Thrombospondin-1 (TSP-1) Stimulates Expression of Integrin alpha6 in Human Breast Carcinoma Cells: A Downstream Modulator of TSP-1-Induced Cellular Adhesion

Abstract

Thrombospondin-1 (TSP-1) is involved in a variety of different cellular processes including cell adhesion, tumor progression, and angiogenesis. This paper reports the novel finding that TSP-1 upregulates integrin alpha6 subunit in human keratinocytes and human breast cancer cells resulting in increased cell adhesion and tumor cell invasion. The effect of TSP-1 on alpha6 subunit expression was examined in human keratinocytes and breast adenocarcinoma cell lines (MDA-MB-231) treated with TSP-1 and in TSP-1 stably transfected breast cancer cells. TSP-1 upregulated alpha6 message and protein in these cells as revealed by differential display, Northern and Western blot analysis and immunohistochemical localization studies. The increased expression of alpha6 was shown to mediate adhesion and invasion of these cells to laminin, a major component of the basement membrane and extracellular matrix (ECM). These data suggest that TSP-1 plays an integral role in the attachment of cells to the ECM facilitating cell motility and angiogenesis.

Figure 1

TSP-1 upregulates integrin α 6 subunit message in human keratinocytes. Keratinocytes were treated with either bis-tris-propane buffer or TSP-1 (60 μg/mL) in serum-free media and mRNA expression was analyzed by (a) differential display analysis and confirmed with (b) northern blot analysis. Experiments were repeated three times and the results of a representative experiment are shown in the figure.

Figure 2

TSP-1 induces integrin α 6 mRNA expression in human breast cancer cells. Two breast cancer cell lines (a) stably transfected TSP- 1 MDA-MB-435 cell lines with variable TSP-1 expression and (b) MDA-MB-231 cells treated with either buffer or TSP-1 (60 μg/mL) were analyzed by northern blot analysis for integrin α6 expression. TSP-1 dose dependent response of integrin α6 was then assessed in the MDA-MB-231 (c). Blots were normalized to a 2.4 β-actin probe. A pancreatic carcinoma cell line, BxPC3, was used as a positive control. (a) TSP-1 stably transfected TSP-1 cells. 1: BxPC3 cell line, 2: TH5 cells (vector control), 3: TH29 cells (intermediate TSP-1 producer), and 4: TH26 cells (high TSP-1 producer). (b) MDA-MB-231 cells. 1: BxPC3 cell line, 2: buffer treatment, 3: TSP-1 (1 hour), 4: TSP-1 (6 hours), 5: TSP-1 (12 hours), and 6: TSP-1 (24 hours). Experiments were repeated three times and the results of a representative experiment are shown in the figure. (c) MDA-MB-231 cells. 1: Buffer treatment, 2: TSP-1 (20 μg/mL), 3: TSP-1 (40 μg/mL), and 4: TSP-1 (60 μg/mL).

Figure 3

TSP-1 induces integrin α 6 protein expression in human breast cancer cells. MD-MBA-231 cells (a) and MD-MBA-435 cells (b) were grown in six well tissue culture plates in serum-free media either with or without 60 μg/mL TSP-1 for 24 hours. Cell extracts were prepared with SDS-sample buffer, reduced with 5%  β-mercaptoethanol, and separated on 10% SDS-PAGE. Blots were probed with 1 μg/mL of mouse α6 integrin IgG and followed by 0.1 μg/mL HRP-coupled rabbit antimouse IgG and developed using enhanced chemoluminesence. Experiments were repeated two times and the results of a representative experiment are shown in the figure.

Figure 4

TSP-1 stably transfected cells express integrin α6. Cells were grown in six well chamber slides, fixed, and stained with rat α6 integrin IgG as described in Section 2. Cells were photographed at 200X magnification. (a) TH5 cells (vector control). (b) TH29 cells, (c) TH50, (d) TH26 cells. Experiments were repeated three times and the results of a representative experiment are shown in the figure.

Figure 5

Anti-TSP-1 antibody inhibition of integrin α6 production in TSP-1 stably transfected cells. Cells were grown in six well chamber slides in either serum-free media or media containing either 10 μg/mL control IgG or 10 μg/mL goat antihuman TSP-1 IgG, fixed, and stained with rat α6 integrin IgG as described in Section 2. Cells were photographed at 200X magnification. (a) TH5 cells (vector control). (b) TH26 cells (high TSP-1 producer). (c) TH26 cells plus anti-TSP-1 antibody. (d) TH26 cells plus control IgG.

Figure 6

High endogenous TSP-1 production increases cell adhesion to laminin. Stably transfected MDA-MB-435 cells were either incubated alone (a), with either 10 μg/mL control IgG or 10 μg/mL goat antihuman TSP-1 IgG (b), or either 10 μg/mL control IgG or 10 μg/mL rat antihuman α6 integrin IgG (c), and assessed for adhesion to laminin as described in Section 2. BSA was used as a negative control. The error bars represent the standard error of the mean of triplicate samples and the experiment was repeated three times with similar results.

Figure 7

High endogenous TSP-1 production increases cell invasion of laminin. Tumor cell invasion of laminin was performed as described in Section 2. Invasion of cells untreated (a), invasion of cells pretreated with either with either 10 μg/mL control IgG or 10 μg/mL goat antihuman TSP-1 IgG or 10 μg/mL rat antihuman α6 integrin IgG (b). The error bars represent the standard error of the mean of triplicate samples and the experiment was repeated three times with similar results.