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

Abstract

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.

Keywords: Akt; CREB; Central sensitization; NMDAR; Spinal cord.

Figure 1. Up-regulation of CREB activity in the spinal cord during cystitis

At 8 or 48 h after CYP treatment, the level of p-CREB was increased in the rostral lumbar L1-L2 and the lumbosacral L6-S1 spinal cord examined by western blot (A). Histogram (B) showed relative levels of p-CREB in spinal segments examined. Immunostaining (C-F) showed that cystitis-induced p-CREB immunoreactivity was increased in the spinal dorsal horn (G) as well as in the cells in deep laminae (H). Results were presented as mean ± SE from 5-6 animals at each time point. *, p < 0.05 vs control. Bar=250 μm.

Figure 2. Up-regulation of p-Akt in the spinal cord during cystitis

At 8 or 48 h after CYP treatment, the level of p-Akt was increased in both L1-L2 and L6-S1 spinal segments examined by western blot (A, summary data B). Immunostaining (C-E) showed that at 8 h post CYP injection, the p-Akt was increased in the region of the superficial dorsal horn (D). At 48 h post CYP injection, the p-Akt immunoreactivity was expressed in both dorsal horn and deep laminae (E). The total number of spinal cells expressing p-Akt was significantly increased in the lumbosacral spinal cord at 48 h post CYP injection (F and G). Results were presented as mean ± SE from 4-6 animals at each time point. *, p < 0.05 vs control. Bar=250 μm.

Figure 3. Attenuation of cystitis-induced CREB phosphorylation by PI3K inhibitor LY294002

Western blot showed that suppression of the endogenous Akt activity (A upper) by LY294002 attenuated cystitis-induced CREB activation (A, bottom; B). Immunohistochemistry showed that inhibition of p-CREB in the spinal cord by LY294002 in vivo occurred in the spinal dorsal horn (compare D to C, circled area) but not in the inner laminae of the spinal cord (red *). Double immunostaining showed that a subpopulation of spinal neurons (E-G, cells indicated by arrows) co-expressed p-CREB (E, red nuclear staining) and p-Akt (F, green). During cystitis, p-Akt was expressed close to the cell membrane of the spinal neurons (F, arrows) indicating that cystitis caused membrane trafficking of Akt, a critical step for Akt activation. Results were presented as mean ± SE from 4 independent experiments. Bar=125 μm in C-D; 20 μm in E-G. *, p<0.05 vs saline+DMSO; #, p<0.05 vs CYP+DMSO.

Figure 4. Up-regulation of p-NR1 Ser⁸⁹⁷ in the spinal cord with cystitis and its interplay with p-Akt

Western blot analysis showed that the levels of p-NR1 Ser⁸⁹⁷ (PKA site) were increased in the L1-L2 and L6-S1 spinal cord during cystitis (A, B). Immunohistochemistry showed that the cystitis-induced p-NR1 Ser⁸⁹⁷ was mainly located in the deep laminae of the spinal cord (C), and in the large neurons at the nucleus dorsalis Clarke's column and the ventral horn regions (C). Different from p-Akt (D), p-NR1 Ser⁸⁹⁷ was not expressed in the dorsal horn region, and was partially co-localized with p-Akt (F, yellow arrows; white arrows indicates p-NR1; blue arrows indicates p-Akt). Inhibition of Akt activity by LY294002 failed to block cystitis-induced up-regulation of pNR1 Ser⁸⁹⁷ examined in L6 spinal cord (G, H). Bar=200 μm in C-E; 15 μm in F. *, p<0.05 vs control. n=4-5 animals for each treatment.

Figure 5. Cystitis-increased p-NR1 Ser⁸⁹⁶ levels were not regulated by the PI3K/Akt pathway

The levels of p-NR1 Ser⁸⁹⁶ (PKC site) were increased in the L1-L2 and L6-S1 spinal cord during cystitis (A, B). Inhibition of Akt activity by LY294002 failed to block cystitis-induced up-regulation of pNR1 Ser⁸⁹⁶ examined in L6 spinal cord (C, D). *, p<0.05 vs control. n=4-5 animals for each treatment.

Figure 6. NMDAR mediated CREB but not Akt activation in the spinal cord during cystitis

The L6 spinal cord was examined for the effects of NMDAR antagonist MK801 on CREB and Akt phosphorylation during cystitis. Cystitis-induced CREB phosphorylation was attenuated by MK801 (3 mg/kg body weight and 1 mg/kg body weight); cystitis-induced Akt phosphorylation was not affected by inhibition of NMDAR. n=4 animals for each treatment. *, p<0.05 vs control.

Figure 7. Interrelationship of p-Akt, p-CREB and p-NR1 in the spinal cord examined in culture

Treatment of spinal cord sections with CGRP increased the phosphorylation levels of Akt, CREB, and NR1 at Ser⁸⁹⁷ (A). Inhibition of Akt activity with the PI3K inhibitor LY294002 attenuated CGRP-induced CREB activation but not p-NR1 phosphorylation (A, C). Treatment with NMDAR antagonists DAP5 or MK801 failed to block CGRP-induced Akt activation but reversed CREB phosphorylation (B, D). Results were from 5 independent experiments. *, p<0.05 vs. control (vehicle); #, p<0.05 vs CGRP (+vehicle)-treatment.

Figure 8. Schematic diagram illustrates the putative mechanism for cystitis- and CGRP-induced spinal plasticity involving novel interrelationship between CREB, Akt and NMDAR pathways

Cystitis induces Akt activation in the dorsal horn region where it activates CREB. Cystitis also triggers NMDAR-mediated signaling in the deep laminae and leads to CREB activation. There is a lack of regulatory relationship between the PI3K/Akt and the NMDAR in the spinal cord during cystitis- and CGRP-induced spinal plasticity.