Collagen XVI Research Articles

Regional Differences in Gene Expression of Proliferating Human Choroidal Endothelial Cells

Choroidal diseases including inflammation and neovascularization seem to have predilection for different vascular beds. In order to improve our understanding of human macular choroidal angiogenic diseases, we investigate the differences in gene expression between matched human macular and peripheral inner choroidal endothelial cells (CEC) and matched human macular inner and outer CEC. The gene expression profiles of matched, unpassaged human macular and peripheral inner CEC and matched human unpassaged macular inner and outer CEC were conducted using Affymetrix GeneChip arrays. Selected differences in gene expression were validated by real-time-PCR and immunohistochemistry. No differences in probeset expression were demonstrated between inner CECs compared with peripheral inner CECs. In comparison, there was a difference of 1.6% of probesets when matched, unpassaged proliferating human macular inner CEC and macular outer CEC from the same donors were compared. Macular inner CECs demonstrated up-regulation of probesets involved in nervous system development, growth factors, PLVAP, and collagen XVI, while macular outer CECs demonstrated up-regulation of probesets involved in immune function and intracellular signalling. There was a marked homogeneity of human macular and peripheral inner CECs. This suggests that gene expression differences in inner CECs are not responsible for the site specific selectivity of choroidal neovascularisation. Variability was noted, however, in the gene expression of matched macular inner and outer CECs. This could be explained by the differences in the roles and microenvironments of the inner and outer choroid.

Increased peri-ductal collagen micro-organization may contribute to raised mammographic density.

BackgroundHigh mammographic density is a therapeutically modifiable risk factor for breast cancer. Although mammographic density is correlated with the relative abundance of collagen-rich fibroglandular tissue, the causative mechanisms, associated structural remodelling and mechanical consequences remain poorly defined. In this study we have developed a new collaborative bedside-to-bench workflow to determine the relationship between mammographic density, collagen abundance and alignment, tissue stiffness and the expression of extracellular matrix organising proteins.MethodsMammographic density was assessed in 22 post-menopausal women (aged 54–66 y). A radiologist and a pathologist identified and excised regions of elevated non-cancerous X-ray density prior to laboratory characterization. Collagen abundance was determined by both Masson’s trichrome and Picrosirius red staining (which enhances collagen birefringence when viewed under polarised light). The structural specificity of these collagen visualisation methods was determined by comparing the relative birefringence and ultrastructure (visualised by atomic force microscopy) of unaligned collagen I fibrils in reconstituted gels with the highly aligned collagen fibrils in rat tail tendon. Localised collagen fibril organisation and stiffness was also evaluated in tissue sections by atomic force microscopy/spectroscopy and the abundance of key extracellular proteins was assessed using mass spectrometry.ResultsMammographic density was positively correlated with the abundance of aligned periductal fibrils rather than with the abundance of amorphous collagen. Compared with matched tissue resected from the breasts of low mammographic density patients, the highly birefringent tissue in mammographically dense breasts was both significantly stiffer and characterised by large (>80 μm long) fibrillar collagen bundles. Subsequent proteomic analyses not only confirmed the absence of collagen fibrosis in high mammographic density tissue, but additionally identified the up-regulation of periostin and collagen XVI (regulators of collagen fibril structure and architecture) as potential mediators of localised mechanical stiffness.ConclusionsThese preliminary data suggest that remodelling, and hence stiffening, of the existing stromal collagen microarchitecture promotes high mammographic density within the breast. In turn, this aberrant mechanical environment may trigger neoplasia-associated mechanotransduction pathways within the epithelial cell population.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0664-2) contains supplementary material, which is available to authorized users.

The NC11 domain of human collagen XVI induces vasculogenic mimicry in oral squamous cell carcinoma cells.

Collagen XVI, a fibril-associated collagen with interrupted triple helix (FACIT) collagen, is involved in oral squamous cell carcinoma (OSCC) and glioblastoma progression. The NC11 domain of collagen XVI has been described previously with a strong implication in physiological processes. We detected the non-collagenous (NC) 11-domain in supernatants of OSCC cells after recombinant expression of full-length collagen XVI and in sera from OSCC patients and healthy individuals. Stable expression of NC11-green fluorescent protein (GFP) fusion protein in OSCC cells initiated proliferation control and block of anchorage-independent growth. Moreover, the NC11 domain triggered the generation of tubular-like net structures on laminin-rich matrix in contrast to mock-GFP control cells and cells expressing full-length collagen XVI. Taqman® quantitative PCR and diaminobenzidine staining in 2D- and 3D cell culture revealed a significantly increased gene and protein expression of VEGFR1, VEGFR2 and uPAR in recombinant NC11-GFP-expressing cells. Specific VEGF receptor inhibition with Axitinib or fetal calf serum heat inactivation prevented formation of tubular-like net structures. Accordantly, NC11-GFP coated culture slides led to an increase of focal adhesion contact formation and the upregulation of VEGFR1 and uPAR in three different non-transfected OSCC cell lines. In summary, we suggest that the NC11 domain of collagen XVI is a potential biomarker for OSCC and triggers vasculogenic mimicry via upregulation of endothelial receptors VEGFR1, VEGFR2 and uPAR in 2D- and 3D OSCC cell culture conditions.

