TGF-β is a potent inducer of nerve growth factor in articular cartilage via the ALK5-Smad2/3 pathway. potential role in OA related pain?

Abstract

Purpose: Pain is the main problem for patients with OA. Unfortunately, pain relief is not effective in every individual. In general, pain is linked to inflammation. However, in a subset of OA patients pain is experienced without obvious inflammation driving the pain. This indicates there must be an additional alternative source of pain besides inflammation. NGF inhibition has been shown very efficient in blocking pain during OA, but the exact source of NGF is unclear. NGF is elevated in human OA,as well as in animal models of OA with cartilage damage without inflammation.TGF-beta is released from cartilage upon damage. We investigated whether TGF-beta could contribute to NGF expression in the joint. Furthermore, we studied the specific Smad-related TGF-beta pathways involved in this process. Methods: Murine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-β1 and/or IL-1β as a positive control for NGF induction. We analyzed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1 (oxozeaenol), Smad2/3 (SB-505124) or Smad1/5/8 (LDN-193189) signaling to identify the TGF-β pathway inducing NGF. Results: We found that TGF-beta was capable of inducing NGF expression, even in low dosage of 1 ng/ml. NGF expression was consistently induced at higher levels by TGF-β than IL-1 in all of our experiments: murine, bovine and human origin, in cell lines, primary chondrocytes and explants cultures. Combining TGF-beta with IL-1 did not result in higher levels of NGF expression than those already reached by a single dose of TGF-beta alone. As TAK1 is a common pathway component of both IL-1 and TGF-beta signaling, we initially expected TAK1 to be dominant and pivotal in NGF induction. TAK1 inhibition consistently reduced (but not blocked) TGF-β-induced NGF whereas it fully blocked IL-1β-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-β-induced NGF expression. Blocking the Smad1/5/8 pathway with LDN did not have an effect on TGF-beta-induced NGF. In human OA primary chondrocytes we found that there was a large variation in basal NGF levels (mRNA and histology). Upon stimulation with TGF-β we found a consistent higher level of NGF induction than upon stimulation with IL-1, which was not related to the basal levels of NGF. In explants of human OA cartilage we observed chondrocytes positive staining for NGF. When stimulating human OA cartilage with TGF-beta NGF expression increased, which could be fully blocked by inhibiting ALK5-Smad2/3 signaling. Conclusions: This is the first time that it is shown that TGF-β induces NGF expression in (primary) chondrocytes. Moreover, we found that TGF-beta consistently induced NGF at higher levels than IL-1. In addition, IL-1 induced NGF could be fully abrogated by TAK1 inhibition, but this only had a reducing effect on TGF-beta induced NGF expression. In contrast blocking signaling via ALK5-Smad2/3 fully abrogated the TGF-beta-induced NGF expression. Clearly, our data show that there are sources of NGF other than inflammation in the joint and that this should be taken into account when studying pain in OA.