Funktionelle Aspekte von Kollagen XVI und seiner NC11-Domäne bei der Progression des oralen Plattenepithelkarzinoms

Kollagen XVI gehört zu der Familie der FACIT Kollagenen (fibril associated collagen with interrupted triple helix). In der oralen Mukosa ist es an der dermal-epidermalen Verbindungszone lokalisiert. Eine verstärket Kollagen XVI Expression konnte im oralen Plattenepitelkarzimom (englisch: oral squamous cell carcinoma (OSCC)) und im Gliom beobachtet werden. Die Induktion der Kollagen XVI Expression in der humanen OSCC Zelllinie PCI13 zeigte eine dosisabhängigen Einfluss auf die Zellproliferation und -invasion. Zudem bewirkt die verstärkte Kollagen XVI Expression eine Hochregulation der MMP-9 Expression und die Invasion der Kollagen XVI Zellen. Bei Kollagen XVI überexprimierenden Zellen bindet Kollagen XVI an β1 Integrine. Wir konnten zeigen, dass die gesteigerte Kollagen XVI Expression in diesen Zellen, abhängig von fokalen Kontakten, eine Interaktion der ILK mit Kinlin-1 verursacht. Des Weiteren führt die erhöhte Kollagen XVI Expression zur Aktivierung des ILK/Akt Signalweges, was wiederum über den Transkriptionsfaktor AP-1 die Aktivierung der MMP-9 Genexpression bewirkt. Neben dem Volllängekollagen XVI konnten wir in vitro und in Plasmaproben von OSCC-Patienten spezifische, trunkierte Formen des Kollagen XVI beobachten. Wir fanden bis zu diesem Zeitpunkt nicht identifizierte, prozessierte Kollagen XVI Fragmente mit einem molekularen Gewicht von 100kDa, 60 kDa und 40 kDa, als auch ein Fragment, das in seiner Größe der NC11-Domäne von Kollagen XVI entspricht (ca.35 kDa, bestätigt durch Massenspektrometrie). Die Überexpression der NC11-Domäne in der humane OSCC Zelllinie PCI 13 (NC11 Zellen) hat bei der Kultivierung auf Matrigel® die Ausbildung von kanalähnlichen Netzstrukturen zur Folge. Gestützt werden diese Daten durch die Beobachtung, dass die NC11 Überexpression auch zu einer verstärkten Gen- und Proteinexpression der Rezeptoren VEGFR1, VEGFR2 und des uPa-Rezeptors führt. Die spezifische VEGFR Inhibition verhindert die Ausbildung der kanalähnlichen Netzstrukturen bei NC11 Zellen. Diese Befunde lassen vermuten, dass die Überexpression der NC11-Domäne von Kollagen XVI, durch die gesteigerte Expression der endothelassoziierten Rezeptoren VEGFR1, VEGFR2 und den uPa Rezeptor, Einfluss auf die vaskulogene Mimikry der OSSC Tumorzellen nimmt

Functional Characterization of Water Transport and Cellular Localization of Three Aquaporin Paralogs in the Salmonid Intestine

Intestinal water absorption is greatly enhanced in salmonids upon acclimation from freshwater (FW) to seawater (SW); however, the molecular mechanism for water transport is unknown. We conducted a pharmacological characterization of water absorption in the rainbow trout intestine along with an investigation of the distribution and cellular localization of three aquaporins (Aqp1aa, -1ab, and -8ab) in pyloric caeca, middle (M), and posterior (P) intestine of the Atlantic salmon. In vitro iso-osmotic water absorption (Jv) was higher in SW than FW-trout and was inhibited by (mmol L−1): 0.1 KCN (41%), 0.1 ouabain (72%), and 0.1 bumetanide (82%) suggesting that active transport, Na+, K+-ATPase and Na+, K+, 2Cl−-co-transport are involved in establishing the driving gradient for water transport. Jv was also inhibited by 1 mmol L−1 HgCl2, serosally (23% in M and 44% in P), mucosally (27% in M), or both (61% in M and 58% in P), suggesting involvement of both apical and basolateral aquaporins in water transport. The inhibition was antagonized by 5 mmol L−1 mercaptoethanol. By comparison, 10 mmol L−1 mucosal tetraethylammonium, an inhibitor of certain aquaporins, inhibited Jv by 20%. In the presence of glucose, mucosal addition of phloridzin inhibited water transport by 20%, suggesting that water transport is partially linked to the Na+-glucose co-transporter. Using polyclonal antibodies against salmon Aqp1aa, -1ab, and -8ab, we detected Aqp1aa, and -1ab immunoreactivity in the brush border and sub-apical region of enterocytes in all intestinal segments. The Aqp8ab antibody showed a particularly strong immunoreaction in the brush border and sub-apical region of enterocytes throughout the intestine and also stained lateral membranes and peri-nuclear regions though at lower intensity. The present localization of three aquaporins in both apical and lateral membranes of salmonid enterocytes facilitates a model for transcellular water transport in the intestine of SW-acclimated salmonids

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

COLXVI overexpression induces MMP9 expression.

