Changes in nerve growth factor signaling in female mice with cyclophosphamide-induced cystitis

Abstract

IC/BPS is a chronic inflammatory pelvic pain syndrome characterized by lower urinary tract symptoms including unpleasant sensation (pain, pressure, or discomfort) in the suprapubic or bladder area, as well as increased urinary frequency and urgency, and decreased bladder capacity. While its etiology remains unknown, increasing evidence suggests a role for changes in nerve growth factor (NGF) signaling. However, NGF signaling is complex and highly context dependent. NGF activates two receptors, TrkA and p75^NTR, which activate distinct but overlapping signaling cascades. Dependent on their coexpression, p75^NTR facilitates TrkA actions. Here, we show effects of CYP treatment and pharmacological inhibition of p75^NTR (via LM11A-31) and TrkA (ARRY-954) on NGF signaling-related proteins: NGF, TrkA, phosphorylated (p)-TrkA, p75^NTR, p-ERK1/2, and p-JNK. Cystitis conditions were associated with increased urothelial NGF expression and decreased TrkA and p75^NTR expression as well as altering their co-expression ratio; phosphorylation of ERK1/2 and JNK were also altered. Both TrkA and p75^NTR inhibition affected the activation of signaling pathways downstream of TrkA, supporting the hypothesis that NGF actions during cystitis are primarily TrkA-mediated. Our findings, in tandem with our recent companion paper demonstrating the effects of TrkA, TrkB, and p75^NTR inhibition on bladder function in a mouse model of cystitis, highlight a variety of potent therapeutic targets and provide further insight into the involvement of NGF signaling in sustained conditions of bladder inflammation.

Keywords: c-Jun N-terminal kinase (JNK); extracellular signal-regulated kinase (ERK1/2); interstitial cystitis/bladder pain syndrome (IC/BPS); lower urinary tract (LUT); nerve growth factor; neurotrophin (NT); p75 NTR; tropomyosin receptor kinase (TrkA).

FIGURE 1

Acute CYP treatment increases urothelial NGF expression. Analysis with a linear model found a significant main effect of condition (F(2,33) = 41.16, p = 1.08x10⁻⁹). Pairwise comparisons using estimated marginal means showed that NGF expression was significantly elevated in the acute CYP condition compared to control (p = 1x10⁻⁴) and chronic CYP (p = 1x10⁻⁴) conditions.

FIGURE 2

Urothelial p75^NTR expression decreased as a consequence of CYP treatment. (A) p75^NTR immunoreactivity (IR) in cryostat sections of urinary bladder from mice in control, acute (4-hour) CYP, and chronic (8-day) CYP conditions. Note the decreasing urothelial IR in CYP conditions. Lumen (L), lamina propria (LP), and urothelium (U) of the bladder as indicated. Calibration bar: 25 μm. (B) Analysis with a linear model found a significant main effect of condition (F(2,34) = 151.05, p = 2x10⁻¹⁶). Pairwise comparisons with estimated marginal means found that p75^NTR expression differed significantly between acute CYP and control conditions (p = 0.000643), acute CYP and chronic CYP conditions (p = 1x10⁻⁴), and chronic CYP and control conditions (p = 1x10⁻⁴).

FIGURE 3

Urothelial TrkA expression decreased as a consequence of chronic CYP treatment. (A) TrkA immunoreactivity (IR) in cryostat sections of urinary bladder from mice in control, acute (4-hour) CYP, and chronic (8-day) CYP conditions. Note the decreased urothelial TrkA IR in the chronic CYP condition. Urothelial hyperplasia is also evident in the CYP conditions. Lumen (L), lamina propria (LP), and urothelium (U) of the bladder as indicated. Calibration bar: 25 μm. (B) Urothelial TrkA expression decreased as a consequence of chronic CYP treatment. Analysis with a linear model found a significant main effect of condition (F(2,34) = 36.49, p = 3.53x10⁻⁹). Pairwise comparisons with estimated marginal means revealed that urothelial TrkA expression in chronic CYP conditions was significantly reduced compared to control (p = 1x10⁻⁴) and acute CYP conditions (p = 1x10⁻⁴). (C) Detrusor TrkA expression increased as a consequence of CYP treatment. Analysis with a linear model found a significant main effect of condition (F(2,34) = 5.37, p = 0.009). Pairwise comparisons with estimated marginal means revealed that detrusor TrkA expression was significantly elevated under acute (p = 0.0447) and chronic (p = 0.01) CYP conditions when compared to the control condition.

