder pain. METHODS: Female C57BL/6 mice received intravesical instillations of protease activated receptor 4 (100 µmol/L, for 1 h) three times every other day and abdominal mechanical hypersensitivity (50% mechanical threshold) was tested on day 0 (baseline), and at days 1, 2, 3, 4, 7 and 9 after the first protease-activated receptor 4 injection. At the end of the experiment, micturition changes were measured and bladders were examined for histological changes. Macrophage migration inhibitory factor antagonist (MIF098; 40 mg/kg i.p. b.i.d.) or high mobility group box 1 inhibitor (glycyrrhizin; 50 mg/kg i.p. daily) was administered from day 2 until day 8. RESULTS: There was a significant and persistent decrease in abdominal mechanical threshold starting from day 3 in the protease-activated receptor 4-treated group that persisted until day 9 (5 days post-last instillation), but not in the control group. Glycyrrhizin fully reversed while MIF098 partially reversed abdominal mechanical hypersensitivity in protease-activated receptor 4-treated mice. The changes started on day 3 after the first protease-activated receptor 4 instillation, and analgesic effects lasted throughout the rest of the testing period. None of the groups had significant micturition changes or overt bladder histological changes. CONCLUSIONS: Repeated intravesical protease activated receptor 4 instillations produce persistent bladder pain without inflammation. Macrophage migration inhibitory factor and high mobility group box 1 are possible effective target molecules for bladder pain alleviation.

Elevated Urine Levels of Macrophage Migration Inhibitory Factor in Inflammatory Bladder Conditions: A Potential Biomarker for a Subgroup of Interstitial Cystitis/Bladder Pain Syndrome Patients.

OBJECTIVE: To investigate whether urinary levels of macrophage migration inhibitory factor (MIF) are elevated in interstitial cystitis/bladder pain syndrome (IC/BPS) patients with Hunner lesions and also whether urine MIF is elevated in other forms of inflammatory cystitis. METHODS: Urine samples were assayed for MIF by enzyme-linked immunosorbent assay. Urine samples from 3 female groups were examined: IC/BPS patients without (N = 55) and with Hunner lesions (N = 43), and non-IC/BPS patients (N = 100; control group; no history of IC/BPS; cancer or recent bacterial cystitis). Urine samples from 3 male groups were examined: patients with bacterial cystitis (N = 50), radiation cystitis (N = 18) and noncystitis patients (N = 119; control group; negative for bacterial cystitis). RESULTS: Urine MIF (mean MIF pg/mL ± standard error of the mean) was increased in female IC/BPS patients with Hunner lesions (2159 ± 435.3) compared with IC/BPS patients without Hunner lesions (460 ± 114.5) or non-IC/BPS patients (414 ± 47.6). Receiver operating curve analyses showed that urine MIF levels discriminated between the 2 IC groups (area under the curve = 72%; confidence interval 61%-82%). Male patients with bacterial and radiation cystitis had elevated urine MIF levels (2839 ± 757.1 and 4404 ± 1548.1, respectively) compared with noncystitis patients (681 ± 75.2). CONCLUSION: Urine MIF is elevated in IC/BPS patients with Hunner lesions and also in patients with other bladder inflammatory and painful conditions. MIF may also serve as a noninvasive biomarker to select IC/BPS patients more accurately for endoscopic evaluation and possible anti-inflammatory treatment.

Macrophage migration inhibitory factor mediates protease-activated receptor 4-induced bladder pain through urothelial high mobility group box 1.

