GPR30 disrupts the balance of GABAergic and glutamatergic transmission in the spinal cord driving to the development of bone cancer pain

Abstract

Cancer induced bone pain is a very complicated clinical pain states that has proven difficult to be treated effectively due to poorly understand of underlying mechanism, but bone cancer pain (BCP) seems to be enhanced by a state of spinal sensitization. In the present study, we showed that carcinoma tibia implantation induced notable pain sensitization and up-regulation of G-protein-coupled estrogen receptor (GPR30) in the spinal cord of rats which was reversed by GPR30 knockdown. Further studies indicated that upregulation of GPR30 induced by cancer implantation resulted in a select loss of γ-aminobutyric acid-ergic (GABAergic) neurons and functionally diminished the inhibitory transmission due to reduce expression of the vesicular GABA transporter (VGAT). GPR30 contributed to spinal cord disinhibition by diminishing the inhibitory transmission via upregulation of α1 subunit and downregulation of γ2 subunits. GPR30 also facilitated excitatory transmission by promoting functional up-regulation of the calcium/calmodulin-dependent protein kinase II α (CaMKII α) in glutamatergic neurons and increasing the clustering of the glutamate receptor subunit 1 (GluR1) subunit to excitatory synapse.Taken together, GPR30 contributed to the development of BCP by both facilitating excitatory transmission and inhibiting inhibitory transmission in the spinal cord. Our findings provide the new spinal disinhibition and sensitivity mechanisms underlying the development of bone cancer pain.

Keywords: GPR30; bone cancer pain; excitatory transmission; inhibitory transmission; spinal cord.

Figure 1. Upregulation of GPR30 in the spinal cord of cancer-bearing rats

A. Immunofluorescent labeling using an antibodies against GPR30(red) in the spinal cord of sham, cancer-bearing, and RNAi-LV treatment rats. Scale bar 100μm, a white arrow marked the location of lentivirus injection. The spinal cord stain showed that GPR30 was increase in the ipsilateral dorsal horn of cancer-bearing rats. B. Immunofluorescent labeling of neuron with an antibody of anti-Neun (red) to show the effect of lentivirus (green) transfection. Scale bar 50μm. C, D. Quantitative analysis of GPR30 by RT-PCR and western blot in the spinal cord of sham, carcinoma, RNAi-LV treatment rats. A blot sample was showed on the right. Values represent the mean±SEM, n=4. *P<0.05 compared with the sham; **P<0.05 compared with the carcinoma.

Figure 2. GPR30 induced the development of BCP

Mechanical allodynia was measure with paw withdrawal threshold (PWT) in sham, cancer-bearing, and RNAi-LV treatment rats. Note that the carcinoma implantation induced decrease of PWT on the ipsilateral paw, but not on the contralateral side which was reversed by RNAi-LV treatment. Values represent the mean±SEM, n=6. *P<0.05 compared with the sham; **P<0.05 compared with the carcinoma (ipsilateral).

Figure 3. GPR30 diminished inhibitory transmission of GABAergic neurons

A. Immunofluorescent labeling using antibodies against GPR30(red) and GABAergic interneuron(green) in the spinal cord of sham, cancer-bearing, and RNAi-LV treatment rats. Scale bar 100μm. The white rectangle outlined the highly magnified cell showing the co-localization of GPR30 and GAD67. B. Immunofluorescent labeling using antibody against GAD67 of sham, cancer-bearing, and RNAi-LV treatment rats C. Quantitative analysis of GABAergic neurons in the spinal cord of sham, carcinoma, carcinoma+siGPR30-LV rats. Values represent the mean±SEM, n=6 in each group. *P<0.01 compared with the sham; ^**P<0.01 compared with the carcinoma. D, E. Quantitative analysis of GAD67 and VGAT by RT-PCR and Western blot respectively in rat spinal cord of sham, carcinoma, carcinoma+siGPR30-LV rats. A blot sample was showed on the right. Values represent the mean±SEM, n=5. *P<0.05 compared with the sham; **P<0.05 compared with the carcinoma.

Figure 4. GPR30 diminished GABAergic inhibitory on excitatory neuron

A. Double immunofluorescent labeling using antibodies against α1 (red) and CaMKII α (green) in the spinal cord of sham, cancer-bearing, and RNAi-LV treatment rats. B. Double immunofluorescent labeling using antibodies against γ2 (red) and CaMKII α(green) in the spinal cord of sham, cancer-bearing, and RNAi-LV treatment rats. Scale bar 100μm. The highly magnified images showed that both α1 and γ2 expressed in the spinal glutamatergic neurons. C. Quantification of α1 and γ2 positive cells in the dorsal horn of sham, carcinoma, RNAi-LV treatment rats. Values represent the mean±SEM, n=6 in each group. *P<0.05 compared with the sham; ^**P<0.05 compared with the carcinoma. D, E. Quantitative analysis of α1 and γ2 subunits using RT-PCR and Western blot respectively of sham, carcinoma and RNAi-LV treatment rats, a blot sample showed on the right. Values represent the mean±SEM, n=5 in each group. *P<0.01 compared with the sham; ^**P<0.01 compared with the carcinoma.

Figure 5. GPR30 enhanced excitatory transmission of glutamatergic neurons

A. Double immunofluorescent labeling using antibodies against GPR30(red) and CaMKII α (green) in the spinal cord, The white rectangle outlined the highly magnified cells (marked by white arrow) showing the co-localization of GPR30 and CaMKII α. B. Immunofluorescent labeling using antibodies against CaMKII α (green) in the spinal cord of sham, cancer-bearing, and RNAi-LV treatment rats. Scale bar 100μm. C. Quantification of CaMKII α positive neurons of sham, carcinoma and RNAi-LV treatment rats. Values represent the mean±SEM, n=6, *P<0.05 compared with the sham; ^**P<0.05 compared with the carcinoma. D, E. Quantitative analysis of CaMKII α by RT-PCR and Western blot in rat spinal cord of sham, carcinoma, RNAi-LV treatment rats. A blot sample showed on the right. Values represent the mean±SEM, n=5. *P<0.05 compared with the sham; ^**P<0.05 compared with the carcinoma.

Figure 6. GPR30 up-regulated GluR1 and PSD95 in the dorsal horn of cancer-bearing rats

A. Double staining of PSD95 (red) and GluR1 (blue) in the dorsal horn of sham, carcinoma and RNAi-LV treatment rats. The white square region was zoomed in (bottom-right) to show the co-localization of GluR1and PSD95. Scale bar 100μm. B. Highly magnified picture of GluR1and PSD95 puncta. Scale bar 20μm. C. Quantitative analysis of co-localization puncta of GluR1 and PSD95. D, E. Quantitative analysis of of GluR1 and PSD95 by RT-PCR and Western blot respectively, a blot sample showed on the right. Values represent the mean±SEM, n=5, *P<0.05 compared with the sham; ^**P<0.05 compared with the carcinoma.