Zu Hauptinhalt springen

Collagen XVI: molecularstructure, localization and interaction

Collagen XVI – Background and State of the Art

Collagen XVI and chronic inflammatory Crohn's disease (CD)

Collagen XVI and Oral Squamous Cell Carcinoma (OSCC)

Relevant publications in context

Collaborations


Collagen XVI – Background and State of the Art

The extracellular matrix of skin and cartilage is supported by fibrillar networks that vary in their molecular composition according to mechanical requirements. The structural diversity of fibrillar matrix aggregates arises from tissue specific incorporation and association of matrix components and different types of collagens.

Collagen XVI, a member of the family of fibril-associated collagens with interrupted triple helices comprises of three identical alpha-chains. It consists of ten multiple triple-helical domains (15-422 amino acids) that are interrupted by short, non-collagenous repeats (11-39 amino acids). The ability of associating with different structures tissue specifically is the remarkable feature of collagen XVI. In the dermo-epidermal junction it is associated with fibrillin-1 containing microfibrils, whereas in cartilage it shows connection to collagen II-containing cartilage fibrils.

We have expressed collagen XVI recombinantly in 293-EBNA-cells. This allows us to investigate and elucidate the influence of collagen XVI on fibrillar structures of the microfibrillar apparatus in D-banded cartilage fibrils. In-vitro binding studies with recombinant collagen XVI show high affinity to fibronectin, however weak interaction with fibrillin-1 and -2. In rotary shadowing, negative staining and atomic force microscopy (AFM) we have identified the structure of recombinant collagen XVI and the molecular size (Fig 1a, b).

Ratfig01ab


Ratfig01c

Close assoziation to alpha 2 integrin in the dermal-epidermal junction zone (Fig. 1c, collagen XVI red, alpha 2 green, cell nuclei blue) was verified by ELISA-based binding assays to integrins alpha 1 and alpha 2 which demonstrated high affinity binding of collagen XVI to integrins.

Arrowup Top


Cell-spreading

Cell spreading properties of ISEMF

ISEMF and CCD18Co cells were cultured with a density of 20 000 cells/ml on coated glass slides for 3 hours. Cell spreading and cell shape were optically evaluated by scanning electron microscopy. Cell spreading was promoted on collagen XVI compared to collagen I and BSA. Representative images were depicted.

Arrowup Top

Collagen XVI in Crohn's Disease

In Crohn’s disease (CD) the stress-shield of intestinal subepithelial myofibroblasts (ISEMF) provided by intact tissue is disturbed due to inflammation and thus, cells start with remodelling activities. This is characterized by increased numbers of collagen-producing ISEMF causing an uncontrolled, irreversible wound-healing response to the chronic inflammation of the gastrointestinal tract. Reconstitution of the original ECM leads ISEMF to exit this cycle. In contrast, during fibrosis, ISEMF persist. It is known that ISEMF produce and deposit collagen types I, III, IV and V; however synthesis and the role of fibrillar peripheral molecules like collagen type XVI have not been addressed yet. Here, we have analyzed the distribution of collagen XVI in the normal and inflamed bowel wall, its gene and protein expression by ISEMF of different inflammation stages, the cell-matrix interactions in different phases of the inflammatory process and their effect on cell spreading, proliferation and migration. Collagen XVI is deposited in the submucosa of the intestinal wall where it co-localizes with fibrillin-1 and integrin alpha 1. ISEMF reveal increasing gene and protein expression of collagen XVI concurrent to increasing inflammation. ISEMF reveal more mature focal adhesion contacts when seeded on collagen XVI resulting in an extensive cell spreading. This involves recruitment of α1b1 integrin, which shows increased cell surface expression on ISEMF in late stages of inflammation. We assume that collagen XVI promotes persistence of ISEMF in the normal and, even stronger in the inflamed bowel wall by stabilizing focal adhesion contacts via cell-matrix interaction preferentially through recruitment of α1ß1 integrin into the tips of the focal adhesion contacts.