<p>(<b>A</b>) Immunoblot analysis of collagen XVI secretion in supernatants of COLXVI cell clones (clones 1-4) and mock control cells (mock 1-2). Only COLXVI cell clones secret the full-length form of COLXVI (213 kDa; black arrow). Clones 3 and 4 exhibit higher COLXVI secretion than clones 1 and 2. COLXVI cell clones also secrete collagen XVI fragments. A Coomassie Blue membrane staining was used as loading control. (<b>B</b>) Quantitative PCR of <i>MMP9</i> expression in COLXVI cell clones and mocks after 24 h incubation with/without recombinant collagen XVI. COLXVI cell clones (1-4) show a significant expression of <i>MMP9</i> that is further enhanced by the addition of recombinant collagen XVI (n = 3). (<b>C</b>) Gelatin zymography of COLXVI cell supernatant and mock controls. The COLXVI cell clones (1-4) show a clear gelatinolytic activity at 92 kDa (pro form). In contrast, the mock control cells (1-2) show very weak MMP9 bands. (<b>D</b>) Immunoblot of total ILK and phosphorylated ILK (P ILK) isolated from membrane fractions of COLXVI cell clones and mock control cells. ILK is activated in COLXVI cell clones whereas in mock control cells P-ILK is lacking. (<b>E</b>) Quantitative PCR of <i>MMP9</i> expression after ILK inhibition with Cpd 22 (ILKi). The expression of <i>MMP9</i> decreased after ILK inhibition in the COLXVI cell clones. (n = 3). (<b>F</b>) Gelatin zymography of the supernatant of the COLXVI cell clone 3 after ILK inhibition with Cpd 22 (ILKi; c  =  300nM). After ILK inhibition the COLXVI cell clone depicts decreased MMP9 secretion. (<b>G</b>) Promoter activity of the COLXVI cell clone 3 after ILK inhibition with Cpd 22 (ILKi; c  =  300 nM). ILK inhibition results in a significant decrease of the MMP9b promoter activity. (p<0.001; n = 3). (<b>H</b>) Immunoblot of total Akt/PKB and phosphorylated Akt/PKB (P-Akt/PKB) in cell lysates of COLXVI cell clones (clones 1-4) and mock control cells (1-2). Akt/PKB activation is increased in COLXVI cell clones compared to mock controls.</p

The AP-1 binding site at –98bp is important for collagen XVI dependent MMP9 induction.

<p>(<b>A</b>) Schematic presentation of the three different MMP9 promoter fragments. MMP9a, MMP9b, and MMP9c contain 0, 1, and 2 AP-1 binding sites, respectively. (<b>B</b>) Comparison of MMP9 promoter activities of MMP9a, MMP9b, and MMP9c in COLXVI and control cells, respectively. The shortest MMP9 promoter fragment (MMP9a) exhibits the lowest activation. In general, COLXVI clones reveal a higher activation of the <i>MMP9</i> promoter than the mock control cells. The luciferase reporter shows increased activation of the <i>MMP9</i> promoter in clone high compared to clone low (n = 3). (<b>C</b>) Schematic presentation of the MMP9b promoter and the MMP9b deletion promoter, respectively. The MMP9b deletion promoter does not contain the AP-1 binding site 98 bp upstream from the start codon. (<b>D</b>) Promoter activity of the MMP9b promoter after deletion of the AP-1 binding site. After deletion of the AP-1 binding site the MMP9b promoter activity decreases significantly (n = 3).</p

Collagen XVI overexpression leads to c-Fos – dyskerin interaction and increased nuclear JunB localization.

