Investig Clin Urol.  2019 Sep;60(5):396-404. 10.4111/icu.2019.60.5.396.

Suppression of CD81 promotes bladder cancer cell invasion through increased matrix metalloproteinase expression via extracellular signal-regulated kinase phosphorylation

Affiliations
  • 1Department of Urology, Eulji University Hospital, Eulji University School of Medicine, Daejeon, Korea. jspark.uro@gmail.com
  • 2Department of Microbiology and Immunology, Eulji University School of Medicine, Daejeon, Korea.

Abstract

PURPOSE
CD81 is a prognostic biomarker for high-grade bladder cancer (BC). In this study, we aimed to determine the functional mechanisms underlying the role of CD81 in BC progression.
MATERIALS AND METHODS
In two invasive BC cell lines (T24, J82), CD81 expression was suppressed by the transfection of lentiviral vectors including CD81-specific shRNAs, and then the migration and invasion of BC cells was analyzed. Enzymatic activity of matrix metalloproteinases (MMPs) was also analyzed by collagen-zymography. The expression of MMPs was confirmed by western blotting using culture supernatants from each cell line. Signaling pathways related to MMPs were investigated using various antibodies.
RESULTS
CD81 was successfully knocked down by shRNAs in T24 and J82 cell lines. While the migration of BC cells was not affected after the knockdown of CD81, the invasive activity was significantly increased in both cell lines. Zymography produced distinct bands using supernatants from CD81-knockdown cells, whereas only faint bands were observed with empty vector-transfected cells. We also observed an increased expression of MMPs, specifically MMP2 and 9, in the conditioned media from CD81-knockdown cells by western blotting. Mechanistically, the phosphorylation of extracellular signal-regulated kinase (ERK) was associated with the invasive activity of BC cells, while U0126 (an ERK inhibitor) reduced the invasive activity of CD81-knockdown BC cells.
CONCLUSIONS
Taken together, CD81 suppression promotes the invasive property of BC cells through MMP signaling via ERK phosphorylation. Our results suggest that the regulation of CD81 expression may have some therapeutic potential in BC.

Keyword

Disease progression; Extracellular signal-regulated MAP kinases; Matrix metalloproteinases; Tetraspanin 28; Urinary bladder neoplasms

MeSH Terms

Antibodies
Antigens, CD81
Blotting, Western
Cell Line
Culture Media, Conditioned
Disease Progression
Extracellular Signal-Regulated MAP Kinases
Matrix Metalloproteinases
Phosphorylation*
Phosphotransferases*
RNA, Small Interfering
Transfection
Urinary Bladder Neoplasms*
Urinary Bladder*
Antibodies
Antigens, CD81
Culture Media, Conditioned
Extracellular Signal-Regulated MAP Kinases
Matrix Metalloproteinases
Phosphotransferases
RNA, Small Interfering

Figure

  • Fig. 1 Knockdown of CD81 in bladder cancer cell lines by shRNA. CD81 shRNAs were transduced into human bladder cancer cell lines, T24 and J82, by lentivirus infection. Virus-infected cells were selected with puromycin for two weeks and the expression of CD81 was analyzed by flow cytometry (A) and western blots (B).

  • Fig. 2 Migration, invasive activity, and proliferation of CD81-knockdown bladder cancer cells. CD81-knockdown T24 and J82 cells were applied to transwell assays with and without matrigel coating to analyze migration (A), invasive activity (B), and cell proliferation (C, D). (A, B) After an overnight incubation, migrated or invasive cells were stained and analyzed (stained with crystal violet, ×400). (C, D) The proliferation of CD81-knockdown T24 (C) and J82 (D) cells was analyzed by MTT assay after 72 hours of culture. Empty vector-transfected cells were used as control. Data are shown as the mean±standard deviation. Cells were counted from three random fields. ns, not significant. *p<0.05, **p<0.01.

  • Fig. 3 Increased expression and activity of MMP2/9 in CD81-knockdown bladder cancer cells. (A) Zymography for MMP activity. (B) Western blot analysis of MMP2 and MMP9. The culture supernatant was concentrated by an Amicon Ultra-2 Centrifugal Filter and MMP2/9 expression was analyzed using each specific antibody.

  • Fig. 4 Screening for MMP2/9-associated signaling pathways in CD81-knockdown bladder cancer cells. MMP2/9-associated signaling pathways were analyzed in CD81-knockdown T24 (A) and J82 (B) bladder cancer cell lines. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping control. p-Akt, phospho-Akt; ERK, extracellular signal-regulated kinase; p-ERK, phospho-ERK; JNK, c-Jun N-terminal kinases; p-JNK, phospho-JNK.

  • Fig. 5 Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation suppressed the invasive activity of CD81-knockdown bladder cancer cells. The ERK inhibitor, U0126, was administered to T24 and J82 cell lines. (A) Inhibition of ERK phosphorylation by U0126 in T24 and J82 bladder cancer cells. (B) Invasive activity of U0126-treated T24 and J82 bladder cancer cells (stained with crystal violet, ×400). DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

  • Fig. 6 Overexpression of CD81 did not induce extracellular signal-regulated kinase (ERK) phosphorylation. CD81 was overexpressed in T24 and J82 bladder cancer cells using lentiviral vectors. CD81 expression and ERK phosphorylation were analyzed by western blot analysis. Ctl, control; p-ERK, phospho-ERK; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


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