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. 2007 Jul;171(1):124-38.
doi: 10.2353/ajpath.2007.051264.

SIKVAV, a laminin alpha1-derived peptide, interacts with integrins and increases protease activity of a human salivary gland adenoid cystic carcinoma cell line through the ERK 1/2 signaling pathway

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SIKVAV, a laminin alpha1-derived peptide, interacts with integrins and increases protease activity of a human salivary gland adenoid cystic carcinoma cell line through the ERK 1/2 signaling pathway

Vanessa M Freitas et al. Am J Pathol. 2007 Jul.

Abstract

Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm. We studied the induction of protease activity by the laminin-derived peptide, SIKVAV, in cells (CAC2) derived from this neoplasm. Laminin alpha1 and matrix metalloproteinases (MMPs) 2 and 9 were immunolocalized in adenoid cystic carcinoma cells in vivo and in vitro. CAC2 cells cultured on SIKVAV showed a dose-dependent increase of MMP9 as detected by zymography and colocalization of alpha3 and alpha6 integrins. Small interfering RNA (siRNA) knockdown of integrin expression in CAC2 cells resulted in decreased adhesion to the peptide. SIKVAV affinity chromatography and immunoblot analysis showed that alpha3, alpha6, and beta1 integrins were eluted from the SIKVAV column, which was confirmed by mass spectrometry and a solid-phase binding assay. Small interfering RNA experiments also showed that these integrins, through extracellular signal-regulated kinase (ERK) 1/2 signaling, regulate MMP secretion induced by SIKVAV in CAC2 cells. We propose that SIKVAV increases protease activity of a human salivary gland adenoid cystic carcinoma cell line through alpha3beta1 and alpha6beta1 integrins and the ERK 1/2 signaling pathway.

