Shanwei Shen

Endogenous PI3K/Akt and NMDAR act independently in the regulation of CREB activity in lumbosacral spinal cord in cystitis ☆

The integral interaction of signaling components in the regulation of visceral inflammation-induced central sensitization in the spinal cord has not been well studied. Here we report that phosphoinositide 3-kinase (PI3K)-dependent Akt activation and N-methyl-d-aspartic acid receptor (NMDAR) in lumbosacral spinal cord independently regulate the activation of cAMP response element-binding protein (CREB) in vivo in a rat visceral pain model of cystitis induced by intraperitoneal injection of cyclophosphamide (CYP). We demonstrate that suppression of endogenous PI3K/Akt activity with a potent PI3K inhibitor LY294002 reverses CYP-induced phosphorylation of CREB, however, it has no effect on CYP-induced phosphorylation of NR1 at Ser(897) and Ser(896); conversely, inhibition of NMDAR in vivo with MK801 fails to block CYP-induced Akt activation but significantly attenuates CYP-induced CREB phosphorylation in lumbosacral spinal cord. This novel interrelationship of PI3K/Akt, NMDAR, and CREB activation in lumbosacral spinal cord is further confirmed in an ex vivo spinal slice culture system exposed to an excitatory neurotransmitter calcitonin gene-related peptide (CGRP). Consistently we found that CGRP-triggered CREB activation can be blocked by both PI3K inhibitor LY294002 and NMDAR antagonists MK801 and D-AP5. However, CGRP-triggered Akt activation cannot be blocked by MK801 or D-AP5; vice versa, LY294002 pretreatment that suppresses the Akt activity fails to reverse CGRP-elicited NR1 phosphorylation. These results suggest that PI3K/Akt and NMDAR independently regulate spinal plasticity in visceral pain model, and target of a single pathway is necessary but not sufficient in treatment of visceral hypersensitivity.

Up-regulation of brain-derived neurotrophic factor is regulated by extracellular signal-regulated protein kinase 5 and by nerve growth factor retrograde signaling in colonic afferent neurons in colitis

Brain-derived neurotrophic factor (BDNF) plays an essential role in sensory neuronal activation in response to visceral inflammation. Here we report that BDNF up-regulation in the primary afferent neurons in the dorsal root ganglia (DRG) in a rat model of colitis is mediated by the activation of endogenous extracellular signal-regulated protein kinase (ERK) 5 and by nerve growth factor (NGF) retrograde signaling. At 7 days of colitis, the expression level of BDNF is increased in conventional neuronal tracing dye Fast Blue labeled primary afferent neurons that project to the distal colon. In these neurons, the phosphorylation (activation) level of ERK5 is also increased. In contrast, the level of phospho-ERK1/2 is not changed in the DRG during colitis. Prevention of the ERK5 activation in vivo with an intrathecal application of the MEK inhibitor PD98059 significantly attenuates the colitis-induced increases in BDNF expression in the DRG. Further studies show that BDNF up-regulation in the DRG is triggered by NGF retrograde signaling which also involves activation of the MEK/ERK pathways. Application of exogenous NGF exclusively to the compartment containing DRG nerve terminals in an ex vivo ganglia-nerve preparation markedly increases the BDNF expression level in the DRG neuronal cell body that is placed in a different compartment; this BDNF elevation is attenuated by U0126, PD98059 and a specific ERK5 inhibitor BIX02188. These results demonstrate the mechanisms and pathways by which BDNF expression is elevated in primary sensory neurons following visceral inflammation that is mediated by increased activity of ERK5 and is likely to be triggered by the elevated NGF level in the inflamed viscera.

Suppression of the PI3K Pathway In Vivo Reduces Cystitis-Induced Bladder Hypertrophy and Restores Bladder Capacity Examined by Magnetic Resonance Imaging

This study utilized magnetic resonance imaging (MRI) to monitor the real-time status of the urinary bladder in normal and diseased states following cyclophosphamide (CYP)-induced cystitis, and also examined the role of the phosphoinositide 3-kinase (PI3K) pathway in the regulation of urinary bladder hypertrophy in vivo. Our results showed that under MRI visualization the urinary bladder wall was significantly thickened at 8 h and 48 h post CYP injection. The intravesical volume of the urinary bladder was also markedly reduced. Treatment of the cystitis animals with a specific PI3K inhibitor LY294002 reduced cystitis-induced bladder wall thickening and enlarged the intravesical volumes. To confirm the MRI results, we performed H&E stain postmortem and examined the levels of type I collagen by real-time PCR and western blot. Inhibition of the PI3K in vivo reduced the levels of type I collagen mRNA and protein in the urinary bladder ultimately attenuating cystitis-induced bladder hypertrophy. The bladder mass calculated according to MRI data was consistent to the bladder weight measured ex vivo under each drug treatment. MRI results also showed that the urinary bladder from animals with cystitis demonstrated high magnetic signal intensity indicating considerable inflammation of the urinary bladder when compared to normal animals. This was confirmed by examination of the pro-inflammatory factors showing that interleukin (IL)-1α, IL-6 and tumor necrosis factor (TNF)α levels in the urinary bladder were increased with cystitis. Our results suggest that MRI can be a useful technique in tracing bladder anatomy and examining bladder hypertrophy in vivo during disease development and the PI3K pathway has a critical role in regulating bladder hypertrophy during cystitis.