Collagen XVI Induces Expression of MMP9 via Modulation of AP-1 Transcription Factors and Facilitates Invasion of Oral Squamous Cell Carcinoma

Collagen XVI belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). It is overexpressed during the progression of oral squamous cell carcinoma (OSCC). The present data show a strong collagen XVI-dependent induction of MMP9 and an increase in OSCC cell invasion. We found activated integrin-linked kinase (ILK) in a complex with kindlin-1 and activation of protein kinase B (PKB/Akt) to be responsible for MMP9 induction. Inhibition of the formation of focal adhesions reduced MMP9 expression. Moreover, collagen XVI overexpressing OSCC cell clones (COLXVI cell clones) transfected with vectors containing different MMP9 promoter fragments adjacent to a luciferase reporter revealed an increase in luciferase signal dependent on AP-1 binding sites. Deletion of the AP-1 binding site 98 bp upstream of the reported transcription start site and inhibition of AP-1 with Tanshinone IIA resulted in decreased MMP9 expression. The AP-1 subunit JunB showed differential expression between COLXVI cell clones and mock control cells. Additionally, mass spectrometric analysis of immunoprecipitates revealed that c-Fos interacted strongly with dyskerin in COLXVI cell clones compared to mock controls.

MicroRNA-152 and -181a participate in human dermal fibroblasts senescence acting on cell adhesion and remodeling of the extra-cellular matrix

Ageing of human skin is associated with phenotypic changes in the cutaneous cells; the major functional markers of ageing occur as consequences of dermal and epidermal cell senescence and of structural and compositional remodeling of normally long-lived dermal extracellular matrix proteins. Understanding the contribution of the dermal cells in skin ageing is a key question, since this tissue is particularly important for skin integrity and its properties can affect the epidermis. Several microRNAs have been shown to be involved in the regulation of pathways involved in cellular senescence and exerted important effects on tissues ageing. In this study, we demonstrate that the expression of miR-152 and miR-181a increased during the human dermal fibroblasts senescence and that their overexpression, is sufficient to induce cellular senescence in early-passage cells. The increase of these miRNAs during cells senescence was accompanied by a decrease in integrin α5 and collagen XVI expression at mRNA and/or protein levels resulting in reduced cellular adhesion and suggesting extracellular matrix remodeling. These findings indicate that changes in miRNAs expression, by modulating the levels of adhesion proteins and extra-cellular matrix components, such as integrin α5 and collagen XVI, could contribute to the compositional remodelling of the dermis and epidermis occurring during skin aging.

Skin benefits of a myconoside‐rich extract from resurrection plant Haberlea rhodopensis

Resurrection plant Haberlea rhodopensis develops molecules to survive drought stress. These molecules allow the plant to resurge from a desiccation state. We have extracted a specific fraction from the plant (Haberlea extract) and found it rich, among other molecules, of a caffeoyl phenylethanoid glycoside called myconoside, a molecule extremely abundant in the plant with a potential role in survival. Peroxide-stressed normal human dermal fibroblasts treated with the Haberlea extract, showed increased collagen VI (+822%), collagen XVI (+928%) and elastin (+144%) mRNA synthesis, measured by RT-qPCR. This effect was superior to those obtained with benchmarks retinoic acid and retinol. When used at 3% in human skin biopsies, Haberlea extract protected against UV-induced dermis oxidation by 100% (P < 0.01), as evidenced by immunohistochemistry. Finally, when tested in human volunteers (n = 20) at 3% in a cream against a placebo, Haberlea extract increased skin elasticity (3× placebo, P < 0.0002) and skin radiance (4× placebo, P < 0.05) after only 15 days of treatment, with the effect sustained after 30 and 60 days of treatment. We demonstrated that by using Haberlea extract (particularly rich in glycoside myconoside), it is possible to strongly stimulate antioxidant skin defences and extracellular matrix protein synthesis. This effect, in turn, will further stimulate skin elasticity and skin radiance significantly in human volunteers. The extract can be suggested for anti-ageing treatments, intended for claims such as protection from oxidation, increased skin elasticity and enhanced skin radiance.