Fibrillin

Figure 1: Morphological distribution of collagen XVI and fibrillin-1 in CD tissue.
Morphological distribution of collagen XVI (green fluorescence) and fibrillin-1 (red fluorescence) is depicted for the bowel wall of CD tissue. Left row represents fibrillin-1 staining (A, D, G, K), middle row collagen XVI staining (B, E, H, L) and right row merge of both (yellow fluorescence; C; F, I, M). Bar indicates 100 µm.
c: crypts, m: circular muscle layer, s: submucosa, bv: blood vessel, e: erythrocytes, ep: epithelial cells, lp: lamina propria

-->


Collagen XVI in Oral Squamous Cell Carcinoma (OSCC)

Type XVI collagen belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). Recently, it has been shown it has a high affinity to integrin alpha1beta1. Cells expressing those integrins attach and spread on recombinant type XVI collagen. Here we show that type XVI collagen is overexpressed in mucosal epithelium from oral squamous cell carcinoma (OSCC) patients. Induction of its expression in OSCC cell lines (COLXVI cells) leads to an increased expression of Kindlin-1. Moreover, we demonstrate a significantly increased Kindlin-1/beta1 integrin interaction. Additionally, we detected a higher number of activated beta1 integrins in COLXVI cells and found a neo-expression of alpha1 integrin subunit on these cells. FACS analysis revealed a significantly higher amount of COLXVI cells in S-phase and G2/M-phase six hours after synchronisation leading to a markedly higher proliferation activity. In summary, we demonstrate that overexpression of type XVI collagen in aberrant oral keratinocytes of OSCC-patients leads to Kindlin-1 induction, increased Kindlin-1/beta1-integrin interaction, integrin activation and subsequently to a proliferative cellular phenotype which leads to OSCC.

Arrowup Top


Figures

Oscc

Figure 1: Immunofluorescent stainings revealed a marked overexpression of type XVI collagen in the superficial layers of tissue from OSCC patients (a, b). On the contrary, there is only weak type XVI collagen expression in normal superficial oral epithelium (c,d). Additionally, a decrease of type XVI collagen basement membrane staining was observed during the progression of OSCC compared to normal mucosa (see white arrows a-d, bar: 600µm, magnification 10X). Images are representative images from 10 OSCC patients.

Ligation

Figure 2: Proximity ligation assay of beta1 integrin and Kindlin-1 in type XVI collagen overexpressing cell lines. Because it is known that Kindlin-1 binds to the C-terminal region of beta1 integrin we tested whether Kindlin-1/integrin interaction is changed in type XVI collagen overexpressing OSCC cells. Indeed our findings showed significantly more Kindlin-1/beta1 integrin interaction in type XVI collagen overexpressing OSCC cells (a,b) than in the control cells (c,d, e: negative control). f: Quantification of Kindlin-1/beta1 integrin interaction. g: Examining the expression of alpha1 integrin subunit on the surface of type XVI collagen overexpressing cell lines revealed a neo-expression of alpha1 integrin on their surface compared to mock control cells.


Selected publications

Kassner A., Tiedemann K., Notbohm H., Ludwig T., Mörgelin M., Reinhardt DP., Chu ML., Bruckner P., Grässel S.: Molecular structure and interaction of recombinant human type XVI collagen. J. Mol. Biol. 339(4): 835-853, 2004

Ratzinger S., Eble J.A., Pasoldt A., Opolka A., Rogler G., Grifka J., Grässel S.: Collagen XVI induces formation of focal contacts on intestinal myofibroblasts isolated from the normal and inflamed intestinal tract. Matrix Biol. 2010 Apr;29 (3):177-933.

Ratzinger S., Grässel S., Dowejko A., Reichert T.E., Bauer R.J.: Induction of type XVI collagen expression facilitates proliferation of oral cancer cells. Matrix Biol. 30(2):118-25. 2011.

Investigators

Prof. Dr. Susanne Grässel, Anja Pasoldt, Orthopedic Surgery, Dr. Richard Bauer, Brigitte Hauer, MKG, University Hospital Regensburg, N.N. (Doktorand und TA)

Funding

Deutsche Krebshilfe

Arrowup Top


  1. Fakultät für Medizin

Forschungsprojekte

Experimentelle Orthopädie
Lab014
Weitere Infos

Zentrum für Medizinische Biotechnologie
im BioPark Regensburg