<p>(<b>A</b>) Immunoblot analyses of JunB and c-Fos nuclear extracts from COLXVI cell clones and mock control cells. COLXVI cell clones show a higher amount of JunB compared to mock control cells. COLXVI cell clones do not differ from mock control cells in c-Fos expression. (<b>B</b>) Immunofluorescence staining of c-Fos (green) in COLXVI cell clones and mock control cells (scale bar equals 50 µm). COLXVI cell clones do not differ from mock control cells in their c-Fos expression. Quantification was performed measuring the fluorescent intensity of 100 COLXVI cells and mock controls, each. (<b>C</b>) Silver stained gel of protein lysates from COLXVI cell clones and mock control cells, respectively, after immune precipitation of c-Fos. In COLXVI cell clones a band with a size of 58 kDa was differentially expressed. Mass spectrometry revealed it as dyskerin. (<b>D</b>) Co-immunoprecipitation of c-Fos and dyskerin of protein extracts isolated from COLXVI clones and mocks. COLXVI cell clones exhibit an increased interaction of c-Fos and dyskerin compared to mock control cells. (E) Dyskerin immunoblot of nuclear extracts from COLXVI cell clones and mock control cells. COLXVI cell clones exhibit a higher protein amount of dyskerin than mock control cells. (<b>F</b>) Immunofluorescence staining of dyskerin (red) in COLXVI cell clones and mock control cells (scale bar equals 50 µm). COLXVI cell clones showed strong dyskerin staining compared to mock control cells (<b>G</b>). Immunofluorescence staining of JunB (red) in COLXVI clones and mock control cells (scale bar equals 50 µm). COLXVI cell clones exhibit stronger JunB staining compared to mock control cells. Quantification of signal intensity demonstrates significantly increased nuclear JunB expression in COLXVI cells compared to mock controls.</p

Collagen XVI overexpression leads to an increase of ILK/kindlin-1 interaction.

<p>(<b>A</b>) Proximity ligation assay for the analysis of ILK/kindlin-1 interaction in COLXVI and mock control cells (scale bar equals 50 µm). COLXVI cell clones exhibit an increased interaction of kindlin-1 and ILK at focal adhesions compared to mock control cells (<b>A+B</b>). After inhibition of focal adhesion formation with soluble RGD peptides (c  =  100 µg/mL) ILK/kindlin-1 interaction was significantly reduced (p<0.001, n = 100) (A+<b>C</b>). (<b>D</b>) Quantitative PCR of <i>MMP9</i> gene expression in COLXVI cell clones and mocks after 24 h incubation with soluble RGD peptides. Inhibition of focal adhesions via soluble RGD-peptides resulted in a dose-dependent decrease in <i>MMP9</i> gene expression (n = 3). (<b>E</b>) Co-Immunoprecipitation of ILK and kindlin-1 of protein extracts isolated from COLXVI clones and mocks. COLXVI cell clones exhibit an increased ILK/kindlin-1 interaction depending on collagen XVI dose. Mock control cells showed least ILK/kindlin-1 interaction.</p

Collagen XVI overexpression leads to increased invasion of OSCC cells.

<p>(<b>A</b>) Quantitative PCR of <i>MMP9</i> gene expression of COLXVI and mock control cells after AP-1 inhibition with Tanshinone IIA (TIIA; c  =  100 ng/mL). The expression of <i>MMP9</i> decreased in the COLXVI cell clones after AP-1 inhibition with Tanshinone IIA. (n = 3) (<b>B</b>) 3D micromass pellets of COLXVI cells compared to mock control cells 24 h after placement. COLXVI cells (high and low expressing) show a significantly wider invasion zone (orange line) compared to mock control cells (p<0.01; n = 10). (<b>C</b>) 3D micromass pellets of mock control cells after 24 h of incubation with 500 ng/mL recombinant collagen XVI. Incubation with recombinant collagen XVI resulted in a significantly increased spreading of the invasion zone (orange line) compared to the control (p<0.001; n = 10).</p

TissueQuest-based characterization of infected macrophages.

<p>Macrophages were cultured in 96-well plates in the presence of <i>L</i>. <i>major</i> parasites (parasite/macrophage ratio 5:1). (A) The infected macrophages were determined automatically by the TissueQuest software. The circle diagrams depict the distribution of infected (black) and not infected macrophages (grey). The infection rate is indicated within the circle diagram. A representative experiment visualizing the infection rate after measuring 310 or 11237 macrophages is shown. (B) The box-plots depict the TissueQuest based quantification of the average number of intracellular parasites per macrophage after analysis of 237 or 8639 macrophages. The gray boxes indicate the range between the first and third quartile. The horizontal lines indicate the median. The whiskers visualize the spread of data. The red bracket highlights the additional information that was generated after measuring 8639 infected macrophages compared to analyzing 237 infected macrophages (***<0.0001). The data shown in (A) and (B) are representative for three experiments. (C) Macrophages were analyzed regarding their number of intracellular parasites and the amount of macrophages (y-axis) harboring the indicated number of parasites (x-axis) are shown. Data are presented in box-blots. The circle diagram highlights the number of macrophages harboring 1–6 (light gray), 7–12 (dark gray) and 12–20 (black) parasites. Data are representative for three experiments. (D) Validation of TissueQuest-based quantification of parasites by real-time PCR was performed. Macrophages were infected with different numbers of parasites per host cell (1:1 and 5:1) and 96 hours post infection the average number of parasites per macrophage (right y-axis) and the number of <i>L</i>. <i>major</i> parasites per β-actin was determined (left y-axis) (*<0,01, *** 0,0001; n = 3; mean +/- SD).</p