Transient down-regulation of beta1 integrin subtypes on kidney carcinoma cells is induced by mechanical contact with endothelial cell membranes

Abstract

Adhesion molecules of the integrin beta1 family are thought to be involved in the malignant progression renal cell carcinoma (RCC). Still, it is not clear how they contribute to this process. Since the hematogenous phase of tumour dissemination is the rate-limiting step in the metastatic process, we explored beta1 integrin alterations on several RCC cell lines (A498, Caki1, KTC26) before and after contacting vascular endothelium in a tumour-endothelium (HUVEC) co-culture assay. Notably, alpha2, alpha3 and alpha5 integrins became down-regulated immediately after the tumour cells attached to HUVEC, followed by re-expression shortly thereafter. Integrin down-regulation on RCC cells was caused by direct contact with endothelial cells, since the isolated endothelial membrane fragments but not the cell culture supernatant contributed to the observed effects. Integrin loss was accompanied by a reduced focal adhesion kinase (FAK) expression, FAK activity and diminished binding of tumour cells to matrix proteins. Furthermore, intracellular signalling proteins RCC cells were altered in the presence of HUVEC membrane fragments, in particular 14-3-3 epsilon, ERK2, PKCdelta, PKCepsilon and RACK1, which are involved in regulating tumour cell motility. We, therefore, speculate that contact of RCC cells with the vascular endothelium converts integrin-dependent adhesion to integrin-independent cell movement. The process of dynamic integrin regulation may be an important part in tumour cell migration strategy, switching the cells from being adhesive to becoming motile and invasive.

1

FACS analysis of integrin beta1 subtype expression on Caki-I, KTC-26 and A498 tumour cells. Cells were washed in blocking solution and then stained with specific monoclonal antibodies as listed in Materials and methods. A mouse IgG1-FITC was used as an isotype control for FITC conjugated antibodies. To evaluate background staining of PE conjugated antibodies, goat, anti-mouse IgG1-PE was used. Fluorescence was analyzed using a FACScan flow cytometer, and a histogram plot was generated to show FITC/PE-fluorescence. One from 6 independent experiments.

2

Down-regulation of alpha integrins on RCC cell lines co-cultivated with HUVEC. Cell cultures were double stained using the HUVEC specific Factor VIII-associated antigen monoclonal antibody (clone F8/86) and monoclonal antibodies directed against alpha2, alpha3 or alpha5 integrins. Both cell populations were defined in the FL-1 versus FL-2 dot blot diagram (Fig. 2A, representative for A498) and tumour cells (VIII-) analyzed separately. Dynamics of integrin expression is depicted in Fig. 2B as MFU values.

3

Confocal images of the distribution pattern of alpha3 integrin receptor molecules on A498 tumour cells.(A) shows distinct alpha3 localization along the tumour cell surface in the monoculture model (arrows).(B) and (C) discriminate HUVEC from A498 in the co-culture model, specimen were stained against Factor VIII-associated antigen, labelled with FITC (green fluorescence), and alpha 3 integrin, labelled with PE (red fluorescence). Overlay images of the red and green fluorescence channels are shown in the right hand corner. Transmission pictures were taken to localize adherent tumour cells (arrow). Note that integrin alpha3 is lost on adherent A498 cells after 60 min (B) but restored after 24 hrs (C).

4

Endothelial membrane fragments but not cell culture supernatant reduce alpha3 and alpha5 surface expression and alpha2, alpha3 and alpha5 mRNA of A498 tumour cells. Surface level was analyzed by flow-cytometry and expressed as MFU values.*indicates significant difference to controls (n = 6). RNA was extracted, reverse-transcribed and submitted to semi-quantitative reverse transcription-PCR using gene-specific primers as described in Materials and methods. The internal control for the RT-PCR reaction was performed by running parallel reaction mixtures with the housekeeping gene GAPDH. One representative of three separate experiments is shown.

5

Endothelial membrane fragments modulate focal adhesion kinase (FAK) and FAK phosphorylation (pFAK) but not integrin-linked kinase (ILK). Analysis was carried out by Western blot technique. Cell lysates were subjected to SDS-PAGE and blotted on the membrane incubated with specific monoclonal antibodies (1:1000 dilution), listed in materials and methods. βactin served as the internal control. The figure shows one representative from three separate experiments.

6

Contact inhibition of A498. A498 cells were treated with endothelial membrane fragments or with monoclonal antibodies directed against alpha2, alpha3 and alpha5 integrins (20μg/ml each). After preincubation, tumour cells were added to immobilized collagen, laminin, or fibronectin. Adherent cells were counted after 60 min and mean values calculated (cells/mm²). *indicates significant difference to controls One representative of 6 experiments is shown.

7

Western blot analysis of intracellular signalling proteins, listed in materials and methods. A498 cells were treated with endothelial membrane fragments for 60 min (control:untreated). Cell lysates were subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies. Concentrations were as follows: 1:250: JNK (total and activated), DGKtheta, PKCbeta, PKCtheta, PKCiota, PKClambda; 1:500: PKCdelta; 1:1000: 14-3-3 epsilon, MEK1, MEK5, p90RSK, PKCalpha, PKCepsilon;1:2500: MEK2, p38 (activated), RACK1;1:5000:ERK1, ERK2, panERK, p38 (total). Beta-actin served as the internal control. Bold types indicate changes to controls. The figure shows one representative from three separate experiments.

8

Western blot analysis of cell cycle proteins, listed in materials and methods. A498 cells were treated with endothelial membrane fragments for 60 min (control: untreated). Cell lysates were subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies. Concentrations were as follows: 1:250: CDK4, cyclin A, RB; 1:1000: p70s6kinase, cyclin B, cyclin D3, RB2, RBBP; 1:2500: CDK1, CDK2. Beta-actin served as the internal control. Bold types indicate changes to controls. The figure shows one representative from three separate experiments.