Extracellular Matrix Component of Joints Reduces Nociceptor Responses and Pain Sensitivity
Joints contain extracellular fluid that buffers the mechanical force imparted to joint tissues by bodily movement. Degradation of the polysaccharide hyaluronan (HA), a key component of the joint synovial fluid and extracellular matrix, has been shown to increase osteoarthritis pain in animal and human studies, but the mechanism underlying this effect has remained unclear. New research from the labs of Elvira De la Peña and Carlos Belmonte, Institute of Neurosciences UMH-CSIC, Alicante, Spain, reveals that intact, healthy HA modulates opening of the transient receptor potential vanilloid type 1 (TRPV1) ion channel on nociceptors to reduce pain in rodents. Along with providing a better understanding of the pathogenesis of osteoarthritis pain, the findings suggest that TRPV1 could be targeted for treatment of this common condition.
“These findings should, and I hope will, provoke a program of research that will seek to exploit HA’s ability to decrease TRPV1-mediated sensitization of primary afferent sensory fibers and provide analgesia,” wrote Gareth Hathway, University of Nottingham, UK, in an email to PRF.
The study was published online August 27 in Nature Communications.
HA modulates TRPV1
Establishing HA’s role in osteoarthritis pain began with the observation that HA is localized to joints. “The presence of intact, high-molecular-weight HA in the synovial fluid of joints has been known for almost one century,” wrote De la Peña and Belmonte in an email to PRF. Based on that knowledge, Endre Balazs, a co-author of the current study who passed away shortly after publication of the new work, pioneered injection of HA into the joint to treat osteoarthritis pain in racehorses. Notably, an analgesic effect of HA has also been observed in humans, as a clinical study of a single injection of the HA derivative hylan G-F 20 into the knee joint was found to relieve osteoarthritic pain for over half a year (Chevalier et al., 2010). Balazs, Belmonte, and colleagues have shown that HA’s analgesic activity likely results from decreased nociceptor activity in joints (Gomis et al., 2004).
Yet precisely how HA calms nociceptor activity was uncertain. One clue came from the study authors’ previous work showing that HA reduced opening of a stretch-activated ion channel in oocytes in vitro (De la Peña et al., 2002). “The dominant idea after these studies was that HA surrounding nociceptive nerve endings in the joint filtered the transmission of movement-evoked mechanical forces to the nerve endings, thereby decreasing their mechanical stimulation,” wrote De la Peña and Belmonte. However, in animal studies, differences in electrophysiological responses to HA observed in inflamed versus normal knee joints suggested another important molecular mediator of joint pain was at work (Gomis et al., 2004). The authors suspected it was TRPV1, since inflammation increases membrane expression of the channel, which contributes to heat hyperalgesia in animals (Ji et al., 2002; Davis et al., 2000).
Lead author Rebeca Caires and colleagues tested for a potential interaction between intact HA and TRPV1 by observing responses to heat and capsaicin, two known activators of TRPV1, in human embryonic kidney (HEK) cells expressing enhanced yellow fluorescent protein (EYFP)-tagged TRPV1 or in cultured mouse dorsal root ganglion (DRG) neurons expressing native TRPV1 channels. They measured responses using calcium imaging while applying a control or HA-containing solution to the cells. In both cell types, intracellular calcium transients evoked by noxious heat or capsaicin were decreased with the HA-containing solution, indicating that HA reduced calcium influx via putative effects on TRPV1 opening. Importantly, heat responses in DRG neurons from TRPV1-null mice were not as strongly reduced as in wild-type animals, suggesting that HA indeed acted through TRPV1. To further test that idea, the authors also performed whole-cell voltage-clamp recordings in TRPV1-EYFP-expressing HEK cells probed with capsaicin. Here, the HA-containing solution reduced TRPV1-mediated currents. By examining the current over time, the investigators discovered that HA slowed TRPV1 activation.
By what mechanism did HA affect TRPV1 activation? Analysis of single-channel kinetics showed that HA selectively lengthened the duration that the channel was closed, with no effect on its open state. Thus, HA seemed to decrease TRPV1 activation by reducing the channel’s open probability.
From TRPV1 to pain
The authors next turned to how inhibition of TRPV1 opening by HA affected action potential firing and ultimately pain behavior. Using cell-attached recordings from mouse DRG neurons, they found that HA-containing solution reduced capsaicin-evoked firing relative to control solution. To test this effect in vivo, the group recorded action potentials in saphenous nerve filaments of anesthetized rats. Units that responded to knee joint forced rotations also responded to intra-arterial injection of capsaicin close to the joint. However, intra-articular HA injection decreased their responses to capsaicin.
Next, to examine pain behavior, the investigators injected a saline or HA-containing solution into the hind paw of mice and tested the animals’ responses to heat using a hot plate test. The HA-containing solution increased the mice’s latency to respond to heat, an effect blocked by TRPV1 knockout. In contrast, injection of the HA-digesting enzyme hyaluronidase decreased response latency; this sensitization to noxious heat was fully reversed with injection of intact HA.
Atomic interactions of hyaluronan tetrasaccharide docked to TRPV1 model. TRPV1 channel model inserted in a lipid bilayer. The proteins are drawn as a ribbon and colored differently for each subunit. The cytosolic C and N-termini have been removed for simplicity. The black square delimits the extracellular loops of TRPV1 and the docked HA. Hyaluronan tetrasaccharide is drawn as sticks and colored in pastel pink. The numbers indicate the sugar rings, starting from the non-reducing end. The loop side chains involved in the interaction are shown as sticks and colored according to the color of their subunit. The dotted lines in red denote hydrogen bonds between atoms closer than 3.2 Å. Image and caption from Caires et al.
Lastly, the authors localized the structural interaction between HA and TRPV1. Because they were unable to isolate the HA-TRPV1 complex, the researchers instead molecularly modeled HA-TRPV1 binding, identifying a tetrasaccharide segment of HA that preferentially bound to a group of positively charged amino acids located in an extracellular loop of TRPV1 apparently involved in the gating of the channel (Cao et al., 2013).
Overall, the new work shows how HA interacts with TRPV1 on nociceptors to protect against aberrant pain sensitivity. Furthermore, “apart from providing a novel mechanistic explanation for the analgesic effects of intra-articularly injected HA, our work shows a hitherto unrecognized mechanism for the control of TRPV1 opening probability,” wrote De la Peña and Belmonte.
However, TRPV1 may not be the only channel involved. Despite showing that HA did not modulate TRPA1 or TRPM8, two other thermosensitive channels expressed by primary sensory neurons, HA still reduced the response of DRG neurons to heat by 26 percent in TRPV1 knockouts. “The results do suggest that HA has targets other than TRPV1. Whether these are other TRPV family members or other thermosensitive channels is still to be determined,” commented Hathway. “The identification of the characteristics of the HA binding site on TRPV1 should provide valuable information to aid in a search for these other potential targets of HA,” he added.
The new findings also have important implications for analgesic drug discovery efforts. “A corollary of this study is that TRPV1 constitutes a good target for osteoarthritic pain treatment,” De la Peña and Belmonte told PRF.
The authors of the study wish to express their gratitude to the late Endre Balazs for his encouragement and support across many years of collaboration and friendship.
Matthew Soleiman is a neuroscientist-turned-science writer currently residing in Nashville, Tennessee. Follow him on Twitter @MatthewSoleiman
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