Molecular Mechanism of Tetraspanin CD9 Mediated Cell Motility

CD9, a member of the tetraspanin superfamily of proteins participates in the regulation of cell adhesive functions such as cell migration. The mechanisms underlying CD9 mediated cell migration are not known. In the current study, we investigated the molecular basis for the CD9 promoted cell migration. Our findings show that the phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortamannin and LY294002 inhibited CD9 promoted cell motility in Chinese hamster ovary (CHO) cells. In contrast, inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect on CD9 driven CHO cell motility. Furthermore, inhibition of PI-3K activity in CHO cells by dominant/negative PI-3K cDNA transfection abolished CD9 mediated pro-migratory effects. Consistent with these observations, CD9 expression in CHO cells and in the rat aortic smooth muscle (RASM) cells induced enhanced phosphorylation of PI-3K substrate, Akt. In CHO cells, CD9 expression also enhanced protein levels and tyrosine phosphorylation of the adaptor protein p130Cas. However, no significant changes in the CD9 enhanced migration were observed in CHO cells upon down regulation of p130Cas CD9, a member of the tetraspanin superfamily of proteins participates in the regulation of cell adhesive functions such as cell migration. The mechanisms underlying CD9 mediated cell migration are not known. In the current study, we investigated the molecular basis for the CD9 promoted cell migration. Our findings show that the phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortamannin and LY294002 inhibited CD9 promoted cell motility in Chinese hamster ovary (CHO) cells. In contrast, inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect on CD9 driven CHO cell motility. Furthermore, inhibition of PI-3K activity in CHO cells by dominant/negative PI-3K cDNA transfection abolished CD9 mediated pro-migratory effects. Consistent with these observations, CD9 expression in CHO cells and in the rat aortic smooth muscle (RASM) cells induced enhanced phosphorylation of PI-3K substrate, Akt. In CHO cells, CD9 expression also enhanced protein levels and tyrosine phosphorylation of the adaptor protein p130Cas. However, no significant changes in the CD9 enhanced migration were observed in CHO cells upon down regulation of p130Cas by siRNA transfection suggesting that p130Cas dependent pathways are not mandatory for CD9 mediated motility. To further understand the mechanisms by which CD9 regulates cell migration, we studied the relative contribution of the fibronectin (FN) receptor integrin, alpha-5-beta-1 in CD9 mediated cell motility. Our findings show that CD9 is in molecular complex with alpha-5-beta-1 and that CD9 promoted migration can be completely blocked by an alpha-5-beta-1 function blocking antibody. Further studies revealed that CD9 expression may stabilize the active conformer of the beta-1 integrin. Taken togeather, our study demonstrates key molecular mechanisms governing CD9 mediated haptotactic cell motility to FN in CHO cells. Our findings indicate that CD9 in concert with integrin alpha-5-beta-1 requires activation of the PI-3K pathway leading to enhanced haptotactic cell migration on FN

Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

<div><p>Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion <i>in vivo</i>. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression.</p></div

Silencing MMP-9 in CD9-HT1080 cells is sufficient to suppress the invasive phenotype.

<p>CD9-HT1080 cells were transiently transfected with short-interfering RNA directed to MMP-9 (CD9+ MMP-9 siRNA). Likewise, Mock- and CD9- HT1080 cells were transfected with a scrambled siRNA as a negative control (Mock+Ctr siRNA or CD9+ Ctr siRNA). (<b>A</b>) qRT-PCR analysis was used to measure changes in MMP-9 mRNA levels among Ctr and MMP-9 siRNA transfected cells. (<b>B</b>) Changes in release of MMP-9 (ng/ml) in to the cleared culture supernatant were examined using a MMP-9 specific ELISA kit. (<b>C</b>) Pro-MMP-9 levels in the supernatants of transiently transfected cells was measured using gelatin zymography and (<b>D</b>) quantified by densitometry. (<b>E</b>) A representative image of cells invading though matrigel after transfection with siRNA. (<b>F</b>) Percent cell invasion through matrigel-coated inserts after 20 hours was quantified and the results were normalized to Mock+Ctr siRNA treated cells *, p<0.05; **, p<0.001.</p

MMP-9 expression and release are greatly enhanced in CD9-HT1080 cells.

<p>(<b>A</b>) Fold changes in MMP and TIMP mRNA expression for Mock- and CD9-HT1080 cells were calculated from cycle threshold values using qRT-PCR. (<b>B, C</b>) Specific ELISA kits were used to measure the concentration (ng/ml) of pro- and active- MMP-1 and MMP-9 in the cleared supernatant of Mock- and CD9-HT1080 cells. (<b>D</b>) A representative gelatin zymogram of Mock- and CD9- HT1080 cell conditioned media. The absence of Coomassie staining at 92 kDa indicates the presence of pro-MMP-9 and at 72 kDa represents pro-MMP-2. (<b>E</b>) Quantification of the relative band intensity was calculated by densitometry analysis as described in the Materials and methods section *, p<0.05; **, p<0.001.</p

CD9-HT1080 cells have stable mRNA expression of integrins though α2 and β1 are reduced at the cell surface.

<p>(<b>A</b>) The fold change in mRNA expression of integrins was compared between Mock- and CD9-HT1080 cells using qRT-PCR. (<b>B</b>) Cell surface expression of the same integrin subunits (shaded histograms) was evaluated by flow cytometry. A non-specific isotype-matched antibody (IgG) was used as a negative control (unshaded histograms).</p

An increase in MMP-9 expression and release and subsequent cell invasion requires the second extracellular loop (EC2) of CD9.

<p>(<b>A</b>) MMP-9 mRNA expression was measured in Mock-, CD9-, and Δ6-HT1080 cells using qRT-PCR (<b>B,C</b>) Release of pro-MMP-9 and pro-MMP-2 was measured by gelatin zymography and quantified using Image J. (<b>D,E</b>) A representative picture of cell invasion through matrigel coated inserts and relative quantification of cell invasion *, p<0.05; **, p<0.001.</p

Characterization of the second extracellular loop (EC2) deletion mutant of CD9 in HT1080 cells.

<p>Upon transfection with the EC2 deletion mutant in HT1080 cells, mRNA expression was measured using primer pairs specific for the nucleotides coding the EC2 (<b>A</b>) and TM1 (<b>B</b>) regions of CD9. (<b>C</b>) Flow cytometric analysis of Mock-, CD9-, and Δ6-HT1080 cells is indicated by each row, and each column indicates CD9 EC2 (mAb7), CD9 TM1 (Rap2), or CD151 binding (shaded histograms) **, p<0.001.</p

The invasive phenotype of HT1080 cells is increased upon CD9 overexpression.

<p>A matrigel invasion assay was used to assess the invasive properties of Mock- and CD9-HT1080 cells. Cells were allowed to invade through matrigel and adhere to the bottom of a cell culture insert as detailed in the Materials and methods section. (<b>A</b>) A representative image of stained cells after 20 hours of invasion though matrigel. (<b>B</b>) Invasion through matrigel-coated inserts was compared to cells that invaded through uncoated inserts and results shown are normalized to Mock-HT1080 cell invasion *, p<0.05.</p