Inhibition of NMDAR Reduces Bladder Hypertrophy and Improves Bladder Function in Cyclophosphamide Induced Cystitis

PURPOSE We examined the role of NMDAR in the regulation of bladder hypertrophy and function in a rat model of cyclophosphamide induced cystitis. MATERIALS AND METHODS Cystitis was induced by intraperitoneal injection of cyclophosphamide (150 mg/kg body weight). NMDAR phosphorylation (activity) and signal transduction pathways were examined by direct measurement and by specific inhibitors in vivo. Bladder hypertrophy was measured by bladder weight/body weight and type I collagen expression. Bladder function was examined by metabolic recording, conscious cystometry and detrusor muscle strip contractility in response to carbachol. RESULTS NMDAR activity measured by the phosphorylation level of the NMDAR1 (NR1) subunit was expressed in the spinal cord but not in the bladder at 48 hours of cystitis. NMDAR inhibition with dizocilpine (MK-801) reduced the cystitis induced increment of bladder weight and type I collagen up-regulation in the bladder. NMDAR regulated type I collagen up-regulation was mediated by the PI3K/Akt pathway. NMDAR inhibition also attenuated cystitis induced urinary frequency measured by metabolic cage and cystometry. Cystitis decreased the responsiveness of detrusor muscle strips to carbachol, which was reversed by MK-801 in vivo. Unlike MK-801 the NMDAR antagonist D-AP5, which could not block central NMDAR activity, had no effect on bladder hypertrophy, type I collagen up-regulation or Akt activation caused by cystitis in the bladder. CONCLUSIONS Findings suggest that NMDAR activity has a role in cystitis induced bladder hypertrophy and overactivity. NMDAR mediated Akt activation may underlie the mechanism of bladder dysfunction.

Inflammation and activity augment brain-derived neurotrophic factor peripheral release

Brain-derived neurotrophic factor (BDNF) release to nerve terminals in the central nervous system is crucial in synaptic transmission and neuronal plasticity. However, BDNF release peripherally from primary afferent neurons has not been investigated. In the present study, we show that BDNF is synthesized by primary afferent neurons located in the dorsal root ganglia (DRG) in rat, and releases to spinal nerve terminals in response to depolarization or visceral inflammation. In two-compartmented culture that separates DRG neuronal cell bodies and spinal nerve terminals, application of 50mM K(+) to either the nerve terminal or the cell body evokes BDNF release to the terminal compartment. Inflammatory stimulation of the visceral organ (e.g. the urinary bladder) also facilitates an increase in spontaneous BDNF release from the primary afferent neurons to the axonal terminals. In the inflamed viscera, we show that BDNF immunoreactivity is increased in nerve fibers that are immuno-positive to the neuronal marker PGP9.5. Both BDNF and pro-BDNF levels are increased, however, pro-BDNF immunoreactivity is not expressed in PGP9.5-positive nerve-fiber-like structures. Determination of receptor profiles in the inflamed bladder demonstrates that BDNF high affinity receptor TrkB and general receptor p75 expression levels are elevated, with an increased level of TrkB tyrosine phosphorylation/activity. These results suggest a possibility of pro-proliferative effect in the inflamed bladder. Consistently we show that the proliferation marker Ki67 expression levels are enhanced in the inflamed organ. Our results imply that in vivo BDNF release to the peripheral organ is an important event in neurogenic inflammatory state.

Colitis-induced bladder afferent neuronal activation is regulated by BDNF through PLCγ pathway.