Macrophage migration inhibitory factor (MIF) mediates pain although the mechanisms are not well understood. Urothelial activation of protease activated receptor 4 (PAR4) results in urothelial MIF release, urothelial high mobility group box 1 (HMGB1) release and bladder pain in mice without bladder inflammation. All three effects are prevented by MIF inhibition while intravesical disulfide HMGB1 alone can induce bladder pain. This study utilizes genetic MIF deletion to determine whether MIF mediates PAR4-induced bladder pain and is upstream of HMGB1-induced bladder pain. Wild type (C57/BL6) and MIF knockout (KO) mice were treated with intravesical PAR4 activating peptide or disulfide HMGB1 and tested for abdominal mechanical hypersensitivity at baseline (before treatment) and 24 h after injection. Micturition parameters and bladder histology were examined after behavioral test. Real-time PCR and western blotting measured HMGB1 mRNA and protein levels in the bladders of naïve wild type and MIF KO mice, while immunofluorescence measured HMGB1 protein levels in the urothelium of both strains. Intravesical PAR4 activation resulted in abdominal mechanical hypersensitivity in wild-type mice but not MIF KO mice. Intravesical disulfide HMGB1 induced abdominal mechanical hypersensitivity in both strains. Neither treatment resulted in significant changes in micturition or bladder histology in either strain. HMGB1 mRNA and protein levels were higher in MIF KO mouse bladders and the urothelium of MIF KO bladder had greater immunostaining than the wild-type strain. MIF is a pivotal molecule mediating PAR4-induced bladder pain and regulating urothelial HMGB1 production and release to elicit bladder pain.

Disulfide high mobility group box-1 causes bladder pain through bladder Toll-like receptor 4.

BACKGROUND: Bladder pain is a prominent symptom in several urological conditions (e.g. infection, painful bladder syndrome/interstitial cystitis, cancer). Understanding the mechanism of bladder pain is important, particularly when the pain is not accompanied by bladder pathology. Stimulation of protease activated receptor 4 (PAR4) in the urothelium results in bladder pain through release of urothelial high mobility group box-1 (HMGB1). HGMB1 has two functionally active redox states (disulfide and all-thiol) and it is not known which form elicits bladder pain. Therefore, we investigated whether intravesical administration of specific HMGB1 redox forms caused abdominal mechanical hypersensitivity, micturition changes, and bladder inflammation in female C57BL/6 mice 24 hours post-administration. Moreover, we determined which of the specific HMGB1 receptors, Toll-like receptor 4 (TLR4) or receptor for advanced glycation end products (RAGE), mediate HMGB1-induced changes. RESULTS: Disulfide HMGB1 elicited abdominal mechanical hypersensitivity 24 hours after intravesical (5, 10, 20 µg/150 µl) instillation. In contrast, all-thiol HMGB1 did not produce abdominal mechanical hypersensitivity in any of the doses tested (1, 2, 5, 10, 20 µg/150 µl). Both HMGB1 redox forms caused micturition changes only at the highest dose tested (20 µg/150 µl) while eliciting mild bladder edema and reactive changes at all doses. We subsequently tested whether the effects of intravesical disulfide HMGB1 (10 µg/150 µl; a dose that did not produce inflammation) were prevented by systemic (i.p.) or local (intravesical) administration of either a TLR4 antagonist (TAK-242) or a RAGE antagonist (FPS-ZM1). Systemic administration of either TAK-242 (3 mg/kg) or FPS-ZM1 (10 mg/kg) prevented HMGB1 induced abdominal mechanical hypersensitivity while only intravesical TLR4 antagonist pretreatment (1.5 mg/ml; not RAGE) had this effect. CONCLUSIONS: The disulfide form of HMGB1 mediates bladder pain directly (not secondary to inflammation or injury) through activation of TLR4 receptors in the bladder. Thus, TLR4 receptors are a specific local target for bladder pain.

Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1.

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 µM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; i.p.) or vehicle, received intravesical PAR4-AP or a control peptide (100 µM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; i.p.) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions.

Macrophage Migration Inhibitory Factor Mediates PAR-Induced Bladder Pain.

INTRODUCTION: Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is constitutively expressed in urothelial cells that also express protease-activated receptors (PAR). Urothelial PAR1 receptors were shown to mediate bladder inflammation. We showed that PAR1 and PAR4 activator, thrombin, also mediates urothelial MIF release. We hypothesized that stimulation of urothelial PAR1 or PAR4 receptors elicits release of urothelial MIF that acts on MIF receptors in the urothelium to mediate bladder inflammation and pain. Thus, we examined the effect of activation of specific bladder PAR receptors on MIF release, bladder pain, micturition and histological changes. METHODS: MIF release was measured in vitro after exposing immortalized human urothelial cells (UROtsa) to PAR1 or PAR4 activating peptides (AP). Female C57BL/6 mice received intravesical PAR1- or PAR4-AP for one hour to determine: 1) bladder MIF release in vivo within one hour; 2) abdominal hypersensitivity (allodynia) to von Frey filament stimulation 24 hours after treatment; 3) micturition parameters 24 hours after treatment; 4) histological changes in the bladder as a result of treatment; 5) changes in expression of bladder MIF and MIF receptors using real-time RT-PCR; 6) changes in urothelial MIF and MIF receptor, CXCR4, protein levels using quantitative immunofluorescence; 7) effect of MIF or CXCR4 antagonism. RESULTS: PAR1- or PAR4-AP triggered MIF release from both human urothelial cells in vitro and mouse urothelium in vivo. Twenty-four hours after intravesical PAR1- or PAR4-AP, we observed abdominal hypersensitivity in mice without changes in micturition or bladder histology. PAR4-AP was more effective and also increased expression of bladder MIF and urothelium MIF receptor, CXCR4. Bladder CXCR4 localized to the urothelium. Antagonizing MIF with ISO-1 eliminated PAR4- and reduced PAR1-induced hypersensitivity, while antagonizing CXCR4 with AMD3100 only partially prevented PAR4-induced hypersensitivity. CONCLUSIONS: Bladder PAR activation elicits urothelial MIF release and urothelial MIF receptor signaling at least partly through CXCR4 to result in abdominal hypersensitivity without overt bladder inflammation. PAR-induced bladder pain may represent an interesting pre-clinical model of Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS) where pain occurs without apparent bladder injury or pathology. MIF is potentially a novel therapeutic target for bladder pain in IC/PBS patients.

Association between macrophage migration inhibitory factor promoter region polymorphism (-173 G/C) and cancer: a meta-analysis.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine upstream of many inflammatory cytokines. MIF is implicated in several acute and chronic inflammatory conditions. MIF's promoter region has functional single nucleotide polymorphisms that controls MIF expression and protein levels. Since increased plasma MIF levels are associated with cancer, studies have examined the association between Mif promoter polymorphisms and cancer. This study is a meta-analysis of the available studies on such an association. RESULTS: A total of 5 studies were included in this meta-analysis to include 1116 cases (cancer patients) and 1728 controls (no cancer). Carrying any C allele in the Mif -173 G/C promoter polymorphism resulted in a significantly greater risk for developing cancer [OR = 1.89 (1.15-3.11), p = 0.012)] when compared to the (G/G) genotype. Subgroup analysis revealed that this association was significant only for "solid" tumors (including gastric and prostate cancers) [OR = 2.67 (1.26-5.65), p = 0.010] but not for "non-solid" tumors (leukemia) [OR = 1.21 (0.95-1.55), p = 0.122]. Furthermore, when only prostate tumor studies were included in the analysis, the association became even stronger [OR = 3.72 (2.55-5.41), p < 0.0001]. CONCLUSIONS: Meta-analysis suggests there is an association between any C allele in the Mif -173 G/C promoter polymorphism and an increased risk of cancer, particularly for solid tumors. The association appeared stronger for prostate cancer, specifically. Future studies that include different types of cancers are needed to support and extend these observations.

Natriuretic peptide receptor a as a novel target for prostate cancer.

