Figure 5

Decreased LKB1 and AMPKα phosphorylation and increased apoptosis and catabolic responses after biomechanical injury. Loss of phosphorylation of liver kinase B1 (LKB1) and AMP-activated protein kinase alpha (AMPKα), in addition to increased caspase-3 activation, nitric oxide (NO) release, and catabolic activities in bovine chondrocytes after biomechanical injury. Bovine knee articular chondrocytes were embedded in alginate disks and cultured for 3 to 4 weeks, followed by the sub-lethal biomechanical injury condition described in Materials and methods. The chondrocyte-alginate constructs were collected at 0, 1, 2 and 5 days after injury. (A) Chondrocytes from both injured and control groups were then released by dissolution of alginate, and subjected to western blot analysis for expression of cleaved caspase-3, phosphorylation of LKB1 and AMPKα, and total AMPKα. Conditioned media were employed for analysis of (B) NO and (C) glucosaminoglycan (GAG) release by Griess reaction and DMMB assay, respectively, and (E) matrix metalloproteinase (MMP)-3 release by western blot analysis. (D) MMP-3 mRNA expression was also examined by quantitative RT-PCR. Data representative of three individual experiments (n = 3 replicates for each condition). **P <0.001, *P <0.01, #P <0.05 relative to the control.