Peripheral Matrix Metalloproteinase-9 Mediates Bone Cancer Pain

Bone cancer pain is one of the most severe chronic pain, which may occur in patients from either bone tumors or more commonly from skeletal metastasize from the most often diagnosed cancers such as breast, prostate and lung carcinomas. Tumors in bone secrete a multitude of factors such as proteinases, cytokines, chemokines,osteoblastic and osteolytic factors, which may excite and sensitize nociceptors leading to peripheral sensitization.MMP-9 is one of the best studied members of MMPs family, with the ability of cleaving extracellular matrix components and activating cytokines and growth factors, such as collagens, gelatin, TNFa, TGF-β and IL-1β. Expression of MMP-9 is low in tissue, however it increases when matrix remodeling is required or inappropriate control of its synthesis, secretion, or activation occurs. MMP-9 is commonly involved in inflammation and various neurodegenerative disease. It’s also implicated in chronic pain, including inflammation pain, neuropathic pain, and cancer pain. However, the role of MMP-9 in bone cancer pain and the mechanism are still unknown.TRPV1 is a non-selective cation channel which can be activated by noxious heat, H+, capsaicin and endogenous ligands. It is predominantly expressed in small-diameter (< 25 μm) dorsal root ganglion (DRG) neurons. TRPV1 channel can be considered as a molecular integrator of pain signals, which is known to be playing important role in inflammatory pain, neuropathic pain, and bone cancer pain.TNFa is an important proinflammatory cytokine produced and secreted as transmembrane precursor. After being cleaved from surface-bound pro-TNFa, it is released from cells as a souble molecule (sTNFa, a homotrimer of 17 KDa monomers). Activated TNFa prefers to interact with TNF receptor 1 (TNFR1) than TNF receptor 2 (TNFR2). TNFa is overexpressed in many diseases, including inflammatory pain, neuropathic pain, and bone cancer pain. TNFa is capable of modulating several ion channels, such as TRPV1 channel, Na+ channel, K+ channel, and Ca2+ channel to sensitize nociceptors.In this study, we investigated the distribution, dynamic changes of peripherial MMP-9, TRPV1 channel and TNFa as well as their roles in bone cancer pain in a rat bone cancer pain model induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells.1. Dynamic changes of peripheral MMP-9, TRPV1, and TNFa during bone cancer painThe expression and activation of MMP-9 in ipsilateral bone increased at day 7,14, and 21, especially at day 7 after inoculation of Walker 256 cells. Not only the expression of MMP-9 but also TRPVl in ipsilateral DRG neurons were upregulated at day 7,14, and 21, especially at day 7 after Walker 256 cells injection. Active-TNFa increased in ipsilateral DRG neurons in poste tumor day 7 (PTD 7), but not in PTD 14 or PTD 21.2. Involvement of peripheral MMP-9 in bone cancer pain Local injection (adjacent to tumor bone) or lumbar puncture injection of TMP-1, an endogenous tissue inhibitor of MMP-9, strongly suppressed bone cancer induced mechanical allodynia and thermal hyperalgesia at PTD 7. Chronic administration of MMP-9 inhibitor I, a MMP-9 synthetic inhibitor, with an osmotic pump attenuated and delayed the progression of bone cancer pain.3. MMP-9 sensitizes TRPVl channel and induces allodynia and hyperalgesiaImmunofluorescence results showed that MMP-9 was colocalized with TRPV1 channel in DRG neurons. We observed a significant increase in the number of MMP-9-and TRPV1-positive DRG neurons at PTD 7 consistent with the dynamic changes of their protein level. Whole-cell patch clamp reveals a potentiate effect of MMP-9 on TRPV1 response. Intraplantar injection of MMP-9 produced rapid mechanical allodynia and thermal hyperalgesia in C57 (wild type) mice, however, intraplantar injection of MMP-9 in TRPVl null mice showed no changes in mechanical or thermal pam.4. MMP-9-induced sensitization of TRPVl is mediated by TNFaActive-TNFa and TRPVl expression levels were increased in acute isolated DRG neurons, after the incubation with MMP-9 for 0.5 h. Intraplantar injection of MMP-9 failed to induce mechanical allodynia and thermal hyperalgesia in TNFR1 null mice. These results suggest that pro-TNFα might be the substrate of MMP-9, involving in the algogenic effect of MMP-9.In summary, this study indicates that there are large amount of MMP-9 produced and released in tumor bone and DRG neurons, which may proteolysise and activate the transmembrane pro-TNFα. The active-TNFa interacts with TNFR1 receptor and influences the expression and function of TRPV1 channel, leading to the development and maintenance of bone cancer pain. Targeting peripheral MMP-9 may provide a good therapeutic strategy against bone cancer pain.