Background:

Osteoarthritis (OA) involves progressive cartilage degradation, and the extracellular matrix (ECM) regulates chondrocyte behavior. In OA, ECM stiffening, driven by age-related matrix changes and mechanical joint injuries, contributes to cartilage damage. Although RhoA/ROCK and ERK1/2 signaling participate in cartilage remodeling, how these pathways interact across ECM stiffness conditions remains unclear.

Purpose:

To clarify how ECM stiffness modulates chondrocyte catabolic/anabolic activity and cartilage degeneration by investigating crosstalk between the RhoA/ROCK and ERK1/2 pathways in vitro and in vivo.

Study Design:

Controlled laboratory study.

Methods:

Chondrocytes were cultured under control conditions or on substrates with varying stiffness values (8, 12, and 25 kPa) and analyzed for viability, gene expression, and signaling activation. Stiffness effects on RhoA/ROCK and ERK1/2 were assessed by Western blotting, quantitative polymerase chain reaction, and immunofluorescence. In vivo, cartilage stiffening was induced by Ad-LOX in rabbits. RhoA/ROCK–ERK1/2 crosstalk was examined via SPRY-4 modulation and co-immunoprecipitation.

Results:

At 25 kPa versus the gel-free control, CCK-8 assay revealed that chondrocyte viability was 0.5905 ± 0.0015 versus 1.0000 ± 0.0111 (P < .0001). Quantitative polymerase chain reaction showed that MMP-13 gene expression was 0.0200 ± 0.0015 versus 0.0109 ± 0.0017 (P = .0003), respectively, and ADAMTS-5 gene expression was 0.0439 ± 0.0033 versus 0.0100 ± 0.0012 (P < .0001), respectively, with the gene expression of COL2A1 and ACAN reduced to 0.0146 ± 0.0008 versus 0.0151 ± 0.0013 (P < .0001), respectively, and 0.0347 ± 0.0041 versus 0.0353 ± 0.0041 (P < .0001), respectively. Western blotting demonstrated that signaling proteins increased under stiffness for p-ROCK and p-ERK1/2: 0.9473 ± 0.1434 versus 0.6788 ± 0.0407 (P = .004), respectively, and 1.1341 ± 0.0542 versus 0.5728 ± 0.0522 (P = .0003), respectively. The pharmacological inhibition of RhoA/ROCK or ERK1/2 reversed the stiffness-induced catabolic shift, and perturbation analysis indicated that RhoA/ROCK lay upstream of ERK1/2 under stiff conditions. SPRY-4 overexpression attenuated stiffness-induced p-ROCK (0.9899 ± 0.0876 vs 0.6312 ± 0.0865, respectively; P = .002) and p-ERK1/2 (1.0780 ± 0.0802 vs 0.3849 ± 0.0745, respectively; P < .0001) activation, whereas SPRY-4 knockdown restored p-ERK1/2 (0.5061 ± 0.0409 vs 0.8108 ± 0.0555, respectively; P = .0019) levels. These results confirm SPRY-4 as a key modulator of ERK1/2 signaling under stiff conditions, mediating crosstalk between the RhoA/ROCK and ERK1/2 pathways. In vivo, atomic force microscopy showed that intra-articular Ad-LOX increased the cartilage elastic modulus to 22.7167 ± 0.6298 versus 18.0600 ± 0.3780 kPa (P = .0004), respectively, and elevated p-ROCK and p-ERK1/2 to 0.9710 ± 0.0077 versus 0.4480 ± 0.0237 kPa (P = .0001), respectively, and 0.9496 ± 0.0879 versus 0.5488 ± 0.0455 kPa (P = .0001), respectively, corroborating the in vitro findings.

Conclusion:

ECM stiffening activates the RhoA/ROCK to ERK1/2 cascade, and SPRY-4 acts as a stiffness-responsive ERK1/2 modulator. These mechanistic insights are pertinent to early OA.

Clinical Relevance:

ECM stiffening acts as an early mechanobiological trigger in OA by promoting a catabolic shift in chondrocytes through activation of a RhoA/ROCK-ERK1/2 signaling cascade. This stiffness-dependent pathway crosstalk is mediated by SPRY-4, positioning SPRY-4 and the RhoA/ROCK-ERK1/2 axis as potential mechanosensitive targets for early OA diagnosis and intervention.