Elasticity provided by elastic fiber formation is essential to maintain tissue flexibility and extensibility for many organs such as the large arteries, lungs, and skin30
. Although fibrillin-1 microfibrils and tropoelastin protein represent the majority of elastic fiber components, it has been poorly understood how microfibrils are built following fibrillin-1 assembly and how microfibrils and tropoelastin interact during elastic fiber organization. Recently, it was shown that a disintegrin and metalloproteinase with thrombospondin motifs-like-6 protein directly binds to fibrillin-1 to promote the formation of fibrillin-1 microfibrils43
. Another recent study revealed that fibulin-4 protein is indispensable for tethering LOX to tropoelastin to facilitate cross-linking44
. Those reports are of great importance to comprehend the mechanisms underlying the construction of elastic fibers, whereas it remains to be determined how many other molecules are involved in elastogenesis to completely understand the process of elastic fiber formation. For example, MAGP-36, a homologue of MFAP-4 detected around elastic fibers in various animals, was reported to have disappeared in photoaged dermis but accumulated in disintegrated elastic fibers in the lesional skin of pseudoxanthoma elasticum, an elastin-related disorder40
. These findings encouraged us to explore the function of MFAP-4 in human skin using an in vivo
photodamaged/photoaged skin model with human skin xenografted on SCID mice in concert with a lentiviral vector to over-express MFAP-4 as well as in vitro
NHDFs combined with siRNA technology.
One of the most significant problems that needed to be addressed in this study was the consideration of how MFAP-4 is involved in elastic fiber formation. It has been previously demonstrated that fibulin-4 and -5 are in charge of recruiting tropoelastins and its cross-linking enzymes onto microfibrils in order to accelerate elastic fiber assembly in collaboration with LTBP-2, which binds to heparin and heparin sulfate proteoglycans45,46,47,48,49
. In addition, the abundance of fibulin-5 has also been documented to be decreased both in photoaged and in intrinsically aged dermis, suggesting that it plays a role in maintaining cutaneous elasticity50
. Given that fibulins play roles in the assembly and cross-linking of tropoelastin monomers, it is reasonable to hypothesize that MFAP-4 contributes to the development of microfibrils rather than the tethering of LOX to tropoelastin which is mainly regulated by fibulins. We have clearly demonstrated that MFAP-4 interacts with fibrillin-1, whose production is also reported to be decreased in photoaged skin51
, and not with tropoelastin for the organization of microfibrils which allows the participation of tropoelastin cross-linked with LOX for the formation of functional mature elastic fibers substantially required for skin homeostasis. Based on our findings, we propose a model for the contribution of MFAP-4 to the development of elastic fibers (). By virtue of the presence of MFAP-4, fibrillin-1 can be assembled to form microfibrils followed by the cross-linking of tropoelastins tethered with LOX to microfibrils, as supported by a previous study44
. In addition to its role in the development of microfibrils, another important role of MFAP-4 in the suppression of MMP-12 activity is illustrated both in our in vivo
and in vitro
analyses. In parallel with previous studies indicating that elastin-derived peptides induce the expression of various kinds of MMPs (including MMP-12) in several types of cells52,53
and that UV radiation alone is sufficient to selectively degrade many elastic fiber associated proteins54
, the enhancement of MFAP-4 is thought to be in charge of the suppression of MMP-12 activity as well as the promotion of microfibril formation which is essential for the organization of functional elastic fibers, resulting in the observed photoprotection.
Schematic representation of the role of MFAP-4 in elastic fiber formation.
It is also of interest to investigate the impact of the elastic fiber formation mediated by MFAP-4 on another major type of age-dependent-decreasing components, collagens, because it has been reported that MAGP-36 also binds to collagen I39
. Our data indicate that over-expression of MFAP-4 protects against UVB-induced degradation of collagen I which is associated with the inhibition of photodamage/photoaging deterioration in xenografted human skin in vivo.
Further, the amount of MMP-1, which is one of the collagenases expressed by dermal fibroblasts55
, was significantly up-regulated in MFAP-4 knockdown NHDFs both at the transcript and the protein levels. Consistently, elevation of MMP-1 in dermal fibroblasts is also observed when the cells are exposed to broken collagens in organ cultures of human skin56
. These findings led us to hypothesize that MFAP-4 has another role in the stabilization of collagen I. Given that the expression of pro-MMP-1 is stimulated by elastin peptides in skin fibroblasts41,42
, it is reasonable to propose another role of MFAP-4 in the maintenance of collagens which is also important for skin homeostasis. In addition to the protection of collagen I by increased MFAP-4 expression, UVB-induced deterioration of collagen IV was also found to be hindered in our photodamage model of xenografted human skin (data not shown).
In conclusion, our data reveal for the first time that the protein levels of MFAP-4 are decreased both in extrinsic photoaged skin and in intrinsic aged skins, and that the enhancement of MFAP-4 expression protects the skin from UVB-induced photodamage/photoaging characterized by the degradation of fibers of elastin and collagens resulting in the aggravation of skin elasticity. More importantly, MFAP-4 plays essential roles not only in microfibril development by direct interaction with fibrillin-1 but also in the maintenance of ECM proteins, including collagen I, by an as yet undisclosed mechanism(s). These findings provide new insights into a fundamental comprehension of the mechanisms underlying microfibril formation resulting in skin homeostasis and also achieve a basis to develop an efficient strategy for treating UV-induced skin disorders.