Transglutaminase-mediated fibronectin multimerization in lung endothelial matrix in response to TNF-alpha

Exposure of lung endothelial monolayers to tumor necrosis factor (TNF)-alph a causes a rearrangement of the fibrillar fibronectin (FN) extracellular ma trix and an increase in protein permeability. Using calf pulmonary artery e ndothelial cell layers, we determined whether these changes were mediated b y FN multimerization due to enhanced transglutaminase activity after TNF-al pha (200 U/ml) for 18 h. Western blot analysis indicated that TNF-alpha dec reased the amount of monomeric FN detected under reducing conditions. Analy sis of I-125-FN incorporation into the extracellular matrix confirmed a two fold increase in high molecular mass (HMW) FN multimers stable under reduci ng conditions (P< 0.05). Enhanced formation of such HMW FN multimers was as sociated with increased cell surface transglutaminase activity (P< 0.05). C alf pulmonary artery endothelial cells pretreated with TNF-alpha also forme d nonreducible HMW multimers of FN when layered on surfaces precoated with FN. Inhibitors of transglutaminase blocked the TNF-alpha-induced formation of nonreducible HMW multimers of FN but did not prevent either disruption o f the FN matrix or the increase in monolayer permeability. Thus increased c ell surface transglutaminase after TNF-alpha exposure initiates the enhance d formation of nonreducible HMW FN multimers but did not cause either the d isruption of the FN matrix or the increase in endothelial monolayer permeab ility.