Human Mast Cell Chymase and Leukocyte Elastase Release Latent Transforming Growth Factor-β1 from the Extracellular Matrix of Cultured Human Epithelial and Endothelial Cells

Abstract

Monolayer cultures of human epithelial and endothelial cells were used to study the association of latent transforming growth factor-beta 1 (TGF-beta 1) to extracellular matrices and its release and activation during matrix degradation. Human umbilical vein endothelial cells and embryonic lung fibroblasts produced relatively high levels of TGF-beta 1, its propeptide (beta 1-latency-associated protein), and latent TGF-beta-binding protein and incorporated latent TGF-beta 1 into their matrices as shown by immunoblotting. Amnion epithelial cells produced lower levels of these proteins. Confluent cultures of epithelial cells were exposed to matrix-degrading proteases and glycosidases. Mast cell chymase, leukocyte elastase, and plasmin efficiently released matrix-bound latent TGF-beta 1 complexes, while chondroitinase ABC and heparitinases were ineffective. The ability of the proteases to activate recombinant latent TGF-beta 1 was tested using growth inhibition assays and a novel sodium deoxycholate-polyacrylamide gel electrophoresis followed by immunoblotting. Sodium deoxycholate solubilized M(r) 25,000 TGF-beta 1 but did not dissociate high M(r) latent TGF-beta 1 complexes, allowing separation of these forms by polyacrylamide gel electrophoresis. Mast cell chymase and leukocyte elastase did not activate latent TGF-beta 1, suggesting that its release from matrix and activation are controlled by different mechanisms. The release of TGF-beta from the matrix by leukocyte and mast cell enzymes may contribute to the accumulation of connective tissue in inflammation.

Apoptosis of human endothelial cells is accompanied by proteolytic processing of latent TGF-β binding proteins and activation of TGF-β

“…medium') was able to induce the release the LTBP proteins from the ECM preparations. Interestingly, essentially a similar pattern of LTBP-1 fragments was released from the ECM by conditioned medium from apoptotic cells as by plasmin (Figure 4b, see also Taipale et al 24 ). The results suggest that certain components of the conditioned medium from apoptotic cells are able to induce proteolytic processing of ECM-associated LL-TGF-b, evidently at the protease sensitive hinge region, in a manner similar to that induced by plasmin.…”

“…Current data suggest that proteolytic release of ECM-bound LTBPs may represent a major mechanism for the activation of TGF-b under physiological conditions. 24,46 Our data are further suggestive of two sources for activation of TGF-b during apoptosis of HUVECs: the secretion of intracellular TGF-b from apoptotic cells and the proteolytic release of stored LL-TGF-b from the ECM. Both of these sources appear to be utilized during apoptosis of HUVECs, as in the anoikis model of apoptosis the activation of TGF-b can be observed despite the lack of endogenous ECM ( Figure 5).…”

“…In addition, apoptotic HUVECs produced protease-like activity capable of releasing truncated LTBPs from isolated ECM in a manner resembling that of plasmin (Figure 4), mast cell chymase or leukocyte elastase. 24 Our model of HUVEC anoikis, induced either with or without endogenous ECM (Figure 8), further illustrates the contribution of both ECM-and cell-derived LL-TGF-b in the activation of TGF-b during apoptosis.…”

“…The results suggest that certain components of the conditioned medium from apoptotic cells are able to induce proteolytic processing of ECM-associated LL-TGF-b, evidently at the protease sensitive hinge region, in a manner similar to that induced by plasmin. 14,24 Analysis of the cell lysates revealed that the total amount of b1-LAP protein, either free or in complex with LTBPs (LL-TGF-b1), was substantially increased in staurosporine-treated HUVECs (Figure 4c). These results suggest that apoptotic cells per se can be the source of TGF-b activation during apoptosis.…”

Expressão e caracterização das quimases recombinantes específicas de mastócitos de camundongos (mMCP4 e 5)

“…Existem indícios de uma atividade mitogênica da quimase sobre fibroblastos da artéria pulmonar bovina e fibroblastos da cápsula de Tenon de cães (PEMBERTON et al, 1997;MARUICHI et al, 2004). Por outro lado, a quimase pode liberar o TGF-β1 que está ligado à matriz extracelular (TAIPALE et al, 1995) e ativar o TGF-β1 latente da matriz extracelular (LINDSTEDT et al, 2001), o que pode explicar a contribuição da quimase na fibrose.…”