NFκB-mediated CXCL1 production in spinal cord astrocytes contributes to the maintenance of bone cancer pain in mice

Abstract

Background: Bone cancer pain (BCP) is one of the most disabling factors in patients suffering from primary bone cancer or bone metastases. Recent studies show several chemokines (for example, CCL2, CXCL10) in the spinal cord are involved in the pathogenesis of BCP. Here we investigated whether and how spinal CXCL1 contributes to BCP.

Methods: Mouse prostate tumor cell line, RM-1 cells were intramedullary injected into the femur to induce BCP. The mRNA expression of CXCL1 and CXCR2 was detected by quantitative real-time PCR. The protein expression and distribution of CXCL1, NFκB, and CXCR2 was examined by immunofluorescence staining and western blot. The effect of CXCL1 neutralizing antibody, NFκB antagonist, and CXCR2 antagonist on pain hypersensitivity was checked by behavioral testing.

Results: Intramedullary injection of RM-1 cells into the femur induced cortical bone damage and persistent (>21 days) mechanical allodynia and heat hyperalgesia. Tumor cell inoculation also produced CXCL1 upregulation in activated astrocytes in the spinal cord for more than 21 days. Inhibition of CXCL1 by intrathecal administration of CXCL1 neutralizing antibody at 7 days after inoculation attenuated mechanical allodynia and heat hyperalgesia. In cultured astrocytes, TNF-α induced robust CXCL1 expression, which was dose-dependently decreased by NFκB inhibitor. Furthermore, inoculation induced persistent NFκB phosphorylation in spinal astrocytes. Intrathecal injection of NFκB inhibitor attenuated BCP and reduced CXCL1 increase in the spinal cord. Finally, CXCR2, the primary receptor of CXCL1, was upregulated in dorsal horn neurons after inoculation. Inhibition of CXCR2 by its selective antagonist SB225002 attenuated BCP.

Conclusion: NFκB mediates CXCL1 upregulation in spinal astrocytes in the BCP model. In addition, CXCL1 may be released from astrocytes and act on CXCR2 on neurons in the spinal cord and be involved in the maintenance of BCP. Inhibition of the CXCL1 signaling may provide a new therapy for BCP management.

Figure 1

RM-1 cell inoculation induces BCP. (A) The animals’ body weight was increased in 21 days in both sham-control and tumor-inoculated animals. (B) Radiography shows cortical bone damage in the distal one-third of the right femur (arrows) at 21 days after inoculation. (C, D) Behavioral tests show that mice displayed both heat hyperalgesia and mechanical allodynia in the ipsilateral paw after RM-1 cell inoculation. The PWL decreased at 7 days and maintained for more than 21 days (C). The PWT progressively decreased from 7 days to 14 days and maintained at 21 days (D). **P <0.01, ***P <0.001 vs. sham-ipsi. One-way ANOVA followed by Newman-Keuls test. n = 6 mice per group.

Figure 2

RM-1 cell inoculation induces CXCL1 upregulation in spinal astrocytes. (A) Real-time PCR results show the increase of CXCL1 mRNA expression in the spinal cord after inoculation. CXCL1 mRNA upregulation was gradually increased from 7 days to 21 days. *P <0.05 vs. sham control. n = 4 mice per group. (B-F) Immunostaining shows the CXCL1-IR was increased in the spinal cord at 7 days (D), 14 days (E), and 21 days (F). ***P <0.001 vs. naive. n = 4 mice per group. (G-I) Double staining shows CXCL1 was colocalized with astrocytic marker, GFAP (G), but not with microglial marker CD11b (H) or neuronal marker NeuN (I).

Figure 3

Intrathecal injection of CXCL1 neutralizing antibody attenuates bone cancer pain. CXCL1 neutralizing antibody at a lower dose (4 μg) had mild effect on RM-1 cell inoculation-induced pain hypersensitivity (A, B), whereas the neutralizing antibody at a higher dose (8 μg) reversed inoculation-induced mechanical allodynia (A) and heat hyperalgesia (B) for more than 6 h. *P <0.05, **P <0.01, ***P <0.001 vs. control serum. n = 6 mice per group.

Figure 4

RM-1 cell inoculation induces GFAP upregulation in the spinal cord. (A-D) GFAP-IR was mild in naïve mice (A), but increased at 7 days (B), 14 days (C), and 21 days (D) in inoculated mice. (E) Statistical analysis shows increased GFAP intensity after RM-1 cell inoculation. **P <0.01, ***P <0.001 vs. sham. n = 4 mice per group.

Figure 5

TNF-α induces NFκB-dependent CXCL1 increase in cultured astrocytes. (A-D) CXCL1 was expressed in control astrocytes (A) and increased at 1 h after TNF-α incubation (B). Double staining of CXCL1 (B) with GFAP (C) shows the expression of CXCL1 by astrocytes (D). (E) ELISA results show TNF-α-induced CXCL1 upregulation was decreased by pretreatment with NFκB inhibitor, BAY11-7082. ***P <0.001 vs. control. #P <0.05, ###P <0.001 vs. TNF-α treatment. (F) Quantitative PCR shows TNF-α-induced CXCL1 mRNA increase was decreased by BAY11-7082. ***P <0.001 vs. control. ##P <0.01, ###P <0.001 vs. TNF-α treatment.

Figure 6

RM-1 cell inoculation increases pNFκB expression in spinal astrocytes. (A,B) Western blot shows that tumor cell inoculation increased pNFκB expression in the spinal cord at 7 days, 14 days, and 21 days. *P <0.05, **P <0.01 vs. naive. One-way ANOVA followed by Newman-Keuls test. n = 4 mice per group. (C, D) Immunostaining shows that pNFκB was expressed in the superficial dorsal horn in sham animals (C), increased at 7 days in inoculated animals (D). (E-G) Double staining showed pNFκB was colocalized with GFAP.

Figure 7

NFκB inhibitor attenuated RM-1 cell inoculation-induced pain hypersensitivity and upregulation of CXCL1 in the spinal cord. (A, B) Intrathecal injection of NFκB inhibitor, BAY11-7082 at the dose of 0.4 μg had no effect on mechanical allodynia or heat hyperalgesia, whereas at the dose of 4 μg attenuated mechanical allodynia (A) and heat hyperalgesia (B) at 1 h and 3 h. *P <0.05, **P <0.01, ***P <0.001 vs. vehicle. One-way ANOVA followed by Newman-Keuls test. (C, D) Immunostaining of CXCL1 in the spinal cord in vehicle and BAY11-7082-treated animals. (E) The CXCL1-IF intensity was decreased by BAY11-7082. ***P <0.001 vs. vehicle. n = 4 mice per group.

Figure 8

RM-1 cell inoculation increases CXCR2 mRNA and protein expression in the spinal cord. (A) Real-time PCR results show the increase of CXCR2 mRNA expression in the spinal cord. CXCR2 mRNA was increased from 7 days to 21 days after inoculation. *P <0.05 vs. sham. n = 4 mice per group. (B) Western blot shows time course of CXCR2 protein expression in the spinal cord after inoculation (B). n = 3 mice per group. (C, D) Immunostaining shows the CXCR2 expression in the spinal cord in sham (C) and inoculated (D) animals. CXCR2-IR was increased at 7 days after inoculation. (E-G) Double staining shows CXCR2 was colocalized with neuronal marker NeuN. H, I. SB2205002 attenuated RM-1 cell inoculation-induced mechanical allodynia (H) and heat hyperalgesia (I). *P <0.05; **P <0.01; ***P <0.001 vs. vehicle.