Lysophospholipid regulates release and activation of latent TGF-β1 from chondrocyte extracellular matrix

Transforming growth factor beta-1 (TGF-β1) is released from the extracellular matrix of rat growth plate chondrocytes and activated by stromelysin-1 (matrix metalloproteinase 3, MMP-3), an enzyme that is stored in matrix vesicles. MMP-3 is released from these extracellular organelles by the direct action of 1α,25(OH)₂D₃ via activation of phospholipase A₂ (PLA₂), resulting in local production of lysophospholipids and matrix vesicle membrane destabilization. This effect of 1α,25(OH)₂D₃ is greater in matrix vesicles from growth zone chondrocyte cultures and PLA₂ activity is higher in the growth zone in vivo, suggesting that it may depend on chondrocyte maturation state in the endochondral lineage. Previous studies have shown that latent TGF-β1 can be activated by mild detergents in vitro, suggesting that lysophospholipids may act in vivo in a similar manner. To test this hypothesis, we determined if rat costochondral growth plate cartilage cells produce lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) in a maturation state-dependent manner and if LPC or LPE could release and activate latent TGF-β1 from the extracellular matrix produced by these cells. Rat growth plate chondrocytes produced both lysophospholipids, with growth zone cells producing higher levels of LPE via PLA₁, and resting zone cells producing higher levels of LPC via PLA₂. LPC and LPE directly increased activation of recombinant human latent TGF-β1 in a biphasic manner with a peak at 2 μg/ml. Phosphatidylcholine, phosphatidylethanolamine, and LPE plasmalogen (LPEP), but not choline, also activated TGF-β1. Latent TGF-β1 incubated with LPC or LPE, but neither lysophospholipid alone, stimulated [³H]-thymidine incorporation of resting zone cells, indicating the TGF-β1 released was biologically active. LPC and LPE also released TGF-β1 in a dose- and time-dependent manner when incubated with cell-free extracellular matrices produced by the cells. These results indicate that LPC and LPE have important roles as regulators of rat growth plate chondrocytes by directly and indirectly activating TGF-β1 stored in the extracellular matrix.