TGF-β Sensitizes TRPVl In Primary Sensory Neurons Via TAK1/P38 And PKC Signalings: A Novel Mechanism Of Bone Cancer Pain

According to the report from the World Health Organization, cancer is the leading cause of death worldwide, accounting for about 13% of all death. The National Cancer Institute estimates that over 1.5 million people were diagnosed with cancer in 2010. Many tumors arising from breast, prostate, thyroid, lung and kidney are easy to spread to bone and induce metastatic cancer. Bone provides a fertile microenvironment for tumor growth and invasion. When cancer metastasizes to bone, severe bone pain and deregulated bone remodeling occurs, greatly diminishing the possibility of cure and reducing patients’quality of life.Transforming growth factor-beta (TGF-β) is a kind of bone-drived growth factor which is secreted in a latent form by bone cells and stored in the bone matrix. When osteoclasts are activated, leading osteoclastic bone resorption, TGF-p can be released and activated from the mineralized bone matrix. In addition to secrete TGF-p which can directly affect cancer cells, metastatic cancer cells can also secrete many other osteoblastic factors and osteolytic factors. The osteoblastic factors can stimulate osteoblasts to proliferation, differentiation and secrete growth factors to enrich the tumor microenvironment. The osteolytic factors can activate ostesclasts and induce osteoclastic bone resorption. Thus, more activated TGF-β is released from this process and further activates cancer cells to release more cytokine driving a feed-forward cycle of tumor growth in bone.Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel that can be activated by an array of chemical and physical signals:noxious heat (>43℃), capsaicin, H+ and endogenous ligands. TRPV1 is considered as a molecular integrator of chemical and physical stimuli that elicit pain. Recent studies have indicated TRPV1 plays a significant role in the model of cancer pain. We know that metastatic bone cancer is always accompanied by severe pain. So whether TGF-β secreted by cancer cells and osteolysis can influence the peripheral nociceptive transmission through TRPV1 channel.By means of electrophysiological recording, calcium imaging, behavior test, immunohistochemistry and western blot, the present study was to investigate the role of TGF-β in the bone cancer pain. The main findings were as followed:1. Identification of DRG neurons innervating the rat tibiaThe fluorogold tracing results showed that L3, L4, and L5 DGR segments main innervated the rat tibia. The immunohistochemistry results showed that the innervating neurons were CGRP-positive neurons, but not IB4-positive neurons, in all the L3-L5 DRG segments.2. Dynamic changes of TGF-β1, TGF-β receptors and TRPV1 during bone cancer painThe western blot and immunohistochemistry results showed that TGF-β1 expression in bone, TGFβ-receptor 1 and TRPV1 receptor expression in dorsal root ganglion (DRG) were increased, but TGFβ-receptor 2 expression had no change after inoculation. The electrophysiology results that the TRPV1 current densities recorded form cancer group were significantly enhanced and the naive group showed more desensitized than cancer group. These results demonstrated that DRG neurons from bone cancer rats were much sensitive.3. TGF-β1 potentiates TRPV1 activity and induces thermal hyperalgesiaThe immunofluorescence results showed that TRPV1 receptor was co-localized well with TGFβ-receptor 1 and TGFβ-receptor 2 whatever in Naive and Cancer group. In addition, electrophysiology and calcium results showed that the TRPV1 currents were sensitive by TGF-β1 perfusion in naive and bone cancer animals. The behavior test results showed that lumbar puncture injection of TGFβR I antagonist SD-208 acutely suppressed bone cancer induced thermal hyperalgesia. Furthermore, TGF-β1 intraplantar injection could induce thermal hyperalgesia in intact rats, which was inhibited by TRPV1 antagonist capsazepine.4. TAK1/p38 and PKC, but not ERK and TAK1/JNK, signaling pathway mediate the TRPV1 sensitization by TGF-β1 TAK1 inhibitor (5Z)-7-Oxozeaenol, p38 inhibitor SB203580 and PKC inhibitor BIM completely abolished the sensitizing effect of TGF-β1 on TRPV1 currents. In contrast, ERK inhibitor PD98059 and U0126 and JNK inhibitor SP600125 was failed to prevent TGF-β1 induced TRPV1 currents sensitization. These results suggested that TAKl/p38 and PKC signaling pathway were involved in sensitization of TRPV1 by TGF-β1.5. Involvement of p38 and PKC in TGF-β1 mediated thermal hyperalgesia after intraplantar injection Intraplantar pretreatment of p38 inhibitor SB203580 and PKC inhibitor BIM dose-dependently blocked TGF-β1 induced heat hyperalgesia. These data suggested that TGF-β1 injection can produce heat hyperalgesia via TAK1/p38 and PKC signaling pathway sensitized TRPV1 receptor.In conclusion, the present work demonstrated that peripheral TGF-β1 secreted by cancer cells and osteolysis can influence the bone cancer pain by sensitizing TRPV1 currents through TAK1/p38 MAPK and PKC signaling pathway in rat primary sensory neurons. Targeting TGF-β1 signaling in primary sensory neurons may provide novel therapeutic strategies against bone cancer pain.