Polyubiquitylated rice stripe virus NS3 translocates to the nucleus to promote cytosolic virus replication via miRNA-induced fibrillin 2 upregulation.

Viruses are encapsidated mobile genetic elements that rely on host cells for replication. Several cytoplasmic RNA viruses synthesize proteins and/or RNAs that translocate to infected cell nuclei. However, the underlying mechanisms and role(s) of cytoplasmic-nuclear trafficking are unclear. We demonstrate that infection of small brown planthoppers with rice stripe virus (RSV), a negarnaviricot RNA virus, results in K63-linked polyubiquitylation of RSV's nonstructural protein 3 (NS3) at residue K127 by the RING ubiquitin ligase (E3) LsRING. In turn, ubiquitylation leads to NS3 trafficking from the cytoplasm to the nucleus, where NS3 regulates primary miRNA pri-miR-92 processing through manipulation of the microprocessor complex, resulting in accumulation of upregulated miRNA lst-miR-92. We show that lst-miR-92 regulates the expression of fibrillin 2, an extracellular matrix protein, thereby increasing RSV loads. Our results highlight the manipulation of intranuclear, cytoplasmic, and extracellular components by an RNA virus to promote its own replication in an insect vector.

Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model.

Mutations in the extracellular matrix gene Fibrillin-2 (FBN2) are related to genetic macular degenerative disorders including age-related macular degeneration (AMD) and early-onset macular degeneration (EOMD). It was reported that the retinal protein expression of FBN2 was reduced in patients with AMD and EOMD. The effect of exogenously supplied fbn2 recombinant protein on fbn2-deficiency-related retinopathy was not known. Here we investigated the efficacy and molecular mechanism of intravitreally applied fibrin-2 recombinant protein in mice with fbn2-deficient retinopathy. The experimental study included groups (all n = 9) of adult C57BL/6J male mice which underwent no intervention, intravitreal injection of adeno-associated virus (AAV) empty vector or intravitreal injection of AAV-sh-fbn2 (adeno-associated virus for expressing short hairpin RNA for fibrillin-2) followed by three intravitreal injections of fbn2 recombinant protein, given in intervals of 8 days in doses of 0.30 µg, 0.75 µg, 1.50 µg, and 3.00 µg, respectively. Eyes with intravitreally applied AAV-sh-fbn2 as compared to eyes with injection of AAV-empty vector or developed an exudative retinopathy with involvement of the deep retinal layers, reduction in axial length and reduction in ERG amplitudes. After additional and repeated application of fbn2 recombinant protein, the retinopathy improved with an increase in retinal thickness and ERG amplitude, the mRNA and protein expression of transforming growth factor-beta (TGF-ß1) and TGF-ß binding protein (LTBP-1) increased, and axial length elongated, with the difference most marked for the dose of 0.75 µg of fbn2 recombinant protein. The observations suggest that intravitreally applied fbn2 recombinant protein reversed the retinopathy caused by an fbn2 knockdown.

Anti-skin-aging effects of human ceramides via collagen and fibrillin expression in dermal fibroblasts.

Intercellular lipids comprise mainly ceramides, known to enhance the barrier function of the stratum corneum. However, the activities of ceramides inside the skin have not yet been fully elucidated. Here we examined how the human ceramide mixture (HC123) functions in the dermis. We treated human skin fibroblasts with HC123-expressed fibroblast growth factor (FGF), transforming growth factor-ß (TGF-ß), collagen I, and fibrillin. We found that HC123 promoted the formation of collagen fibers and microfibrils (fibrillin) which affect the elasticity of the skin. We also confirmed that the gene expression of collagen and fibrillin is promoted via TGF-ß and FGF2, respectively. We then investigated the permeability of HC123 for external use, in pursuit of evidence that HC123 may exert an anti-aging effect by penetrating into the dermis, activating fibroblasts, and promoting the production of collagen fibers and elastin-related microfibrils.

Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development.

The embryonic extracellular matrix (ECM) undergoes transition to mature ECM as development progresses, yet few mechanisms ensuring ECM proteostasis during this period are known. Fibrillin microfibrils are macromolecular ECM complexes serving structural and regulatory roles. In mice, Fbn1 and Fbn2, encoding the major microfibrillar components, are strongly expressed during embryogenesis, but fibrillin-1 is the major component observed in adult tissue microfibrils. Here, analysis of Adamts6 and Adamts10 mutant mouse embryos, lacking these homologous secreted metalloproteases individually and in combination, along with in vitro analysis of microfibrils, measurement of ADAMTS6-fibrillin affinities and N-terminomics discovery of ADAMTS6-cleaved sites, identifies a proteostatic mechanism contributing to postnatal fibrillin-2 reduction and fibrillin-1 dominance. The lack of ADAMTS6, alone and in combination with ADAMTS10 led to excess fibrillin-2 in perichondrium, with impaired skeletal development defined by a drastic reduction of aggrecan and cartilage link protein, impaired BMP signaling in cartilage, and increased GDF5 sequestration in fibrillin-2-rich tissue. Although ADAMTS6 cleaves fibrillin-1 and fibrillin-2 as well as fibronectin, which provides the initial scaffold for microfibril assembly, primacy of the protease-substrate relationship between ADAMTS6 and fibrillin-2 was unequivocally established by reversal of the defects in Adamts6-/- embryos by genetic reduction of Fbn2, but not Fbn1.

Fibrillin2 in chloroplast plastoglobules participates in photoprotection and jasmonate-induced senescence.

Fibrillins (FBNs) are the major structural proteins of plastoglobules (PGs) in chloroplasts. PGs are associated with defense against abiotic and biotic stresses, as well as lipid storage. Although FBN2 is abundant in PGs, its independent function under abiotic stress has not yet been identified. In this study, the targeting of FBN2 to PGs was clearly demonstrated using an FBN2-YFP fusion protein. FBN2 showed higher expression in green photosynthetic tissues and was upregulated at the transcriptional level under high-light stress. The photosynthetic capacity of fbn2 knockout mutants generated using CRISPR/Cas9 technology decreased rapidly compared with that of wild-type (WT) plants under high-light stress. In addition to the photoprotective function of FBN2, fbn2 mutants had lower levels of plastoquinone-9 and plastochromanol-8. The fbn2 mutants were highly sensitive to methyl jasmonate (MeJA) and exhibited root growth inhibition and a pale-green phenotype due to reduced chlorophyll content. Consistently, upon MeJA treatment, the fbn2 mutants showed faster leaf senescence and more rapid chlorophyll degradation with decreased photosynthetic ability compared with the WT plants. The results of this study suggest that FBN2 is involved in protection against high-light stress and acts as an inhibitor of jasmonate-induced senescence in Arabidopsis (Arabidopsis thaliana).

LTBP1 promotes fibrillin incorporation into the extracellular matrix.

LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillin-microfibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGFß growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and -2 into the extracellular matrix in vitro. We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGFß-independent LTBP1 function potentially contributing to the development of connective tissue disorders.

Latent TGFß complexes are transglutaminase cross-linked to fibrillin to facilitate TGFß activation.

TGFß superfamily members are potent growth factors in the extracellular matrix with essential roles in all aspects of cellular behaviour. Latent TGFß binding proteins (LTBPs) are co-expressed with TGFß, essential for correct folding and secretion of the growth factor, to form large latent complexes. These large latent complexes bind extracellular proteins such as fibrillin for sequestration of TGFß in the matrix, essential for normal tissue function, and dysregulated TGFß signalling is a hallmark of many fibrillinopathies. Transglutaminase-2 (TG2) cross-linking of LTBPs is known to play a role in TGFß activation but the underlying molecular mechanisms are not resolved. Here we show that fibrillin is a matrix substrate for TG2 and that TG2 cross-linked complexes can be formed between fibrillin and LTBP-1 and -3, and their latent TGFß complexes. The structure of the fibrillin-LTBP1 complex shows that the two elongated proteins interact in a perpendicular arrangement which would allow them to form distal interactions between the matrix and the cell surface. Formation of the cross-link with fibrillin does not change the interaction between latent TGFß and integrin αVß6 but does increase TGFß activation in cell-based assays. The activating effect may be due to direction of the latent complexes to the cell surface by fibrillin, as competition with heparan sulphate can ameliorate the activating effect. Together, these data support that TGFß activation can be enhanced by covalent tethering of LTBPs to the matrix via fibrillin.

Association between anti-fibrillin-2 protein induced retinal degeneration via intravitreous delivery and activated TGF-ß signalling in mice.

Fibrillin-2 (FBN2) is a major component of tissue microfibrils, and the decrease of FBN2 perturbs the signalling events mediated by transforming growth factor-ß (TGF-ß), thereby playing a role in macular degeneration. However, the association between the retinal degeneration resulting from the abnormality of FBN2 and the activation of TGF-ß signalling has not been fully addressed. In the present study, the mice were divided into a normal control group (NC group), a phosphate-buffered saline (PBS) injection group (PBS group), and an anti-FBN2 protein injection group (anti-FBN2 group), and the mice in PBS and anti-FBN2 groups received the relevant treatment via the intravitreal injection once a week for three consecutive weeks. One week later after injection, the retinal morphology and visual function of the fundus were detected. Further, the expression of FBN2, TGF-ß1, TGF-ß2 and TGF-ß3 in retina was measured using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. As a result, fundus examination suggests that after intravitreous injection of anti-FBN2 protein, there were a large patchy yellow white degeneration region and numerous pigmentations in the retina in anti-FBN2-treated mice; by contrast, there was no apparent change in mice from the NC and PBS groups. The retina suffered markedly damage, and the thickness of whole retina and outer nuclear layer markedly thinned. The expression of FBN2 was decreased whereas the levels of TGF-ß1, TGF-ß2 and TGF-ß3 were upregulated. Together, our findings indicate that the intravitreous delivery of anti-FBN2 protein could induce retina degeneration in mice, accompanied by the higher activated TGF-ß. The retinal degeneration mouse model established will provide a platform for the investigation of the retinal diseases.

POGLUT2 and POGLUT3 O-glucosylate multiple EGF repeats in fibrillin-1, -2, and LTBP1 and promote secretion of fibrillin-1.

Fibrillin-1 (FBN1) is the major component of extracellular matrix microfibrils, which are required for proper development of elastic tissues, including the heart and lungs. Through protein-protein interactions with latent transforming growth factor (TGF) ß-binding protein 1 (LTBP1), microfibrils regulate TGF-ß signaling. Mutations within the 47 epidermal growth factor-like (EGF) repeats of FBN1 cause autosomal dominant disorders including Marfan Syndrome, which is characterized by disrupted TGF-ß signaling. We recently identified two novel protein O-glucosyltransferases, Protein O-glucosyltransferase 2 (POGLUT2) and 3 (POGLUT3), that modify a small fraction of EGF repeats on Notch. Here, using mass spectral analysis, we show that POGLUT2 and POGLUT3 also modify over half of the EGF repeats on FBN1, fibrillin-2 (FBN2), and LTBP1. While most sites are modified by both enzymes, some sites show a preference for either POGLUT2 or POGLUT3. POGLUT2 and POGLUT3 are homologs of POGLUT1, which stabilizes Notch proteins by addition of O-glucose to Notch EGF repeats. Like POGLUT1, POGLUT2 and 3 can discern a folded versus unfolded EGF repeat, suggesting POGLUT2 and 3 are involved in a protein folding pathway. In vitro secretion assays using the N-terminal portion of recombinant FBN1 revealed reduced FBN1 secretion in POGLUT2 knockout, POGLUT3 knockout, and POGLUT2 and 3 double-knockout HEK293T cells compared with wild type. These results illustrate that POGLUT2 and 3 function together to O-glucosylate protein substrates and that these modifications play a role in the secretion of substrate proteins. It will be interesting to see how disease variants in these proteins affect their O-glucosylation.

Fibrillin 2 gene knockdown inhibits invasion and migration of lung cancer cells.

To investigate the effect of Fibrillin 2 (FBN2) expression on the invasion and migration of lung cancer cells, and the underlying mechanism. Protein and mRNA expressions of FBN2 were assayed. The relationship between FBN2 protein expression and clinical characteristics of lung cancer patients was analyzed. Correlation between FBN2 expression level and patient survival time was analyzed. Moreover, the mRNA and protein expression of FBN2 in lung cancer cells and human normal lung epithelial cells were assayed. After constructing low-expressing FBN2 cells, the cell proliferation, clone formation, migration and invasion capabilities were tested. Lung cancer cells proliferation with low FBN2 expression in nude mice was measured with a nude mouse tumorigenic experiment. The mRNA and protein expressions of FBN2 in lung cancer tissues were significantly higher than those in para-cancerous tissues (p<0.05).  FBN2 protein expression was significantly correlated with TNM stage, lymph node metastasis and histological type (p<0.05). Survival time was markedly reduced in patients with high FBN2 expression (p<0.001). The expressions of FBN2 mRNA and protein were markedly higher in lung cancer cells than in human normal lung epithelial cells. The proliferation, migration and invasion of lung cancer cells were significantly inhibited by FBN2 knockdown. The FBN2 knockdown significantly inhibited the protein expressions of p-FAK, p-MEK and p-ERK. FBN2 is highly expressed in lung cancer tissues, and as an oncogene, it affects the pathogenesis of lung cancer. The knockdown of the expression of FBN2 significantly inhibits the proliferation, invasion and migration abilities of lung cancer cells.

Role of fibrillin-2 in the control of TGF-ß activation in tumor angiogenesis and connective tissue disorders.

Fibrillins constitute a family of large extracellular glycoproteins which multimerize to form microfibrils, an important structure in the extracellular matrix. It has long been assumed that fibrillin-2 was barely present during postnatal life, but it is now clear that fibrillin-2 molecules form the structural core of microfibrils, and are masked by an outer layer of fibrillin-1. Mutations in fibrillins give rise to heritable connective tissue disorders, including Marfan syndrome and congenital contractural arachnodactyly. Fibrillins also play an important role in matrix sequestering of members of the transforming growth factor-ß family, and in context of Marfan syndrome excessive TGF-ß activation has been observed. TGF-ß activation is highly dependent on integrin binding, including integrin αvß8 and αvß6, which are upregulated upon TGF-ß exposure. TGF-ß is also involved in tumor progression, metastasis, epithelial-to-mesenchymal transition and tumor angiogenesis. In several highly vascularized types of cancer such as hepatocellular carcinoma, a positive correlation was found between increased TGF-ß plasma concentrations and tumor vascularity. Interestingly, fibrillin-1 has a higher affinity to TGF-ß and, therefore, has a higher capacity to sequester TGF-ß compared to fibrillin-2. The previously reported downregulation of fibrillin-1 in tumor endothelium affects the fibrillin-1/fibrillin-2 ratio in the microfibrils, exposing the normally hidden fibrillin-2. We postulate that fibrillin-2 exposure in the tumor endothelium directly stimulates tumor angiogenesis by influencing TGF-ß sequestering by microfibrils, leading to a locally higher active TGF-ß concentration in the tumor microenvironment. From a therapeutic perspective, fibrillin-2 might serve as a potential target for future anti-cancer therapies.

Adamts17 is involved in skeletogenesis through modulation of BMP-Smad1/5/8 pathway.

Fibrillin microfibrils are ubiquitous elements of extracellular matrix assemblies that play crucial roles in regulating the bioavailability of growth factors of the transforming growth factor beta superfamily. Recently, several "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) proteins were shown to regulate fibrillin microfibril function. Among them, ADAMTS17 is the causative gene of Weill-Marchesani syndrome (WMS) and Weill-Marchesani-like syndrome, of which common symptoms are ectopia lentis and short stature. ADAMTS17 has also been linked to height variation in humans; however, the molecular mechanisms whereby ADAMTS17 regulates skeletal growth remain unknown. Here, we generated Adamts17-/- mice to examine the role of Adamts17 in skeletogenesis. Adamts17-/- mice recapitulated WMS, showing shorter long bones, brachydactyly, and thick skin. The hypertrophic zone of the growth plate in Adamts17-/- mice was shortened, with enhanced fibrillin-2 deposition, suggesting increased incorporation of fibrillin-2 into microfibrils. Comprehensive gene expression analysis of growth plates using laser microdissection and RNA sequencing indicated alteration of the bone morphogenetic protein (BMP) signaling pathway after Adamts17 knockout. Consistent with this, phospho-Smad1 levels were downregulated in the hypertrophic zone of the growth plate and in Adamts17-/- primary chondrocytes. Delayed terminal differentiation of Adamts17-/- chondrocytes, observed both in primary chondrocyte and primordial metatarsal cultures, and was prevented by BMP treatment. Our data indicated that Adamts17 is involved in skeletal formation by modulating BMP-Smad1/5/8 pathway, possibly through inhibiting the incorporation of fibrillin-2 into microfibrils. Our findings will contribute to further understanding of disease mechanisms and will facilitate the development of therapeutic interventions for WMS.

Unique cartilage matrix-associated protein regulates fibrillin-2 expression and directly interacts with fibrillin-2 protein independent of calcium binding.

Unique cartilage matrix-associated protein (UCMA) is a secretory γ-carboxyglutamate (Gla) containing protein that is mainly expressed in the cartilage. Ucma, a downstream gene of both Runx2 and Osterix, has recently been described to promote osteoblast differentiation and matrix mineralization. However, till date, no studies have focused on the role of downstream target genes of Ucma in osteogenesis. Here, by Affymetrix GeneChip microarray analysis, we determined 45 differentially expressed genes in response to Ucma stable overexpression or knockdown in osteoblast cells, which provided insight into molecular mechanisms underlying osteoblast differentiation. In particular, we showed that fibrillin-2 (FBN2) expression was proportional to Ucma expression in osteoblasts as validated by quantitative PCR. We also showed that even though Gla-containing UCMA and calcium-binding EGF-like domain-containing FBN2 are known to have a high affinity for calcium, FBN2 whose expression was regulated by UCMA directly interacted with the UCMA protein, independent of calcium.

Limb- and tendon-specific Adamtsl2 deletion identifies a role for ADAMTSL2 in tendon growth in a mouse model for geleophysic dysplasia.

Geleophysic dysplasia is a rare, frequently lethal condition characterized by severe short stature with progressive joint contractures, cardiac, pulmonary, and skin anomalies. Geleophysic dysplasia results from dominant fibrillin-1 (FBN1) or recessive ADAMTSL2 mutations, suggesting a functional link between ADAMTSL2 and fibrillin microfibrils. Mice lacking ADAMTSL2 die at birth, which has precluded analysis of postnatal limb development and mechanisms underlying the skeletal anomalies of geleophysic dysplasia. Here, detailed expression analysis of Adamtsl2 using an intragenic lacZ reporter shows strong Adamtsl2 expression in limb tendons. Expression in developing and growing bones is present in regions that are destined to become articular cartilage but is absent in growth plate cartilage. Consistent with strong tendon expression, Adamtsl2 conditional deletion in limb mesenchyme using Prx1-Cre led to tendon anomalies, albeit with normal collagen fibrils, and distal limb shortening, providing a mouse model for geleophysic dysplasia. Unexpectedly, conditional Adamtsl2 deletion using Scx-Cre, a tendon-specific Cre-deleter strain, which does not delete in cartilage, also impaired skeletal growth. Recombinant ADAMTSL2 is shown here to colocalize with fibrillin microfibrils in vitro, and enhanced staining of fibrillin-1 microfibrils was observed in Prx1-Cre Adamtsl2 tendons. The findings show that ADAMTSL2 specifically regulates microfibril assembly in tendons and that proper microfibril composition in tendons is necessary for tendon growth. We speculate that reduced bone growth in geleophysic dysplasia may result from external tethering by short tendons rather than intrinsic growth plate anomalies. Taken together with previous work, we suggest that GD results from abnormal microfibril assembly in tissues, and that ADAMTSL2 may limit the assembly of fibrillin microfibrils.

Fibrillin-2 is a key mediator of smooth muscle extracellular matrix homeostasis during mouse tracheal tubulogenesis.

Epithelial tubes, comprised of polarised epithelial cells around a lumen, are crucial for organ function. However, the molecular mechanisms underlying tube formation remain largely unknown. Here, we report on the function of fibrillin (FBN)2, an extracellular matrix (ECM) glycoprotein, as a critical regulator of tracheal tube formation.We performed a large-scale forward genetic screen in mouse to identify regulators of respiratory organ development and disease. We identified Fbn2 mutants which exhibit shorter and narrowed tracheas as well as defects in tracheal smooth muscle cell alignment and polarity.We found that FBN2 is essential for elastic fibre formation and Fibronectin accumulation around tracheal smooth muscle cells. These processes appear to be regulated at least in part through inhibition of p38-mediated upregulation of matrix metalloproteinases (MMPs), as pharmacological decrease of p38 phosphorylation or MMP activity partially attenuated the Fbn2 mutant tracheal phenotypes. Analysis of human tracheal tissues indicates that a decrease in ECM proteins, including FBN2 and Fibronectin, is associated with tracheomalacia.Our findings provide novel insights into the role of ECM homeostasis in mesenchymal cell polarisation during tracheal tubulogenesis.

Adamts10 inactivation in mice leads to persistence of ocular microfibrils subsequent to reduced fibrillin-2 cleavage.

Mutations in the secreted metalloproteinase ADAMTS10 cause recessive Weill-Marchesani syndrome (WMS), comprising ectopia lentis, short stature, brachydactyly, thick skin and cardiac valve anomalies. Dominant WMS caused by FBN1 mutations is clinically similar and affects fibrillin-1 microfibrils, which are a major component of the ocular zonule. ADAMTS10 was previously shown to enhance fibrillin-1 assembly in vitro. Here, Adamts10 null mice were analyzed to determine the impact of ADAMTS10 deficiency on fibrillin microfibrils in vivo. An intragenic lacZ reporter identified widespread Adamts10 expression in the eye, musculoskeletal tissues, vasculature, skin and lung. Adamts10-/- mice had reduced viability on the C57BL/6 background, and although surviving mice were slightly smaller and had stiff skin, they lacked brachydactyly and cardiovascular defects. Ectopia lentis was not observed in Adamts10-/- mice, similar to Fbn1-/- mice, most likely because the mouse zonule contains fibrillin-2 in addition to fibrillin-1. Unexpectedly, in contrast to wild-type eyes, Adamts10-/- zonule fibers were thicker and immunostained strongly with fibrillin-2 antibodies into adulthood, whereas fibrillin-1 staining was reduced. Furthermore, fibrillin-2 staining of hyaloid vasculature remnants persisted post-natally in Adamts10-/- eyes. ADAMTS10 was found to cleave fibrillin-2, providing an explanation for persistence of fibrillin-2 at these sites. Thus, analysis of Adamts10-/- mice led to identification of fibrillin-2 as a novel ADAMTS10 substrate and defined a proteolytic mechanism for clearance of ocular fibrillin-2 at the end of the juvenile period.

Fibrillin-2 and Tenascin-C bridge the age gap in lung epithelial regeneration.