BMP activated Smad signaling strongly promotes migration and invasion of hepatocellular carcinoma cells

Several of the different bone morphogenetic proteins (BMPs) are involved in development and progression of specific tumors. For hepatocellular carcinoma (HCC) only BMP4 and BMP6 are described to be important for carcinogenesis. However, up to now neither the influence of other BMPs on tumor progression, nor the responsible signaling pathways to mediate target gene expression in HCC are known.In order to characterize BMP expression pattern in HCC cell lines, we performed RT-PCR analysis and revealed enhanced expression levels of several BMPs (BMP4, 6, 7, 8, 9, 10, 11, 13 and 15) in HCC. Thus, we treated HCC cells with the general BMP inhibitors chordin and noggin to determine the functional relevance of BMP overexpression and observed decreased migration and invasion of HCC cells. A cDNA microarray of noggin treated HCC cells was performed to analyze downstream targets of BMPs mediating these oncogenic functions. Subsequent analysis identified collagen XVI as ‘Smad signaling specific’ and nidogen-2 as ‘MAPK/ERK signaling specific’ BMP-target genes. To examine which signaling pathway is mainly responsible for the oncogenic role of BMPs in HCC, we treated HCC cells with dorsomorphin to determine the influence of BMP activated Smad signaling. Interestingly, also migratory and invasive behavior of dorsomorphin treated HCC cells was diminished.In summary, our findings demonstrate enhanced expression levels of several BMPs in HCC supporting enhanced migratory and invasive phenotype of HCC cells mainly via activation of Smad signaling.

Inhibition of Collagen XVI Expression Reduces Glioma Cell Invasiveness

Glioblastomas are characterized by an intense local invasiveness that limits surgical resection. One mechanism by which glioma cells enforce their migration into brain tissue is reorganization of tumour associated extracellular matrix (ECM). Collagen XVI is a minor component of connective tissues. However, in glioblastoma tissue it is dramatically upregulated compared to the ECM of normal cortex. The aim of this study is to delineate tumour cell invasion and underlying mechanisms involving collagen XVI by using a siRNA mediated collagen XVI knockdown model in U87MG human glioblastoma cells. Knockdown of collagen XVI resulted in decreased invasiveness in Boyden chamber assays, and in a reduction of focal adhesion contact numbers per cell. Gene expression was upregulated for protocadherin 18 and downregulated for kindlin-1 and -2. Proliferation was not affected while flow cytometric analysis demonstrated reduced β1-integrin activation in collagen XVI knockdown cells. We suggest that in glioblastoma tissue collagen XVI may impair the cell-cell interaction in favour of enhancement of invasion. The modification of the β1-integrin activation pattern through collagen XVI might be a molecular mechanism to further augment the invasive phenotype of glioma cells. Elucidating the underlying mechanisms of glioma cell invasion promoted by collagen XVI may provide novel cancer therapeutic approaches in neurooncology.

Collagen XVI induces formation of focal contacts on intestinal myofibroblasts isolated from the normal and inflamed intestinal tract

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 α1. 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 α1β1 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.

Collagen XVI expression is upregulated in glioblastomas and promotes tumor cell adhesion

The poor prognosis of glioblastoma patients is related to diffuse brain invasion and interaction of tumor cells with extracellular matrices (ECM). We describe expression and function of the FACIT-collagen XVI in glioblastomas. We found upregulation of collagen XVI mRNA as well as protein in glioblastomas as compared to normal cortex. SiRNA knockdown resulted in decreased cell adhesion whereas increased adhesion was observed on surfaces coated with collagen XVI. The migration of glioblastoma cells on this substrate remained unchanged. Our results demonstrate de-novo expression of collagen XVI in glioblastomas as part of the tumor specific remodeling of the ECM. Structured summaryMINT-6743179:Collagen IV (uniprotkb:P02462-1) and Collagen XVI (uniprotkb:Q07092) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-6743170:GFAP (uniprotkb:P14136) and Collagen XVI (uniprotkb:Q07092) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Collagen XVI is a neural component of the developing and regenerating dorsal root ganglia extracellular matrix

Collagen XVI is a neural component of the developing and regenerating dorsal root ganglia extracellular matrix