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Figures

Figure 1
Figure 1
Laminin α1, MMP2, and MMP9 are detected in adenoid cystic carcinomas. Laminin α1 appears as diffuse staining throughout the cytoplasm in cribriform and solid subtypes (A and B), with some cell surface staining that appears as a linear structure that is likely in the basement membrane in the cribriform subtype (C). Negative serum controls (D). MMP2 (E and F) and MMP9 (G and H) are observed in pseudocystic (PS), solid (S), and tubular subtypes of the tumor. MMP2 also fills tubular spaces (asterisk in F). MMP9 is present in pseudocystic spaces (G) and in stromal regions (arrow in H). Magnifications: ×200 (A, B, D, and H); ×400 (E and G); and ×600 (C and F).
Figure 2
Figure 2
CAC2 cells also express laminin α1, MMP2, and MMP9. Laminin α1 appears as punctate staining on the cell membrane (A). MMP2 appears in the cytoplasm and is prominent on the cell periphery (B). MMP9 appears in the cytoplasm (C). Nuclei are counterstained with Sytox green. Magnification, ×630.
Figure 3
Figure 3
SIKVAV induces MMP9 in a dose-dependent manner. The conditioned medium of CAC2 cells cultured on SIKVAV or a scrambled (IVSKVA) control peptide (5, 20, 50, and 100 μg) were analyzed by zymography (A). MMP2- and MMP9-positive controls (Std) are included, and treatments with either 1,10-phenantroline or EDTA (negative controls) demonstrate that the bands are MMPs (A, top). The volume of conditioned medium loaded on the zymogram gel was normalized to the amount of protein in the CAC2 cell lysate. Gel densitometry of the zymograms shows the dose-dependent increase of MMP9 compared with control (B). Results represent a mean ± SEM of six experiments.
Figure 4
Figure 4
CAC2 adhesion to SIKVAV is sensitive to EDTA. CAC2 cells adhere to laminin and SIKVAV but not to BSA and a scrambled peptide (IVSKVA) in a cell adhesion assay (A). EDTA decreases CAC2 adhesion to SIKVAV (B). The results (±SEM) are triplicate experiments performed at least three times.
Figure 5
Figure 5
Integrins α3 and α6 colocalize on CAC2 cells cultured on SIKVAV and laminin-111. Immunofluorescence staining of CAC cells cultured on SIKVAV, IVSKVA, and laminin-111 shows α6 integrin (green in A, E, and I), α3 integrin (red in B, F, and J), and nuclei (blue in C, G, and K) counterstained with 4,6-diamidino-2-phenylindole. Colocalization appears yellow in the overlay image (D, H, and L). Cells grown on SIKVAV show discrete areas of colocalization (white arrowheads in D), whereas cells on laminin-111 show extensive overlap of α3 and α6 integrins (L). Magnification, ×630.
Figure 6
Figure 6
siRNA knockdown of α3, α6, and β1 integrins and syndecan-1 in CAC2 cells results in decreased protein and mRNA expression. CAC2 cells were cultured for 30 hours after siRNA treatment, and protein levels were analyzed by Western blot (A) of the integrin subunit as well as β-actin to show equal protein loading in the gel. There was at least 50% decrease in protein levels for all proteins targeted. After 30 hours of siRNA treatment, the less glycosylated β1 integrin isoform, which is faster migrating on a gel, is not detected whereas some fully glycosylated β1 remains. Quantitative PCR (B) confirms the knockdown of gene expression. The control is transfected with the nonsilencing siRNA. Quantitative PCR results (±SEM) are triplicates repeated at least three times.
Figure 7
Figure 7
There is decreased CAC2 cell adhesion to SIKVAV after siRNA knockdown of α3, α6, and β1 integrins but not syndecan-1. The adhesion of CAC2 cells with decreased levels of α3, α6, and β1 integrins and syndecan-1 were compared with SIKVAV, laminin-111, type I collagen, and fibronectin. Decreasing either α3 or α6 integrins results in decreased adhesion to SIKVAV and laminin-111, whereas knockdown of β1 integrin reduces cell adhesion to all substrates. Decreasing syndecan-1 has no effect on adhesion to SIKVAV, type I collagen, or fibronectin. The control is transfected with the nonsilencing siRNA. Results (±SEM) are triplicates repeated at least three times.
Figure 8
Figure 8
SIKVAV affinity chromatography of CAC2 cell membranes identifies putative SIKVAV ligands. SIKVAV affinity columns were compared with columns with either IVSKVA (scrambled control) or BSA, and fractions eluted from the columns were visualized by silver staining after SDS-polyacrylamide gel electrophoresis. The fraction eluted with SIKVAV appears as a broad high-molecular weight smear with discrete bands of ∼130 kd (top gel, lane SIK). Similar bands are observed in fractions eluted with either EDTA or ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (top gel). Fractions are also eluted with high salt (NaCl) and glycine (Gly). ST, molecular weight standards.
Figure 9
Figure 9
Integrins α3, α6, and β1 are present in the fraction eluted by SIKVAV from the peptide affinity column (A). Western blot analysis identified α3, α6, and β1 integrins in the membrane preparation, in the SIKVAV-eluted fraction, and in EDTA-eluted fraction. The same membranes were reprobed for integrin α1 integrin, which is more predominant in the membrane fraction. Mass spectrometry also identified the α6 integrin in the fraction eluted by SIKVAV from the peptide affinity column (B). Coomassie-stained band eluted from SIKVAV affinity column was submitted to in-gel digestion with trypsin and analyzed by mass spectrometry, the tryptic peptides matched the α6 integrin sequence.
Figure 10
Figure 10
SIKVAV binds to integrin antibody IP complexes in a solid-phase binding assay. Integrin α3, α6, and β1 were immunoprecipitated from CAC2 cell lysates. A: A silver-stained gel and subsequent Western blot of lanes 2, 3, and 4 with anti-integrin antibodies. Lane Std, molecular weight marker; lane 1, whole-cell lysate; lane 2, IP with α3 integrin antibody; lane 3, IP with α6 integrin antibody; lane 4, IP with β1 integrin antibody. B: The IP complexes were adsorbed to plastic wells (5 μg/well), and a solid-phase assay with biotinylated SIKVAV was used to show that SIKVAV binds α3, α6, or β1 integrin antibody IP complexes and does not bind the plastic well. Biotinylated IVSKVA or BSA did not bind to integrin IP complexes. Solid-phase results (±SEM) are triplicates repeated at least three times.
Figure 11
Figure 11
There is a significant decrease in SIKVAV-induced MMP9 activity with knockdown of α3, α6, and β1 integrins. CAC2 cells treated with siRNA to integrins were cultured with either SIKVAV (A) or the control peptide IVSKVA (B), and the MMP9 activity was measured by zymography. These results suggest that α3β1 and α6β1 signaling affects protease activity in CAC2 cells. The control is transfected with the nonsilencing siRNA. Densitometric results (±SEM) are six lanes of zymography combined and repeated at least three times. NS, not significant.
Figure 12
Figure 12
SIKVAV-induced MMP secretion in CAC2 cells is dependent on ERK signaling, and decreasing α3 and α6 integrin expression reduces SIKVAV-induced p-ERK. A: CAC2 cells were pretreated with U0126 (10, 25, and 50 μmol/L) for 2 hours before the addition of 100 μg/ml SIKVAV to the medium. A dose-dependent decrease of MMP activity and p-ERK levels are observed by zymography and Western blot analysis with U0126 treatment. A carrier control group (A) was treated with methanol. B: SIKVAV induces p-ERK twofold in CAC2 cells compared with cells cultured on plastic or scrambled peptide IVSKVA. The p-ERK Western blot analysis was quantitated and normalized to total ERK levels. The graph represents triplicates (±SEM) repeated at least three times. C: Decreased levels of α3 and α6 integrins result in reduced p-ERK levels after SIKVAV treatment. Cells transfected with nonsilencing siRNA are the controls.

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