Independent regulation of short and long forms of latent TGF-beta binding protein (LTBP)-4 in cultured fibroblasts and human tissues.

Transforming growth factor (TGF)-beta is secreted and targeted into the extracellular matrix (ECM) in association with one of the latent TGF-beta binding proteins (LTBPs). Activation of these latent complexes is an important regulatory step in TGF-beta signaling. LTBPs target the growth factor into the ECM and expose it to activating mechanisms. Disruption of LTBP-4 gene causes severe developmental abnormalities in both humans and mice. Transcripts for two N-terminally distinct LTBP-4 variants, LTBP-4S (short) and -4L (long), have been identified. In the current work, we have characterized differences in the expression, processing, and ECM targeting of these LTBP-4 variants. Heart and skeletal muscle displayed expression of both variants, while liver expressed mainly LTBP-4L and lung as well as small intestine LTBP-4S. This tissue-specific expression pattern was found to originate from control of transcription by two independent promoters. Furthermore, LTBP-4S and -4L proteins were secreted and processed differently. During secretion, LTBP-4L was complexed with TGF-beta1, whereas the majority of LTBP-4S was secreted in a free form. In addition, LTBP-4S was incorporated into the ECM, while full-length LTBP-4L was not readily detectable in the ECM. These data suggest that LTBP-4 functions are modified by tissue-specific expression of the two N-terminally distinct variants, which in addition exhibit significant differences in cellular processing and targeting, that is, this provides a basis for understanding molecular diversity in LTBP-4 structure and function.

Disruption of LTBP-4 function reduces TGF-beta activation and enhances BMP-4 signaling in the lung.

Disruption of latent TGF-beta binding protein (LTBP)-4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-beta, whereas secretion of latent TGF-beta was significantly increased. Expression and secretion of TGF-beta2 and -beta3 increased considerably. These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the -/- fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4-mediated targeting and activation of TGF-beta1 leads to enhanced BMP-4 signaling in mouse lung.

Fibronectin and heparin binding domains of latent TGF-beta binding protein (LTBP)-4 mediate matrix targeting and cell adhesion.

Latent transforming growth factor (TGF)-beta binding proteins are extracellular matrix (ECM) proteins involved in the regulation of TGF-beta sequestration and activation. In this study, we have identified binding domains in LTBP-4, which mediate matrix targeting and cell adhesion. LTBP-4 was found to possess heparin binding activity, especially in its N-terminal region. The C-terminal domain of LTBP-4 supported fibroblast adhesion, a property reduced by soluble heparin. In addition, we found that LTBP-4 binds directly to fibronectin (FN), which was indispensable for the matrix assembly of LTBP-4. The FN binding sites were also located in the N-terminal region. Interestingly, heparin was able to reduce the binding of LTBP-4 to FN. In fibroblast cultures, LTBP-4 colocalized first with FN and subsequently with fibrillin-1, pointing to a role for FN in the early assembly of LTBP-4. In FN -/- fibroblasts, LTBP-mediated ECM targeting was disturbed, resulting in increased TGF-beta activity. These results revealed new molecular interactions which are evidently important for the ECM targeting, but which also are evidence of novel functions for LTBP-4 as an adhesion molecule.

Induction of human LTBP-3 promoter activity by TGF-beta1 is mediated by Smad3/4 and AP-1 binding elements.

Latent TGF-beta binding proteins (LTBPs) are extracellular matrix glycoproteins, which are essential for the targeting and activation of TGF-betas. LTBP-3 regulates the bioavailability of TGF-beta especially in the bone. To understand the regulation of LTBP-3 expression, we have isolated and characterized the promoter region of human LTBP-3 gene. The GC-rich TATA-less promoter contained several transcription initiation sites and putative binding sites for multiple sequence specific transcription factors including Sp1, AP-1, c-Ets, MZF-1, Runx1 and members of the GATA-family. Reporter gene analyses of the promoter indicated that it was more active in MG-63 than in Saos-2 osteosarcoma cells, suggesting that it is regulated as the endogenous gene. TGF-beta1 stimulated the transcriptional activity of LTBP-3 promoter in MG-63 cells, while certain other bone-derived growth factors and hormones were ineffective. TGF-beta1 increased LTBP-3 mRNA levels accordingly. Analyses of deletion constructs of the promoter and mutational deletion of specific transcription factor binding sites indicated that Smad3/4 and AP-1 binding sites mediated the TGF-beta1 response. The involvement of AP-1 activity was further indicated by decreased TGF-beta responsiveness of the LTBP-3 promoter in the presence of a MEK/Erk signaling pathway inhibitor. Our results suggest an important new role for TGF-beta1 in the regulation of its binding protein, LTBP-3.