Sequential Deposition of Latent TGF-beta Binding Proteins (LTBPs) During Formation of the Extracellular Matrix in Human Lung Fibroblasts

Latent TGF-beta binding proteins (LTBPs) mediate the targeting of latent TGF-beta complexes into ECM structures, which is important for TGF-beta activation and functions. LTBPs-1, -3 and -4 associate with and regulate the bioavailability of TGF-betas. We investigated whether LTBP-3 and -4 are associated with pericellular fibrillar structures of human lung fibroblast ECM, and which of their domains are important for this function. Immunoblotting analyses of isolated insoluble matrices as well as immunofluorescence analyses and confocal microscopy indicated that both LTBP-3 and -4 get assembled into the ECM. Interestingly, LTBP-4 was not detected until 7-10 days of culture and LTBP-3 until 14 days of culture. This was a major difference from the deposition kinetics of LTBP-1, which was detected already within 2 days of culture. Expression analyses by real time RT-PCR indicated that the slow appearance of LTBP-3 and -4 was due to the low expression levels soon after subculture. Recombinant N-terminal fragments of LTBP-3 and -4 bound readily to fibroblast ECM. The C-terminal domain of LTBP-4, but not of LTBP-3, also associated with the matrix structures. The levels of ECM-associated latent complexes of TGF-beta1 increased in parallel with the increased production and deposition of the LTBPs. The amount of active TGF-beta in the conditioned medium decreased during extended culture. Our results suggest that ECM is an important site of deposition also for LTBP-3 and -4 and that the temporal and spatial targeting of the TGF-beta complexes are associated with ECM maturation.