Role of the latent transforming growth factor beta binding protein 1 in fibrillin-containing microfibrils in bone cells in vitro and in vivo

Latent transforming growth factor beta-binding proteins (LTBPs) are extrace llular matrix (ECM) proteins that bind latent transforming growth factor be ta (TGF-beta) and influence its availability in bone and other connective t issues, LTBPs have homology,vith fibrillins and may have related functions as microfibrillar proteins. However at present little is known about their structural arrangement in the ECM, By using antibodies against purified LTB P1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 cons tructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibri llar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-mm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early col ocalization of LTBP1 with fibronectin suggested a role for fibronectin in t he initial assembly of LTBP1 into the matrix; however, in more differentiat ed osteoblast cultures, LTBP1 and fibronectin 1 were! found in distinct fib rillar networks. Overexpression of LTBP1 deletion constructs in osteoblast- like cells showed that N-terminal amino acids 67-467 were sufficient for in corporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation, In emb ryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone, However, LTBP1-positive fibrils, which did not contain fibrillin 1, mere present in cartilage matrix, These studie s show that in addition to regulating TGF beta 1, LTBP1 may function as a s tructural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded.