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The extracellular matrix protein fibulin-4 has been shown to be indispensable for elastic fiber assembly, but there is also evidence from human mutations that it is involved in controlling skeletal development and bone stability. Fibulin-4 mutations were identified in patients suffering from vascular abnormality and/or cutis laxa, and some of these patients exhibited bone fragility, arachnodactyly and joint laxity. In order to elucidate the role of fibulin-4 in bone structure and skeletal development, we analyzed structural changes in skeletal tissues of Fbln4-/- mice. Immunostaining confirmed that fibulin-4 is highly expressed in cartilage, bone, ligaments and tendons. No morphological abnormalities were found in the skeleton of Fbln4-/- mice as compared to wild type littermates except forelimb contractures as well as unusually thick collagen fibrils. Furthermore, fibulin-4 deficiency caused enhanced susceptibility of bone collagen for acid extraction, consistent with significantly reduced lysylpyridinoline and hydroxylysylpyridinoline cross-links in bone. In accordance with that, the amount of lysyl oxidase in long bones and calvaria was strongly decreased and proteolytic activation of lysyl oxidase was reduced in fibulin-4 deficient osteoblasts, while addition of recombinant fibulin-4 rescued the activation. The finding suggested that fibulin-4 is important for the proteolytic activation of lysyl oxidase which has a pivotal role in cross-linking of collagen and elastin. © 2015 International Society of Matrix Biology.

Three distinct alpha chains form the collagen VI monomer, the alpha 3(VI) chain being much larger than the alpha 1(VI) and alpha 2(VI) chains. The alpha 3(VI) chain has 10 von Willebrand Factor type A domains of approximately 200 amino acids at the N-terminus (N1-N10) compared with only one such domain in the alpha 1(VI) and alpha 2(VI) chains. Domains N10, N9, N7 and N3 of the alpha 3(VI) chain are subject to alternative splicing in chick and/or human tissues, indicating the possibility of isoforms that have different functions depending on which N-terminal domains are included or excluded. In this study we have PCR amplified and sequenced mouse alpha 3(VI) cDNA encoding the N2-N10 domains. By reverse transcription-PCR using oligonucleotides spanning different regions of the cDNA we have undertaken a comprehensive analysis of alternative splicing of the alpha 3(VI) mRNA in embryonic and adult mouse tissues. We demonstrate that domains N10, N9 and N7 are also subject to alternative splicing in mouse tissues and in addition identify an abundant novel variant transcript that lacks all four N-terminal domains (N7-N10) in mouse tissues and human cells. We also identify less abundant transcripts that lack a large part of the N3 domain, and transcripts lacking the entire N5 domain. Using specific RNase protection assays we show that the shorter transcripts containing domains (N8+N7+N6), (N8+N6) and N6 are present at higher levels than transcripts containing the N10 and/or N9 domains, with tissue-specific variation in the levels of variant transcripts. These studies demonstrate a larger range of collagen VI protein variants than previously described.

The alpha 1(XVI) collagen chain, recently identified by cDNA cloning, exhibits structural similarity to a subgroup of collagens that associate with collagen fibrils. Recombinant alpha 1(XVI) collagen chains produced in embryonic kidney cells are able to form stable homotrimers, which are rapidly converted into smaller polypeptides after secretion into the culture medium. In this study, we investigated the biosynthesis of native type XVI collagen by immunoprecipitation of metabolically labeled human cells. Dermal fibroblasts and arterial smooth muscle cells were precipitated with three antibodies raised against distinct regions in the N- and C-terminal part of the human alpha 1(XVI) collagen chain. A disulfide-bonded polypeptide of 220 kDa was obtained from the culture medium, cells and extracellular matrix with all three antibodies. This polypeptide is sensitive to bacterial collagenase digestion and partially resistant to pepsin digestion, suggesting that it is the endogenous alpha 1(XVI) collagen chain. Pulse/chase experiments showed that the newly synthesized alpha 1(XVI) chains are secreted into the medium and deposited in the extracellular matrix in a time-dependent manner. Unlike the recombinant chain, the native type XVI collagen does not undergo extensive proteolytic processing upon secretion. Both cell types deposit a substantial amount of the newly synthesized alpha 1(XVI) chain into the extracellular matrix, in which the 220-kDa polypeptide is the only product immunoprecipitated. There is little evidence for the presence of another constituent chain. The data are consistent with a nomotrimeric chain composition for type XVI collagen. No apparent difference exists in the rate of synthesis and secretion between fibroblasts and smooth muscle cells. Indirect immunofluorescence microscopy showed an extracellular distribution of type XVI collagen, which is located close to cells but not associated with fibrillar structures.

The expression of a recently identified collagen, alpha 1 (XVI), in adult mouse tissue and developing mouse embryo was examined by immunohistochemistry and in situ hybridization. A polyclonal antiserum was raised against a recombinant fusion protein, which contained a segment of 161 amino acids in the N-terminal noncollagenous domain of the human alpha 1 (XVI) collagen. Immunoprecipitation of metabolically labelled human or mouse fibroblast cell lysates with this antibody revealed a major, bacterial collagenase sensitive polypeptide of approximately 210 kDa. The size agrees with the prediction from the full-length cDNA. Immunofluorescence examination of adult mouse tissues using the affinity purified antibody revealed a rather broad distribution of the protein. The heart, kidney, intestine, ovary, testis, eye, arterial walls and smooth muscles all exhibited significant levels of expression, while the skeletal muscle, lung and brain showed very restricted and low signals. During development, no significant expression of the mRNA or protein was observed in embryo of day 8 of gestation, but strong signals was detected in placental trophoblasts. Expression in embryos was detectable first after day 11 of gestation with weak positive signals appearing in the heart. In later stages of development, stronger RNA hybridizations were observed in a variety of tissues, particularly in atrial and ventricular walls of the developing heart, spinal root neural fibers and skin. These data demonstrate that type XVI collagen represents another collagenous component widely distributed in the extracellular matrix and may contribute to the structural integrity of various tissues.