Background/Purpose:

Cleavage of aggrecan in the interglobular domain (E³⁷³–³⁷⁴A) by ADAMTS-4/5 is an early event in osteoarthritis pathogenesis. Further cleavage by MMPs (N³⁴¹–³⁴²F) releases a 32-amino-acid fragment, FFGVGGEDDITIQTVTWPDLELPLPRNVTEGE. We recently reported that this 32-mer acts as a TLR2-dependent damage-associated molecular pattern on chondrocytes and synovial fibroblasts. Here, we hypothesized that this aggrecan fragment may directly excite nociceptors.

Methods:

DRG cells (L3-L5) were isolated from adult C57BL/6 wild-type, Tlr4-null, or Tlr2-null mice and cultured. Direct effects of synthetic 32-mer on DRG neurons were monitored by examining intracellular calcium (Ca)i mobilization or production of  MCP-1. For Ca mobilization assays, cells were loaded with Fura-2 and responses to 32-mer or scrambled peptide recorded in >100 neurons. For MCP-1 stimulation assays, cells were treated overnight with 32-mer (0.3-30 μM) or with scrambled control. Supernatants were collected for MCP-1 ELISA, because we have previously shown that MCP-1 is a key mediator of pain in experimental OA.

For ex vivo imaging assays, intact DRG were isolated from Pirt-GCaMP3 mice, which express the fluorescent calcium indicator, GCaMP3, in 90% of sensory neurons through the Pirt promoter. Explants, placed in a perfusion chamber and imaged using a spinning disk confocal microscope, were stimulated by injecting 10 µL of 1 mM 32-mer into a continuously running perfusion chamber with a 1 mL-volume. ImageJ analysis was performed to determine change in fluorescence intensity with time.

Results:

Cultured DRG neurons rapidly responded to 32-mer, but not scrambled control peptide, as indicated by increased (Ca)i in 20% of neurons. This suggests that DRG neurons express excitatory receptors for this protein fragment. Responses were mostly seen in small-to-medium-diameter neurons (nociceptors) that were also responsive to capsaicin, which demonstrates that TRPV1-expressing nociceptors are capable of responding to 32-mer. In order to show that 32-mer responses are not an artifact of cell culture, calcium imaging was also performed using intact DRG from Pirt-GCaMP3 mice. Within DRG explants, 7% of neurons responded to 32-mer peptide, while scrambled peptide elicited no responses.

Overnight stimulation of cultured DRG cells with either 3 or 30 μM 32-mer peptide resulted in significant upregulation in MCP-1 protein production compared to unstimulated cells (3.1-fold (3 μM) and 3.5-fold (30 μM), p<0.001). The highest concentration of scrambled peptide (30 μM) did not induce MCP-1 production (0.8-fold, p=0.09 vs. unstimulated).

In order to investigate which receptor mediates the upregulation of MCP-1, DRG cells were cultured from Tlr2 null or Tlr4 null mice. Stimulation with 32-mer peptide (3 μM) produced increased MCP-1 in Tlr4 null DRG cells compared to unstimulated cells (3.3-fold, p<0.01), but not in Tlr2null cells (0.9-fold, p=0.5), suggesting that these effects are mediated through TLR2.

Conclusion:

Nociceptors can respond to a specific 32-mer cleavage product of aggrecan through TLR2. This pathway may contribute to the development of OA-associated pain, a hypothesis currently being tested in in vivo models.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-32-mer-aggrecan-fragment-generated-through-adamts-45-and-mmp-mediated-cleavage-can-directly-excite-nociceptive-neurons/