Extracellular Matrix Stiffness as a Key Molecular Event in Early Osteoarthritis: Crosstalk Between the RhoA/ROCK and...

Area of Science:

• Biomedical Engineering
• Cell Biology
• Biochemistry

Background:

• Osteoarthritis (OA) is characterized by progressive cartilage degradation, where the extracellular matrix (ECM) plays a crucial role in regulating chondrocyte behavior.
• ECM stiffening, resulting from aging and joint injuries, contributes significantly to cartilage damage in OA.
• While RhoA/ROCK and ERK1/2 signaling pathways are implicated in cartilage remodeling, their interaction under varying ECM stiffness conditions remains poorly understood.

Purpose of the Study:

• To elucidate how ECM stiffness influences chondrocyte catabolic and anabolic activity, leading to cartilage degeneration.
• To investigate the crosstalk between RhoA/ROCK and ERK1/2 signaling pathways in response to ECM stiffness.
• To explore the role of SPRY-4 as a modulator in this stiffness-dependent signaling cascade.

Main Methods:

• Chondrocytes were cultured on substrates with controlled stiffness (8, 12, and 25 kPa) to assess viability, gene expression (MMP-13, ADAMTS-5, COL2A1, ACAN), and signaling activation (p-ROCK, p-ERK1/2) via Western blotting and qPCR.
• In vivo studies utilized an Ad-LOX induced cartilage stiffening model in rabbits, with cartilage elastic modulus measured by atomic force microscopy.
• RhoA/ROCK-ERK1/2 crosstalk was examined through SPRY-4 modulation (overexpression and knockdown) and co-immunoprecipitation.

Main Results:

• Increased substrate stiffness (25 kPa) significantly reduced chondrocyte viability and promoted a catabolic phenotype, evidenced by increased MMP-13 and ADAMTS-5 expression and decreased COL2A1 and ACAN expression.
• Stiffness significantly upregulated RhoA/ROCK and ERK1/2 signaling pathways (p-ROCK and p-ERK1/2).
• Pharmacological inhibition of RhoA/ROCK or ERK1/2 reversed the stiffness-induced catabolic shift, with RhoA/ROCK identified as upstream of ERK1/2.
• SPRY-4 overexpression attenuated stiffness-induced p-ROCK and p-ERK1/2 activation, while SPRY-4 knockdown restored p-ERK1/2 levels, confirming SPRY-4 as a key stiffness-responsive modulator.
• In vivo, Ad-LOX induced cartilage stiffening and elevated p-ROCK and p-ERK1/2 levels, corroborating in vitro findings.

Conclusions:

• ECM stiffening triggers a catabolic shift in chondrocytes by activating the RhoA/ROCK-ERK1/2 signaling cascade.
• SPRY-4 acts as a critical stiffness-responsive modulator, mediating crosstalk between RhoA/ROCK and ERK1/2 signaling.
• These findings highlight the RhoA/ROCK-ERK1/2-SPRY-4 axis as a potential mechanosensitive target for early OA diagnosis and intervention.