Ten-nm microfibrils were initially described in and around amorphous elastin and were later found to exist independent of elastin as well. These fibers were shown by Sakai et al. to be composed mainly of fibrillin. Fibrillin exists in three isoforms (fibrillin-1, -2, and -3) encoded by separate genes. The ultrastructural appearance of microfibrils suggests they are important structural components of the ECM. However, it is increasingly evident that fibrillin assemblies, through their interaction with LLC and other GFs, perform what Ramirez, Sakai, Rifkin, and Dietz have termed an “instructive” role by presenting GFs to cells in the correct amounts, places and times (Ramirez et al. 2007
; Ramirez and Sakai 2010
Fibrillin microfibrils are string-like structures containing distinct bead-like shapes. The beads are separated by about 50–150 nm depending on the source of the fibril and how it was isolated. Sakai and colleagues, using epitope-specific antibodies, showed that the beads contain amino and carboxyl termini of fibrillins, indicating that the fibrillin molecules are arranged in a head-to-tail manner (Reinhardt et al. 1996
). Disulfide bond formation between fibrillin molecules is required for fibril formation. Also, microfibril formation depends on the presence of fibronectin fibers, which are assembled on cell surfaces in an integrin-dependent manner. However, other details of the process such as the roles of the amino and carboxyl termini in self-assembly and the role of processing by furin-type proteases, remain unsettled. Schemes of organization that could account for the EM observations, involving different types of staggering of fibrillin molecules among themselves and possible folding or “pleating” of the fibrillin molecules, have been proposed, but this issue remains unsettled (Ramirez and Sakai 2010
Fibrillin-1 also is produced by epithelial cells in a nonfibrillar form deposited in the lamina densa of basement membranes (Dzamba et al. 2001
). The epithelial cells in these studies secreted fibrillin-1 into the cell layer, but secreted fibrillin-2 into the medium, indicating that the cells can discriminate between these similar proteins. It is not known to what extent fibrillar and nonfibrillar assemblies of fibrillin-1 differ in their “instructive” functions. Sakai and colleagues make the interesting point that fibrillin functions dependent on polymerized fibrillin may be susceptible to dominant-negative mutations that affect the polymerized structure, as in MFS, but the same mutations may not be dominant-negative for functions related to nonfibrillar fibrillin.
LTBPs associate with fibrillin assemblies, for example in perichondrium and in cultures of osteoblasts (Dallas et al. 2000
; Isogai et al. 2003
). A host of other proteins also associate with fibrillin, e.g., fibulins, microfibril-associated glycoproteins (MAGP-1 and -2), perlecan, versican, and emilin1 (Ono et al. 2009
). Emilin1 modulates extracellular pro-TGF-β processing, as noted above, and is a ligand for integrin α4β1 (Spessotto et al. 2003
). Biglycan and decorin are proteoglycans that inhibit active TGF-β, and they associate with other components of elastic fibers in close proximity to fibrillin and may regulate fibrillin-1 expression (Trask et al. 2000
; Reinboth et al. 2002
; Schaefer et al. 2004
Experiments using multiday cultures of normal and fibrillin-1-null dermal fibroblasts show that LTBP-1 and LTBP-4 incorporation into ECM requires fibrillin-1 (Ono et al. 2009
). Other experiments show that in early cultures, LTBP-1 is colocalized with fibronectin and fibrillin-1, whereas in longer-term cultures LTBP-1 dissociates from fibronectin but remains colocalized with fibrillin-1 (Hyytiainen et al. 2004
). Matrix incorporation of LTBP-2 is also dependent on a fibrillin-1 network (Vehvilainen et al. 2009
The protein–protein interaction sites between LTBPs and fibrillins have been determined using recombinant protein fragments and surface plasmon resonance (SPR) (Ono et al. 2009
). LTBP-4 binds to the first hybrid domain of fibrillin-1 (Hyb1), whereas LTBP-1 binds to a site involving both Hyb1 and adjacent EGF-like domains 2 and 3. Previous studies showed that LTBP-1’s carboxyl terminus binds to fibrillin-1, whereas the amino terminus of LTBPs is mainly responsible for binding ECM made in cell culture, generally, and fibronectin, specifically (Kantola et al. 2008
). Under experimental conditions, competition for binding sites occurs. For example, fibulin-2, -4, and -5 compete with LTBP-1 for binding to fibrillin-1. Also, LTBP-2 (which cannot link to SLC) binds the amino terminus of fibrillin-1 and competes for this binding with LTBP-1. This raises the possibility that the amounts of latent TGF-β associated with microfibrils might be affected by physiological competition with fibulins and LTBP-2.
Proteases (e.g., plasmin, chymase, neutrophil elastase, and MMPs) can cleave LTBP-1 in the “hinge” region near the amino terminus and release LLC from ECM formed by cultured cells. The carboxyl terminus of LTBP-1 also contains a protease-sensitive region (Unsold et al. 2001
). Thus, proteases may be able to release a truncated form of LLC from associations with fibronectin or other components of ECM that bind LTBP’s amino terminus, and from fibrillin-1 interacting at LTBP’s carboxyl terminus. A model of muscular dystrophy suggests proteolytic LLC release may be physiologically important (see below).
TGF-β belongs to a large superfamily of GFs, all with prodomains loosely related to LAP. In mammals, this TGF-β superfamily includes BMPs, activins/inhibins, GDFs, nodal, and myostatin. In general, the prodomains of these superfamily members do not cause latency of the GFs (myostatin is an exception), but many associate noncovalently with their cognate GF after proteolytic processing.
Notably, fibrillin-1 and -2 can bind propeptides of multiple members of the TGF-β superfamily directly (specifically, BMP2, -4, -7, -10, and GDF5, as determined by SPR) (Sengle et al. 2008
). Prodomain binding to fibrillins is noncovalent and occurs near the amino terminus of fibrillin in a region that includes Hyb1 and the third and fourth EGF-like domains. By SPR, the dissociation constants are about 20 nM. It is not reported whether prodomain binding competes with LTBP, or other molecules, for binding to fibrillin.
BMP4 colocalizes with fibrillin-1 in several tissues, suggesting that BMP-fibrillin interactions are physiologically relevant. Nistala et al. analyzed bone formation in mice lacking either filbrillin-1 or -2 and found that fibrillin-1 and -2 differentially control both TGF-β and BMP bioavailability (Nistala et al. 2010
mice have reduced bone mass caused by increased TGF-β activation and signaling, which reduces expression of a transcriptional regulator of osteoblast differentiation (osterix). Fbn1–/–
mice also fail to restrain TGF-β activation by osteoclasts; however, lack of fibrillin-1 also leads to enhanced BMP bioavailability and signaling that promotes osterix expression, overriding the inhibitory effect of TGF-β signaling. Interestingly, Fbn2–/–
mice have reduced BMP signaling in the distal developing limb (Arteaga-Solis et al. 2001
), in contrast to increased BMP signaling in growing bones in Fbn1–/–
mice, indicating that fibrillins can control BMP effects positively or negatively depending on the cellular context.