BACKGROUND: The receptor for the cardiac hormone atrial natriuretic peptide (ANP), natriuretic peptide receptor A (NPRA), is expressed in cancer cells, and natriuretic peptides have been implicated in cancers. However, the direct role of NPRA signaling in prostate cancer remains unclear. RESULTS: NPRA expression was examined by western blotting, RT-PCR and immunohistochemistry. NPRA was downregulated by transfection of siRNA, shRNA and NPRA inhibitor (iNPRA). Antitumor efficacy of iNPRA was tested in mice using a TRAMP-C1 xenograft. Here, we demonstrated that NPRA is abundantly expressed on tumorigenic mouse and human prostate cells, but not in nontumorigenic prostate epithelial cells. NPRA expression showed positive correlation with clinical staging in a human PCa tissue microarray. Down-regulation of NPRA by siNPRA or iNPRA induced apoptosis in PCa cells. The mechanism of iNPRA-induced anti-PCa effects was linked to NPRA-induced expression of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine over-expressed in PCa and significantly reduced by siNPRA. Prostate tumor cells implanted in mice deficient in atrial natriuretic peptide receptor A (NPRA-KO) failed to grow, and treatment of TRAMP-C1 xenografts with iNPRA reduced tumor burden and MIF expression. Using the TRAMP spontaneous PCa model, we found that NPRA expression correlated with MIF expression during PCa progression. CONCLUSIONS: Collectively, these results suggest that NPRA promotes PCa development in part by regulating MIF. Our findings also suggest that NPRA is a potential prognostic marker and a target for PCa therapy.

Antagonism of macrophage migration inhibitory factor decreases cyclophosphamide cystitis in mice.

AIMS: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine found pre-formed in the urothelium. During inflammation, MIF is released into the bladder lumen and bladder MIF mRNA is upregulated. Since MIF also has tautomerase activity and blocking tautomerase activity also blocks MIF's biological activity, we hypothesized that blocking MIF's tautomerase activity would prevent bladder inflammation. Therefore, we examined the effects of a MIF tautomerase inhibitor (ISO-1; also blocks biological activity) on cyclophosphamide (CYP)-induced cystitis in mice. METHODS: Mice receiving CYP (300 mg/kg; i.p.) to induce cystitis or saline (control) were treated either with ISO-1 (20 mg/kg; i.p.; daily) or vehicle (20% DMSO; i.p.; daily) for 2 days. After 2 days, micturition volume and frequency in awake mice were recorded and also mechanical sensitivity to abdominal stimulation using von Frey monofilaments. Bladders were collected under anesthesia and examined histologically, nerve growth factor levels were assayed in bladder homogenates, and production of inflammatory cytokines in the bladder was determined using a targeted array. RESULTS: CYP treatment resulted in decreased micturition volume, increased frequency, decreased threshold, increased histological signs of cystitis, increased bladder NGF levels and production of inflammatory cytokines when compared to the control group. Treatment with ISO-1 prevented or greatly decreased all these changes. CONCLUSION: Antagonizing MIF's activity with a systemic MIF tautomerase inhibitor was able to prevent or greatly reduced chemical cystitis in mice, thus indicating the MIF mediates bladder inflammation in this model. MIF represents a novel and important modulator of cystitis.

Thrombin induces macrophage migration inhibitory factor release and upregulation in urothelium: a possible contribution to bladder inflammation.