Patients with inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS) often experience increased sensory responsiveness in the urinary bladder reflecting neurogenic bladder overactivity. Here we demonstrate that colitis-induced up-regulation of the phospholipase C gamma (PLCγ) pathway downstream of brain-derived neurotrophic factor (BDNF) in bladder afferent neurons in the dorsal root ganglia (DRG) plays essential roles in activating these neurons thereby leading to bladder hyperactivity. Upon induction of colitis with 2,4,6-trinitrobenzenesulfonic acid (TNBS) in rats, we found that the phosphorylation (activation) level of cAMP responsive element-binding (p-CREB) protein, a molecular switch of neuronal plasticity, was increased in specifically labeled bladder afferent neurons in the thoracolumbar and lumbosacral DRGs. In rats having reduced levels of BDNF (BDNF+/-), colitis failed to elevate CREB protein activity in bladder afferent neurons. Physiological examination also demonstrated that colitis-induced urinary frequency was not shown in BDNF+/- rats, implicating an essential role of BDNF in mediating colon-to-bladder sensory cross-sensitization. We further implemented in vivo and in vitro studies and demonstrated that BDNF-mediated colon-to-bladder sensory cross-activation involved the TrkB-PLCγ-calcium/calmodulin-dependent protein kinase II (CaMKII) cascade. In contrast, the PI3K/Akt pathway was not activated in bladder afferent neurons during colitis and was not involved in BDNF action in the DRG. Our results suggest that colon-to-bladder sensory cross-sensitization is regulated by specific signal transduction initiated by the up-regulation of BDNF in the DRG.

The urinary bladder of spontaneously hypertensive rat demonstrates bladder hypertrophy, inflammation, and fibrosis but not hyperplasia

AIMS The present study aims to systemically characterize the factors that are associated with urinary bladder organ enlargement in spontaneously hypertensive rats (SHR). MAIN METHODS We compared the SHR to age-matched normotensive Wistar-Kyoto (WKY) control rats in the levels of bladder pro-inflammatory factors, collagen expression (type I), and detrusor smooth muscle growth. KEY FINDINGS Our results showed that enhanced inflammatory responses and fibrosis were key factors that were closely associated with bladder wall thickening in SHR. Specifically the mRNA levels of inflammatory factors interleukin (IL)-1α, IL-6 and TNFα were significantly higher in SHR than those in WKY rats. The SHR also had a higher number of mast cells in the suburothelium space. Type I collagen production was also significantly higher in SHR when compared to that in control rats. However, the smooth muscle content stayed the same in SHR and WKY rats. This was shown by the results that the ratio of α-smooth muscle actin (SMA) to the nuclear protein histone H3 had no difference between these two rat strains. The mRNA and protein levels of proliferating cell nuclear antigen (PCNA) also showed no change in the urinary bladder of SHR and WKY rats. Further study showed that the phosphorylation level of Akt in the urinary bladder was not changed in SHR when compared to WKY rats. In contrast, the phosphorylation level of ERK1/2 was significantly higher in SHR bladder when compared to that of WKY rats. SIGNIFICANCE These results suggest that inflammation and fibrosis are primary factors that may lead to urinary bladder hypertrophy in SHR.

Regulation of transient receptor potential cation channel subfamily V1 protein synthesis by the phosphoinositide 3-kinase/Akt pathway in colonic hypersensitivity

Abstract The transient receptor potential cation channel subfamily V member 1 (TRPV1), also known as the capsaicin receptor or vanilloid receptor 1 (VR1), is expressed in nociceptive neurons in the dorsal root ganglia (DRG) and participates in the transmission of pain. The present study investigated the underlying molecular mechanisms by which TRPV1 was regulated by nerve growth factor (NGF) signaling pathways in colonic hypersensitivity in response to colitis. We found that during colitis TRPV1 protein levels were significantly increased in specifically labeled colonic afferent neurons in both L1 and S1 DRGs. TRPV1 protein up‐regulation in DRG was also enhanced by NGF treatment. We then found that TRPV1 protein up‐regulation in DRG was regulated by activation of the phosphoinositide 3‐kinase (PI3K)/Akt pathway both in vivo and in vitro. Suppression of endogenous PI3K/Akt activity during colitis or NGF treatment with a specific PI3K inhibitor LY294002 reduced TRPV1 protein production in DRG neurons, and also reduced colitis‐evoked TRPV1‐mediated visceral hypersensitivity tested by hyper‐responsiveness to colorectal distention (CRD) and von Frey filament stimulation of abdomen. Further studies showed that TRPV1 mRNA levels in the DRG were not regulated by either colitis or NGF. We then found that an up‐regulation of the protein synthesis pathway was involved by which both colitis and NGF caused a PI3K‐dependent increase in the phosphorylation level of eukaryotic translation initiation factor 4E‐binding protein (4E‐BP)1. These results suggest a novel mechanism in colonic hypersensitivity which involves PI3K/Akt‐mediated TRPV1 protein, not mRNA, up‐regulation in primary afferent neurons, likely through activation of the protein synthesis pathways. HighlightsTRPV1 protein, not mRNA, up‐regulation in DRG is associated with colonic hypersensitivity.The PI3K/Akt pathway mediates colitis‐ and NGF‐induced TRPV1 protein up‐regulation.PI3K/Akt activation up‐regulates the protein synthesis pathway contributing to TRPV1 protein up‐regulation in DRG.

