BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) reveals the involvement of the transforming growth factor-beta family in pain modulation

Abstract

Transforming growth factors-beta (TGF-betas) signal through type I and type II serine-threonine kinase receptor complexes. During ligand binding, type II receptors recruit and phosphorylate type I receptors, triggering downstream signaling. BAMBI [bone morphogenetic protein (BMP) and activin membrane-bound inhibitor] is a transmembrane pseudoreceptor structurally similar to type I receptors but lacks the intracellular kinase domain. BAMBI modulates negatively pan-TGF-beta family signaling; therefore, it can be used as an instrument for unraveling the roles of these cytokines in the adult CNS. BAMBI is expressed in regions of the CNS involved in pain transmission and modulation. The lack of BAMBI in mutant mice resulted in increased levels of TGF-beta signaling activity, which was associated with attenuation of acute pain behaviors, regardless of the modality of the stimuli (thermal, mechanical, chemical/inflammatory). The nociceptive hyposensitivity exhibited by BAMBI(-/-) mice was reversed by the opioid antagonist naloxone. Moreover, in a model of chronic neuropathic pain, the allodynic responses of BAMBI(-/-) mice also appeared attenuated through a mechanism involving delta-opioid receptor signaling. Basal mRNA and protein levels of precursor proteins of the endogenous opioid peptides proopiomelanocortin (POMC) and proenkephalin (PENK) appeared increased in the spinal cords of BAMBI(-/-). Transcript levels of TGF-betas and their intracellular effectors correlated directly with genes encoding opioid peptides, whereas BAMBI correlated inversely. Furthermore, incubation of spinal cord explants with activin A or BMP-7 increased POMC and/or PENK mRNA levels. Our findings identify TGF-beta family members as modulators of acute and chronic pain perception through the transcriptional regulation of genes encoding the endogenous opioids.

Figure 1.

Targeted disruption of the BAMBI gene by homologous recombination. A, Gene-targeting strategy. Top, Wild-type BAMBI locus showing exons 1–3 (filled boxes). Bottom, Targeting vector in which exons 2 and 3 were replaced with the neomycin (neo) resistance marker. Arrows indicate the position of the primers used for genotyping. B, PCR analysis of genomic DNA from BAMBI^+/+, BAMBI^+/−, and BAMBI^−/− mice. C, RT-PCR analysis of BAMBI mRNA expression in the brain cortices (Cx) and spinal cords (Sc) of BAMBI^+/+ and BAMBI^−/− mice. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. D, Representative Western blot of phosphorylated Smad1 and Smad2 proteins in the spinal cord of BAMBI^+/+ and BAMBI^−/− mice.

Figure 2.

In situ hybridization showing the distribution of BAMBI mRNA in the spinal cord (A) and brain (B) and type I receptors ALK-4 (C), ALK-3 (D), and ALK-6 (E) mRNA in the spinal cord. One-hundred-micrometer coronal sections were hybridized with digoxigenin-labeled riboprobes. Immunohistochemical staining evidenced the presence of BAMBI immunoreactivity in the superficial layers of the spinal cord dorsal horn (F). Representative immunofluorescence images from squash preparations of dissociated neurons and glia from dorsal horn (G–G″) and dorsal root ganglion (H–H″). The nuclei were counterstained with DAPI (G, H). BAMBI immunoreactivity (green) was detected in some neurons within dorsal horn (G′) with negligible presence in astrocytes stained (red) with GFAP (G″). In dorsal root ganglion, BAMBI signal was present in neurons (H′) but absent in their surrounding satellite glial cells. Confocal high-magnification immunofluorescence images indicate a peripheral expression pattern of BAMBI immunoreactivity (H″), consistent with its transmembrane localization. HP, Hippocampus; PG, mesencephalic periaqueductal gray; DH, dorsal horn of the spinal cord.

Figure 3.

Responses to acute painful stimuli. A, Warm-water tail-flick assay assessed at 45, 47, and 49°C. B, Effect of the pretreatment with NLX (1 mg/kg, i.p.) on tail-flick latencies. C, Hotplate licking and jump latencies at 50°C. D, Cumulative time spent licking the hindpaw after subcutaneous injection of Formalin (20 μl of 2% Formalin) into the plantar surface from 0–5 min (first phase) and 20–30 min after injection (second phase). E, Mechanical stimulation test (von Frey monofilaments). Values are the percentage of hindpaw withdrawals in response to stimuli of increasing strength. F, Effect of NLX (1 mg/kg, i.p.) on the mechanical sensitivity threshold. Note that, after naloxone treatment, BAMBI^−/− mice showed a normalization in the withdrawal response to both thermal and mechanical stimuli, whereas no changes were evident in wild-type mice. Data are means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 versus BAMBI^+/+ mice (two-tailed Student's t test).

Figure 4.

Development of neuropathic pain in response to crush injury of sciatic nerve. Representative graphs showing the behavioral manifestations of neuropathic pain (mechanical allodynia) evaluated with the von Frey monofilaments, on day 14 after nerve injury and the effect of naloxone pretreatment before performing the von Frey test. Values are the mean ± SEM percentage of hindpaw withdrawals elicited by mechanical stimuli of increasing strength, in wild-type (top graph) and BAMBI^−/− (bottom graph) mice subjected to sham operation (triangles) or sciatic nerve injury (circles). Two-way ANOVA indicates that BAMBI^−/− mice were significantly less sensitive to mechanical stimuli compared with their wild-type littermates. Naloxone treatment (filled circles) before performing the mechanical test, on day 14 after nerve injury triggered mechanical allodynia in BAMBI^−/− mice, whereas no significant changes in mechanical sensitivity were evidenced in wild-type mice.

Figure 5.

Development of neuropathic pain in response to spared nerve injury. Representative graphs showing the behavioral manifestations of neuropathic pain (mechanical allodynia) evaluated with the von Frey monofilaments, on day 14 after SNI surgery in BAMBI^−/− (filled circles) and wild-type (filled squares) mice. Values are the mean ± SEM percentage of hindpaw withdrawals elicited by mechanical stimuli of increasing strength, in wild-type (triangles) and BAMBI^−/− mice (circles), subjected to sham operation (open symbols) or SNI surgery (filled symbols). Two-way ANOVA indicates that BAMBI^−/− mice were significantly less sensitive to mechanical stimuli compared with their wild-type littermates. Note also the difference in mechanical sensitivity between sham-operated BAMBI^+/+ and BAMBI^−/− mice.

Figure 6.

Effects of specific opioid antagonists on neuropathic pain behavior in BAMBI^−/− mice. Representative graphs showing the behavioral manifestations of neuropathic pain (mechanical allodynia) evaluated with von Frey monofilaments, on day 14 after sciatic nerve crush injury (triangles), and the effect of pretreatment with selective opioid antagonists before performing the von Frey test (open circles). The antagonistic effect achieved by subsequent naloxone administration is also shown (filled circles). Values are the mean ± SEM percentage of hindpaw withdrawals elicited by mechanical stimuli of increasing strength. The selective antagonist of δ-opioid receptors, naltrindole (top graph), fully reversed the anti-allodynic phenotype of BAMBI^−/− mice, whereas the μ-opioid (β-FNA) and κ-opioid (nor-BNI) selective antagonists were much less effective.