Collagen XVI Harbors an Integrin α1β1 Recognition Site in Its C-terminal Domains

Collagen XVI is integrated tissue-dependently into distinct fibrillar aggregates, such as D-banded cartilage fibrils and fibrillin-1-containing microfibrils. In skin, the distribution of collagen XVI overlaps that of the collagen-binding integrins alpha1 beta1 and alpha2 beta1. Basal layer keratinocytes express integrin alpha2 beta1, whereas integrin alpha1 beta1 occurs in smooth muscle cells surrounding blood vessels, in hair follicles, and on adipocytes. Cells bearing the integrins alpha1 beta1 and alpha2 beta1 attach and spread on recombinant collagen XVI. Furthermore, collagen XVI induces the recruitment of these integrins into focal adhesion plaques, a principal step in integrin signaling. Of potential physiological relevance, these integrin-collagen XVI interactions may connect cells with specialized fibrils, thus contributing to the organization of fibrillar and cellular components within connective tissues. In cell-free binding assays, collagen XVI is more avidly bound by alpha1 beta1 integrin than by alpha2 beta1 integrin. Both integrins interact with collagen XVI via the A domain of their alpha subunits. A tryptic collagen XVI fragment comprising the collagenous domains 1-3 is recognized by alpha1 beta1 integrin. Electron microscopy of complexes of alpha1 beta1 integrin with this tryptic collagen XVI fragment or with full-length collagen XVI revealed a unique alpha1 beta1 integrin-binding site within collagen XVI located close to its C-terminal end.

Molecular Structure and Interaction of Recombinant Human Type XVI Collagen

Collagen XVI is a minor component of at least two different extracellular fibrillar networks of specialized regions of skin and cartilage. In skin, collagen XVI is integrated into particular fibrillin-rich microfibrils lacking an amorphous elastin core. In cartilage, collagen XVI is a component of small heterotypic D-banded fibrils, mainly occurring in the territorial matrix of chondrocytes. Here, we present the first direct evidence for the molecular structure and functional properties of these fibril-associated collagens with interrupted triple helices (FACIT). We have expressed recombinantly the full-length α1 chain of human collagen XVI in HEK 293 EBNA cells in large quantities using an episomal expression system. Secreted full-length recombinant collagen XVI forms stable disulfide-bonded homotrimers and is rapidly proteolytically processed to distinct fragments at specific protease sequence motifs, one resembling an aggrecanase recognition site. Limited trypsin digestion assays and thermal transition curves imply sequential thermal denaturation of individual triple helical domains of this recombinant collagen, similar to authentic collagen XVI. Molecular images of collagen XVI reveal rod-like molecules which harbor multiple sharp kinks attributing a highly flexible structure presumably introduced by non-collagenous (NC) regions. Terminally located cloverleaf-shaped nodules correspond to the large NC NC11 domain of trimeric collagen XVI. The total length of individual trimeric recombinant collagen XVI molecules constitutes about 240 nm as calculated by atomic force and negative staining electron microscopy. Recombinant collagen XVI interacts with fibrillin-1 and with fibronectin indicating multiple molecular interactions in which this ubiquitously expressed and versatile FACIT-collagen can participate. In vitro generated collagen XVI provides an indispensable tool for future determination of its function during supramolecular assembly of matrix aggregates and its role in maintenance, organization and interaction of fibrillar structures.

Discrete integration of collagen XVI into tissue-specific collagen fibrils or beaded microfibrils.

The structural and functional diversity of extracellular matrices is determined, not only by individual macromolecules, but even more decisively, by the alloyed aggregates they form. Although quantitatively major matrix molecules can occur ubiquitously, their organization varies from one tissue to another due to their amalgamation with specific sets of minor components. Here, we show that the fibril-associated collagen with interrupted triple helices collagen XVI is unique in that, depending on the tissue context, it can be incorporated into distinct suprastructural aggregates. In papillary dermis, the protein unexpectedly does not occur in banded collagen fibrils, but rather, is a component of specialized fibrillin-1-containing microfibrils. In territorial cartilage matrix, however, collagen XVI is not a component of aggregates containing fibrillin-1. Instead, the protein resides in a discrete population of thin, weakly banded collagen fibrils also containing collagens II and XI. Collagen IX also occurs in this population of fibrils, but at longitudinal locations discrete from those of collagen XVI. This suprastructural versatility of a collagen is without precedent and highlights pivotal differences in the tissue-specific organization of matrix aggregate structures.