Phospholipase C gamma mediates endogenous brain-derived neurotrophic factor-regulated calcitonin gene-related peptide expression in colitis-induced visceral pain

Background Visceral hypersensitivity is a complex pathophysiological paradigm with unclear mechanisms. Primary afferent neuronal plasticity marked by alterations in neuroactive compounds such as calcitonin gene-related peptide is suggested to underlie the heightened sensory responses. Signal transduction that leads to calcitonin gene-related peptide expression thereby sensory neuroplasticity during colitis remains to be elucidated. Results In a rat model with colitis induced by 2,4,6-trinitrobenzene sulfonic acid, we found that endogenously elevated brain-derived neurotrophic factor elicited an up-regulation of calcitonin gene-related peptide in the lumbar L1 dorsal root ganglia. At seven days of colitis, neutralization of brain-derived neurotrophic factor with a specific brain-derived neurotrophic factor antibody reversed calcitonin gene-related peptide up-regulation in the dorsal root ganglia. Colitis-induced calcitonin gene-related peptide transcription was also inhibited by brain-derived neurotrophic factor antibody treatment. Signal transduction studies with dorsal root ganglia explants showed that brain-derived neurotrophic factor-induced calcitonin gene-related peptide expression was mediated by the phospholipase C gamma, but not the phosphatidylinositol 3-kinase/Akt or the mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway. Application of PLC inhibitor U73122 in vivo confirmed that colitis-induced and brain-derived neurotrophic factor-mediated calcitonin gene-related peptide up-regulation in the dorsal root ganglia was regulated by the phospholipase C gamma pathway. In contrast, suppression of the phosphatidylinositol 3-kinase activity in vivo had no effect on colitis-induced calcitonin gene-related peptide expression. During colitis, calcitonin gene-related peptide also co-expressed with phospholipase C gamma but not with p-Akt. Calcitonin gene-related peptide up-regulation during colitis correlated to the activation of cAMP-responsive element binding protein in the same neurons. Consistently, colitis-induced cAMP-responsive element binding protein activation in the dorsal root ganglia was attenuated by brain-derived neurotrophic factor antibody treatment. Conclusion These results suggest that colitis-induced and brain-derived neurotrophic factor-mediated calcitonin gene-related peptide expression in sensory activation is regulated by a unique pathway involving brain-derived neurotrophic factor-phospholipase C gamma-cAMP-responsive element binding protein axis.

Urinary bladder organ hypertrophy is partially regulated by Akt1-mediated protein synthesis pathway

Aims: The present study aims to investigate the role of Akt in the regulation of urinary bladder organ hypertrophy caused by partial bladder outlet obstruction (pBOO). Main methods: Male rats were surgically induced for pBOO. Real‐time PCR and western blot were used to examine the levels of mRNA and protein. A phosphoinositide 3‐kinase (PI3K) inhibitor LY294002 was used to inhibit the activity of endogenous Akt. Key findings: The urinary bladder developed hypertrophy at 2 weeks of pBOO. The protein but not mRNA levels of type I collagen and &agr;‐smooth muscle actin (&agr;SMA) were increased in pBOO bladder when compared to sham control. The phosphorylation (activation) levels of Akt1 (p‐Ser473), mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and 4E‐BP1 were also increased in pBOO bladder. LY294002 treatment reduced the phosphorylation levels of Akt1 and 4E‐BP1, and the protein levels of type I collagen and &agr;SMA in pBOO bladder. The mRNA and protein levels of proliferating cell nuclear antigen (PCNA) were increased in pBOO bladder, and PCNA up‐regulation occurred in urothelial not muscular layer. LY294002 treatment had no effect on the mRNA and protein levels of PCNA in pBOO bladder. LY294002 treatment partially reduced the bladder weight caused by pBOO. Significance: pBOO‐induced urinary bladder hypertrophy is attributable to fibrosis, smooth muscle cellular hypertrophy, and urothelium cell hyper‐proliferation. Akt1‐mediated protein synthesis in pBOO bladder contributes to type I collagen and &agr;SMA but not PCNA up‐regulation. Target of Akt1 is necessary but not sufficient in treatment of urinary bladder hypertrophy following pBOO.