Organ engineering based on native matrix scaffolds involves combining regenerative cell populations with corresponding biological matrices to form functional grafts on-demand. The extracellular matrix (ECM) that is retained following lung decellularization provides essential structure and biophysical cues for whole organ regeneration after recellularization. The unique ECM composition in the early post-natal lung, during active alveologenesis, may possess distinct signals that aid in driving cell adhesion, survival, and proliferation. We evaluated the behavior of basal epithelial stem cells (BESCs) isolated from adult human lung tissue, when cultured on acellular ECM derived from neonatal (aged < 1 week) or adult lung donors (n = 3 donors per group). A significant difference in cell proliferation and survival was found. We next performed in-depth proteomic analysis of the lung scaffolds to quantify proteins significantly enriched in the neonatal ECM, and identified the glycoproteins Fibrillin-2 (FBN-2) and Tenascin-C (TN-C) as potential mediators of the observed effect. BESCs cultured on Collagen Type IV coated plates, supplemented with FBN-2 and TN-C demonstrated significantly increased proliferation and decreased cellular senescence. No significant increase in epithelial-to-mesenchymal transition was observed. In vitro migration was also increased by FBN-2 and TN-C treatment. Decellularized lung scaffolds treated with FBN-2 and TN-C prior to re-epithelialization supported greater epithelial proliferation and tissue remodeling. BESC distribution, matrix alignment, and overall tissue morphology was improved on treated lung scaffolds, after 3 and 7 days of ex vivo lung culture. These results demonstrate that scaffold re-epithelialization is enhanced on neonatal lung ECM, and that supplementation of FBN-2 and TN-C to the native scaffold may be a valuable tool in lung tissue regeneration.

GABA promotes elastin synthesis and elastin fiber formation in normal human dermal fibroblasts (HDFs).

The multiple physiological effects of γ-aminobutyric acid (GABA) as a functional food component have been recently reported. We previously reported that GABA upregulated the expression of type I collagen in human dermal fibroblasts (HDFs), and that oral administration of GABA significantly increased skin elasticity. However, details of the regulatory mechanism still remain unknown. In this study, we further examined the effects of GABA on elastin synthesis and elastin fiber formation in HDFs. Real-time PCR indicated that GABA significantly increased the expression of tropoelastin transcript in a dose-dependent manner. Additionally, the expression of fibrillin-1, fibrillin-2, and fibulin-5/DANCE, but not lysyl oxidase and latent transforming factor-ß-binding protein 4, were also significantly increased in HDFs. Finally, immunohistochemical analysis confirmed that treatment with GABA dramatically increased the formation of elastic fibers in HDFs. Taken together, our results showed that GABA improves skin elasticity in HDFs by upregulating elastin synthesis and elastin fiber formation.

Unusual life cycle and impact on microfibril assembly of ADAMTS17, a secreted metalloprotease mutated in genetic eye disease.

Secreted metalloproteases have diverse roles in the formation, remodeling, and the destruction of extracellular matrix. Recessive mutations in the secreted metalloprotease ADAMTS17 cause ectopia lentis and short stature in humans with Weill-Marchesani-like syndrome and primary open angle glaucoma and ectopia lentis in dogs. Little is known about this protease or its connection to fibrillin microfibrils, whose major component, fibrillin-1, is genetically associated with ectopia lentis and alterations in height. Fibrillin microfibrils form the ocular zonule and are present in the drainage apparatus of the eye. We show that recombinant ADAMTS17 has unique characteristics and an unusual life cycle. It undergoes rapid autocatalytic processing in trans after its secretion from cells. Secretion of ADAMTS17 requires O-fucosylation and its autocatalytic activity does not depend on propeptide processing by furin. ADAMTS17 binds recombinant fibrillin-2 but not fibrillin-1 and does not cleave either. It colocalizes to fibrillin-1 containing microfibrils in cultured fibroblasts and suppresses fibrillin-2 (FBN2) incorporation in microfibrils, in part by transcriptional downregulation of Fbn2 mRNA expression. RNA in situ hybridization detected Adamts17 expression in specific structures in the eye, skeleton and other organs, where it may regulate the fibrillin isoform composition of microfibrils.