Collagen XVI is expressed by human dermal fibroblasts and keratinocytes and is associated with the microfibrillar apparatus in the upper papillary dermis.

Indirect immunofluorescence staining of normal skin with affinity-purified antibodies revealed a conspicuous presence of collagen XVI at the dermo-epidermal interface where it occurs in close vicinity to collagen VII. In addition, the protein co-localizes with fibrillin 1 at the cutaneous basement membrane zone and the adjacent papillary dermis, but not in deeper layers of the dermis. Both fibronectin and collagen XVI are distributed throughout smooth muscles of hair follicles but do not co-localize. These data suggest, therefore, that collagen XVI contributes to the structural integrity of the dermo-epidermal junction zone by interacting with components of the anchoring complexes and the microfibrillar apparatus. A strong immunofluorescence signal associated with the extracellular matrix of individual cells was observed for keratinocytes or fibroblasts in monolayer cultures. Therefore, both cell types are likely sources of the protein also in situ. The rate of expression of collagen XVI mRNA in keratinocytes is about half of that in normal human skin fibroblasts. In both cell types, TGF-beta2 treatment results in an up-regulation of the collagen XVI-mRNA by approximately 50%. In keratinocytes, synthesis of collagen XVI protein and deposition to the cell layer and the extracellular matrix is stimulated fivefold and twofold, respectively. Since TGF-beta2 also upregulates the biosynthesis of other matrix macromolecules in the subepidermal zone the factor is likely to contribute to the stabilization of matrix zones near basement membranes in healing wounds.

Recombinant Analysis of Human alpha1(XVI) Collagen. Evidence for Processing of the N-Terminal Globular Domain

The N-terminal non-collagenous domain NC11 of the human collagen α1(XVI) chain was obtained as a recombinant 35-kDa protein from stably transfected kidney cell clones. This form had undergone proteolytic trimming at a basic cleavage motif indicating a similar release in vivo. Domain NC11 showed a globular shape after rotary shadowing and was resistant to neutral proteases. Specific antibodies could be raised against recombinant NC11 and were used for the analysis of other cell clones transfected with the full-length α1(XVI) chain. Immunoprecipitation of detergent extracts of metabolically labelled cells demonstrated the presence of disulfide-bonded 200-kDa polypeptides possessing NC11 epitopes. This material was partially resistant to pepsin, indicating the formation of α1(XVI) chain homotrimers with a triple-helical conformation. Yet a substantial proportion of these homotrimers was degraded to fragments of variable size (35–150 kDa) when secreted into the culture medium. Several of these fragments could be obtained on a semi-preparative scale from cells grown in hollow fiber cassettes and showed substantial hydroxylation of proline, consistent with triple-helix formation. Edman degradation demonstrated the origin of some from the N-terminal and of one from a more C-terminal position of collagen XVI. This extensive degradation may be explained by the release of NC11 and by further cleavages within some of the nine interruptions of the triple-helical domain of the α1(XVI) chain. Whether this process also occurs in situ remains to be shown.

Recombinant analysis of human alpha 1 (XVI) collagen. Evidence for processing of the N-terminal globular domain.

The N-terminal non-collagenous domain NC11 of the human collagen alpha 1 (XVI) chain was obtained as a recombinant 35-kDa protein from stably transfected kidney cell clones. This form had undergone proteolytic trimming at a basic cleavage motif indicating a similar release in vivo. Domain NC11 showed a globular shape after rotary shadowing and was resistant to neutral proteases. Specific antibodies could be raised against recombinant NC11 and were used for the analysis of other cell clones transfected with the full-length alpha 1 (XVI) chain. Immunoprecipitation of detergent extracts of metabolically labelled cells demonstrated the presence of disulfide-bonded 200-kDa polypeptides possessing NC11 epitopes. This material was partially resistant to pepsin, indicating the formation of alpha 1 (XVI) chain homotrimers with a triple-helical conformation. Yet a substantial proportion of these homotrimers was degraded to fragments of variable size (35-150 kDa) when secreted into the culture medium. Several of these fragments could be obtained on a semi-preparative scale from cells grown in hollow fiber cassettes and showed substantial hydroxylation of proline, consistent with triple-helix formation. Edman degradation demonstrated the origin of some from the N-terminal and of one from a more C-terminal position of collagen XVI. This extensive degradation may be explained by the release of NC11 and by further cleavages within some of the nine interruptions of the triple-helical domain of the alpha 1(XVI) chain. Whether this process also occurs in situ remains to be shown.