Divergent responses of chondrocytes and endothelial cells to shear stress: cross-talk among COX-2, the phase 2 response, and apoptosis

Abstract

Fluid shear exerts anti-inflammatory and anti-apoptotic effects on endothelial cells by inducing the coordinated expression of phase 2 detoxifying and antioxidant genes. In contrast, high shear is pro-apoptotic in chondrocytes and promotes matrix degradation and cartilage destruction. We have analyzed the mechanisms regulating shear-mediated chondrocyte apoptosis by cDNA microarray technology and bioinformatics. We demonstrate that shear-induced cyclooxygenase (COX)-2 suppresses phosphatidylinositol 3-kinase (PI3-K) activity, which represses antioxidant response element (ARE)/NF-E2 related factor 2 (Nrf2)-mediated transcriptional response in human chondrocytes. The resultant decrease in antioxidant capacity of sheared chondrocytes contributes to their apoptosis. Phase 2 inducers, and to a lesser extent COX-2-selective inhibitors, negate the shear-mediated suppression of ARE-driven phase 2 activity and apoptosis. The abrogation of shear-induced COX-2 expression by PI3-K activity and/or stimulation of the Nrf2/ARE pathway suggests the existence of PI3-K/Nrf2/ARE negative feedback loops that potentially interfere with c-Jun N-terminal kinase 2 activity upstream of COX-2. Reconstructing the signaling network regulating shear-induced chondrocyte apoptosis may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic strategies for arthritic disorders.

Fig. 1.

Phenotype-specific effects of shear stress duration and intensity on phase 2 response. HUVECs and T/C28a2 chondrocytic cells were subjected to either static conditions or laminar shear flow (5, 20, or 40 dyn/cm²) for 24 or 48 h, and NQO1 specific activities (A) and total glutathione levels (per mg protein) (B) were determined. Data are relative to static controls. Bars are mean ± SEM (n = 4-7, *, P < 0.01, and §, P < 0.05, with respect to static controls). (C) ARE-driven NQO1 promoter activity in response to shear stress stimulation and phase 2 inducers. (Left) T/C28a2 cells were transfected with pNQO1/ARE-luc vector and exposed to either static conditions or laminar shear flow (5 or 20 dyn/cm²) for 24 or 48 h.(Right) To determine the efficacy of phase 2 inducers, transfected cultures were treated with solvent (0.1%), SFN (1.25 μM), or D3T (5 μM) for 24 h under static conditions. ARE-driven firefly luciferase activity was normalized to Renilla luciferase and GFP expression. Data are relative to static controls (n = 4, *, P < 0.01, and §, P < 0.05, with respect to the static control)

Fig. 2.

Effects of phase 2 induction on shear-dependent phase 2 response in chondrocytes. Cells were treated with DMSO (0.1%), SFN (1.25 μM), or D3T (5μM) for 24 h, subjected to either static conditions or laminar shear flow (20 or 40 dyn/cm²) for 24 or 48 h in the presence of the agent, and NQO1 enzyme activity (A) and GSH levels (B) were determined. Data are relative to static controls (n = 3-9, *, P < 0.01 with respect to shear stress-paired solvent-treated controls)

Fig. 3.

Effects of the phase 2-inducer D3T on COX-2 protein levels in shear-activated chondrocytes. Cells, treated with either solvent (0.1% DMSO) or D3T (5 μM) were subjected to either static or laminar shear (5 or 20 dyn/cm²) for 48 h in the presence of the agent. Fluorescence intensity is proportional to COX-2 expression. COX-1 expression remains unchanged (n = 3)

Fig. 4.

Effects of phase 2 induction on shear-induced COX-2-dependent prostaglandin E₂ (PGE₂) production in chondrocytes. Cells were treated with either solvent (0.1% DMSO) or D3T (5 μM) or transfected with pCMV-null or pCMV-mNrf2 (24 h), and then exposed to fluid shear (48 h) in the presence of the agent. PGE₂ levels were determined in culture media at the indicated times. Data are relative to paired static controls at t = 0 (n = 4, transfection efficiency = 32.8 ± 4.5%)

Fig. 5.

Effects of inhibition of COX-2 activity on shear-dependent phase 2 response in chondrocytes. Cells were treated with CAY10404 (6.75 μM) or control solvent (0.1% DMSO) for 2 h, exposed to static conditions or laminar shear flow (20 dyn/cm²) for 48 h in the presence of agent, and NQO1 enzyme activities (A) and total GSH levels (B) were determined. Data obtained with NS398 (30 μM) were indistinguishable from those with CAY10404. Data are relative to static controls (n = 3-9, *, P < 0.01 with respect to static controls)

Fig. 6.

Effects of phase 2 inducers and COX-2 inhibitors on shear-mediated DNA fragmentation, mitochondrial membrane permeabilization, and caspase-9 protein levels. T/C-28a2 cells were treated with the solvent (0.1% DMSO) or D3T (5 μM), for 24 h or solvent CAY10404 (6.75 μM) or NS398 (30 μM) for 2 h, and then exposed to either static or laminar flow (20 dyn/cm²) for 48 h in the presence of the agent. Cells were examined for markers of apoptosis by using DNA fragmentation (TUNEL, A), mitochondrial membrane potential (MMP, B), and caspase-9 expression (C)

Fig. 7.

Effects of PI3-K activity on shear-dependent phase 2 response in chondrocytes. Cells were transfected with pBJ M·p110^* (constitutively active PI3-K), pBJ M·p110·UR (Δkinase mutant), or pBJ-null vector, subjected to static conditions or laminar flow (20 dyn/cm²) for 48 h, and NQO1 enzyme activity (A) and total GSH levels (B) were determined. Data are relative to null-transfected static cultures (n = 4, *, P < 0.01 with respect to static controls; ‡, P < 0.05 with respect to null-transfected shear)

Fig. 8.

In chondrocytes, high shear flow (20 dyn/cm²) induces COX-2 expression through a JNK2/c-jun-dependent pathway, leading to PGE₂ accumulation and suppression of PI3-K activity and Nrf2/ARE-mediated phase 2 response. The resultant decrease in antioxidant capacity permits disruption of mitochondrial integrity, caspase-9 activation, and apoptosis. Phase 2 inducer, PI3-K activity, and COX-2 inhibitors restore phase 2 activity. Moreover, activation of PI3-K and/or the Nrf2/ARE pathway abrogates COX-2 expression and apoptosis through negative feedback, potentially by suppressing upstream JNK2/c-jun signaling