PURPOSE: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine expressed by urothelial cells that mediates bladder inflammation. We investigated the effect of stimulation with thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and MIF mRNA upregulation in urothelial cells. MATERIALS AND METHODS: MIF and PAR1 expression was examined in normal human immortalized urothelial cells (UROtsa) using real-time RT-PCR, Western blotting and dual immunostaining. MIF and PAR1 immunostaining was also examined in rat urothelium. The effect of thrombin stimulation (100 nM) on urothelial MIF release was examined in UROtsa cells (in vitro) and in rats (in vivo). UROtsa cells were stimulated with thrombin, culture media were collected at different time points and MIF amounts were determined by ELISA. Pentobarbital anesthetized rats received intravesical saline (control), thrombin, or thrombin +2% lidocaine (to block nerve activity) for 1 hr, intraluminal fluid was collected and MIF amounts determined by ELISA. Bladder or UROtsa MIF mRNA was measured using real time RT-PCR. RESULTS: UROtsa cells constitutively express MIF and PAR1 and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium. Thrombin stimulation of urothelial cells resulted in a concentration- and time-dependent increase in MIF release both in vitro (UROtsa; 2.8-fold increase at 1 hr) and in vivo (rat; 4.5-fold) while heat-inactivated thrombin had no effect. In rats, thrombin-induced MIF release was reduced but not abolished by intravesical lidocaine treatment. Thrombin also upregulated MIF mRNA in UROtsa cells (3.3-fold increase) and in the rat bladder (2-fold increase) where the effect was reduced (1.4-fold) by lidocaine treatment. CONCLUSIONS: Urothelial cells express both MIF and PAR1. Activation of urothelial PAR1 receptors, either by locally generated thrombin or proteases present in the urine, may mediate bladder inflammation by inducing urothelial MIF release and upregulating urothelial MIF expression.

Macrophage migration inhibitory factor anti-thrombin III complexes are decreased in bladder cancer patient serum: Complex formation as a mechanism of inactivation.

Mounting evidence suggests that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) may serve as an important link between chronic inflammation and carcinogenesis as evidenced by the increase in serum MIF found in patients with various cancers. The present study identifies anti-thrombin III (ATIII) as an endogenous MIF binding protein, which reduces MIF biological activity. Serum MIF in bladder cancer patients (TCC stage II, n=50) was increased when compared to normal patients (n=50), while ATIII-MIF complexes were decreased in bladder cancer patient serum. These data suggest that increased circulating levels of bioactive MIF are present in bladder cancer patient serum.

Intraluminal blockade of cell-surface CD74 and glucose regulated protein 78 prevents substance P-induced bladder inflammatory changes in the rat.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine constitutively expressed by urothelial cells. During inflammatory stimuli, MIF is released into the lumen complexed to other proteins and these complexes can bind to urothelial cell-surface receptors to activate signaling pathways. Since MIF is complexed to alpha1-inhibitor III (A1-I3; a member of the alpha2-macroglubulin family) and glucose regulated protein 78 (GRP78) is a receptor for A1-I3 the goals of this study were to determine if substance P elicits urothelial cell-surface expression of GRP78 and to assess the functional role of CD74 (receptor for MIF) or GRP78 in substance P-induced bladder inflammatory changes. METHODOLOGY/PRINCIPAL FINDINGS: Anesthetized male Sprague-Dawley rats received either saline or substance P (s.c.), bladders were collected 1 hour after treatment and processed for histology or protein/mRNA. The expression of GRP78 at urothelial cell-surface was determined by performing in vivo biotinylation of urothelial cell-surface proteins. Finally, in order to determine the effects of receptor blockade on substance P-induced MIF release and inflammatory changes, rats received either intraluminal antibodies to CD74, GRP78, both, or non-specific IgG (as a control). GRP78 and MIF immunostaining was simultaneously visualized in umbrella cells only after substance P treatment. Immunoprecipitation studies showed GRP78-MIF complexes increased after substance P while in vivo biotinylation confirmed substance P-induced GRP78 cell-surface expression in urothelial cells. Intraluminal blockade of CD74 and/or GRP78 prevented substance P-induced changes, including bladder edema, intraluminal MIF release by urothelial cells and production of inflammatory cytokines by urothelial cells. CONCLUSIONS/SIGNIFICANCE: GRP78 is expressed on the surface of urothelial cells after substance P treatment where it can bind MIF complexes. Blocking CD74 (receptor for MIF) and/or GRP78 prevented substance P-induced inflammatory changes in bladder and urothelium, indicating that these urothelial receptors are effective targets for disrupting MIF-mediated bladder inflammation.