FIGURE 4

AR treatment significantly reduced TrkA phosphorylation in CYP conditions. Analysis with a linear model found a significant main effect of treatment (F(2,24) = 4.16, p = 0.029). Pairwise comparisons with estimated marginal means revealed that p-TrkA expression was significantly reduced following AR treatment when compared to saline under acute (p = 0.022) and chronic (p = 0.022) CYP conditions.

FIGURE 5

Urothelial TrkA:p75^NTR expression ratio was significantly altered in the chronic CYP condition. (A) Merged TrkA and p75^NTR immunoreactivity (IR) in cryostat sections of urinary bladder from mice in control, acute (4-hour) CYP, and chronic (8-day) CYP conditions. Note the dominance of TrkA IR in the urothelium in the chronic CYP condition. Urothelial hyperplasia is especially evident in the chronic CYP condition. Lumen (L), lamina propria (LP), and urothelium (U) of the bladder as indicated. Calibration bar: 25 μm. (B) Analysis with a linear model found a significant main effect of condition (F(2,34) = 11.81, p = 0.00013). Pairwise comparisons with estimated marginal means revealed that TrkA:p75^NTR expression ratio was significantly elevated in the chronic CYP condition when compared to control (p = 0.014) and acute CYP (p = 0.0005) conditions.

FIGURE 6

ERK1/2 phosphorylation is significantly increased under acute CYP conditions, but not following LM or AR treatment. (A) p-ERK1/2 immunoreactivity (IR) in cryostat sections of urinary bladder from mice in control, acute (4-hour) CYP, and chronic (8-day) CYP conditions. Note the increased pERK1/2 IR in the urothelium in the chronic CYP condition. Lumen (L), lamina propria (LP), detrusor (D), and urothelium (U) of the bladder as indicated. Calibration bar: 25 μm. (B) Analysis with a linear model found significant main effects of condition (F(2,32) = 32.77, p = 1.79x10⁻⁸) and treatment (F(2,32) = 2.97, p = 0.046), and the interaction was significant (F(2,32) = 8.49, p = 0.0011). Pairwise comparisons with estimated marginal means revealed that p-ERK1/2 expression was significantly elevated under acute CYP conditions when compared to control (p < 0.0001) and chronic CYP (p < 0.0001) conditions when treated with saline; however, under acute CYP conditions, p-ERK1/2 expression was significantly reduced following both AR (p = 0.0001) and LM (p = 0.046) treatment when compared to saline.

FIGURE 7

Urothelial and detrusor JNK phosphorylation changed as a consequence of condition and treatment. (A) p-JNK immunoreactivity (IR) in cryostat sections of urinary bladder from mice in control, acute (4-hour) CYP, and chronic (8-day) CYP conditions. Note the increased p-JNK IR in the urothelium in the acute CYP condition. Urothelial hyperplasia is especially evident in the chronic CYP condition. Lumen (L), lamina propria (LP), and urothelium (U) of the bladder as indicated. Calibration bar: 25 μm. (B) Analysis with a linear model found a significant main effect of condition (F(2,32) = 13.27, p = 6.39x10⁻⁵) on urothelial p-JNK expression. Pairwise comparisons with estimated marginal means revealed that urothelial p-JNK expression was significantly elevated when compared to control (p = 0.0058) and chronic CYP (p = 0.00028) conditions. (C) Analysis with a linear model found significant main effects of condition (F(2, 33) = 20.63, p = 1.55x10⁻⁶) and treatment (F(3,33) = 6.01, p = 0.0022) on detrusor p-JNK expression. The interaction was not significant. Pairwise comparisons with estimated marginal means revealed that detrusor p-JNK expression was significantly elevated under chronic CYP conditions when compared to control (p = 0.012) and acute CYP (p = 1x10⁻⁴) conditions, but p-JNK expression was significantly reduced following LM treatment when compared to saline (p = 0.016) and AR (p = 0.0018) treatments.