ARTICLE |
Correspondence to: Douglas R. Keene, Shriners Hospital for Children, 3101 S.W. Sam Jackson Park Road, Portland OR 97201.
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Summary |
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Immunolocalization studies demonstrate that Type VI collagen forms a flexible network that interweaves among collagen fibrils in the dermis of skin as well as in other loose connective tissues. Although binding of Type VI collagen with other matrix components has been suggested, no structural evidence supporting these studies has been reported. In this study, we demonstrate that Type VI microfilaments consistently crossbanded collagen fibrils near the "d" band, indicating that the interaction of Type VI collagen with banded fibrils is not passive. This "d" band is also the location of the binding domain of decorin to banded fibrils, suggesting that decorin mediates the interaction of Type VI microfilaments with banded fibers. Examination of the architecture of the Type VI network in a decorin nullizygous mouse demonstrates a continuance of this specific interaction, indicating that the association is not entirely dependent on the presence of decorin. At least one other component, whose identity is uncertain, persists near the "d" band, which may also serve to mediate the attachment of Type VI collagen to collagen fibrils. (J Histochem Cytochem 46:215220, 1998)
Key Words: collagen, Type VI microfilaments, decorin, banded fibers, human skin, decorin-deficient mouse, immunocytochemistry, electron microscopy, proteoglycan
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Introduction |
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Immunolocalization STUDIES of Type VI collagen in skin have previously demonstrated that this collagen forms an extensive, flexible network that anchors large interstitial structures such as myelinated nerves, blood vessels, and collagen fibers to the surrounding connective tissue matrices (
The banded collagen fibrils of skin are heteropolymers composed primarily of Type I collagen, with lesser amounts of collagen Types III and V. Type III collagen is limited to the outside of the fibrils and may participate in the regulation of fibril diameter. The bulk of Type V collagen is within the interior of the fibril, though a portion of the Type V molecule may protrude from the fibril surface (for review see
Decorin (
Immunocytochemical studies in fetal and neonate cornea suggest an association of decorin with Type VI collagen microfilaments (
In this study we sought to determine if there is a consistent site of interaction between Type VI collagen microfilaments and banded collagen fibers, and to establish whether or not the presence of decorin is required for this biological interaction. We show that Type VI microfilaments interact near the "d" band of both human and mouse dermal collagen fibrils. Surprisingly, the association of Type VI microfilaments with collagen fibrils persists in decorin-deficient animals, suggesting that additional components, likely to be other members of the SLRP gene family, also mediate an attachment of Type VI collagen to banded collagen fibrils.
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Materials and Methods |
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Antibodies
The rabbit PAb 1909 recognizing human decorin was purchased from Chemicon (Temecula, CA). The rabbit PAb LF113 recognizing the amino terminal peptide region of mouse decorin was a generous gift from Dr. Larry Fisher (
Immunocytochemistry
En bloc immunolabeling of tissues was carried out using a previously described protocol (
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Results |
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Labeling of human skin with MAb 3C4 (specific for Type VI collagen) resulted in the decoration of Type VI microfilaments at 100-nm intervals (Figure 1C, inset), consistent with the length of the Type VI collagen molecule and the known periodicity of Type VI microfilaments (
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To gain further insight into the nature of the moiety interacting near the "d" band with Type VI collagen and considering that decorin may be implicated in performance of this function, we analyzed the interaction of Type VI microfilaments and collagen fibrils in the dermis of both wild-type and decorin-deficient mice. The architecture of the Type VI collagen network in mouse, as determined by immunocytochemical staining, closely resembles that reported in human skin (data not shown). En bloc immunolabeling of normal mouse skin with antibody specific for decorin (LF 113) results in periodic labeling of the fibers to a region near the "d" band (Figure 2A and Figure 2C). Also consistent with human skin are the presence of knobs on the surface of the fibrils, near the "d" band, after exposure to RHT-containing fixatives (Figure 2B, arrowheads). As in human tissue, RHT-stained Type VI microfilaments are consistently seen to intersect banded collagen fibrils near the "d" band (Figure 2C, open arrow). As expected, exposure of decorin-deficient skin (Dcn-/-) to the decorin-specific antibody results in a complete lack of secondary gold labeling (Figure 2D). However, the presence of small knobs on the surface of the fibrils after RHT staining persists in the Dcn-/- mouse (Figure 2D, arrowheads), suggesting that another component is present near the "d" band in addition to decorin. Interestingly, fields could still be commonly found in which Type VI collagen microfilaments interacted at the "d" band of banded collagen in the decorin nullizygous mouse (Figure 2E, arrows). Collectively, we interpret these data as an indication that decorin is not the only component associated with collagen fibrils that mediate an interaction with Type VI collagen.
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Discussion |
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Type VI collagen microfilaments are composed of three different polypeptide chains, which form short triple helical regions and large carboxyl and amino terminal domains. Monomers align anti-parallel to form dimers, and dimers align with their ends in register to form covalently crosslinked tetramers. Tetramers associate via the globular domains to form microfilaments (Furthmayer 1983;
A mechanism for anchoring the Type VI collagen network to collagen fibrils has not been previously demonstrated in tissue. Significant in this study is the identification of a definitive site at which Type VI collagen microfibrils interact with the surface of banded fibrils, indicating that the role of Type VI in tissue integrity is more than passive. The location of this interactive site is near the "d" band of the collagen fibrils, which is demonstrated here and elsewhere to be the site of decorin binding.
Solid-phase binding assays have demonstrated an affinity between decorin and Type VI collagen (
Other small, leucine-rich proteoglycans are known to be present within the matrix including fibromodulin, biglycan, and lumican (
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Acknowledgments |
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Supported in part by grants from the Shriners Hospital for Children (DRK,CCR) and by National Institutes of Health grants RO1 CA39481 and RO1 CA47282 (RVI).
Electron microscopy facilities were provided in part by the Fred Meyer and R. Blaine Bramble Charitable Trust Foundations. We are deeply grateful to E. Engvall and L. Fisher for providing precious immunological reagents.
Received for publication May 9, 1997; accepted September 24, 1997.
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Literature Cited |
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