Latent effects of fibronectin, alpha5beta1 integrin, alphaVbeta5 integrin and the cytoskeleton regulate pancreatic carcinoma cell IL-8 secretion

Abstract

Interactions between tumour cells and the microenvironment are increasingly recognised to have an influence on cancer progression. In pancreatic carcinoma, a highly desmoplastic stroma with abnormal extracellular matrix (ECM) protein and interleukin-8 (IL-8) expression is seen. To investigate whether the ECM may further contribute to abnormalities in the microenvironment by influencing IL-8 secretion, we cultured the Mia PaCa2 pancreatic carcinoma cell line on fibronectin. This resulted in a dose-dependent increase in IL-8 secretion, which was RGD dependent and accompanied by cell spreading and proliferation. The role of spreading was assessed by disruption of the cytoskeleton with cytochalasin D, resulting in a large increase in IL-8 secretion, which was reduced from 31- to 24-fold by fibronectin. This remarkable response was associated with inhibition of spreading and proliferation and represents a novel cytoskeletal function. To investigate whether it could be accounted for by the loss of integrin-mediated signalling, the expressed alpha5beta1, alphaVbeta5 and alpha3beta1 integrins were inhibited. alpha5beta1 inhibition prevented spreading and proliferation but produced a much smaller rise in IL-8 secretion than cytochalasin D. alphaVbeta5 inhibition alone had only minor effects but when inhibited in combination with alpha5beta1 completely abolished the response to fibronectin. These results reveal latent stimulatory effects of the alphaVbeta5 integrin on IL-8 secretion and suggest that integrin crosstalk may limit the induction of IL-8 secretion by fibronectin. However, the magnitude of IL-8 secretion induced by cytochalasin cannot be accounted for by integrin signalling and may reflect the influence of another signalling pathway or a novel, intrinsic cytoskeletal function.

Figure 1

Effect of fibronectin and type I collagen on Mia PaCa2 cell IL-8 secretion, number and spreading. Culture of Mia PaCa2 cells on fibronectin for 72 h produced a dose-dependent increase in IL-8 and cell number (A). However, when expressed as IL-8 per cell, this was not significant (B). Values are expressed as a percentage of those obtained for culture on BSA. When cultured on fibronectin cells took on a fusiform, spread appearance (C) in comparison with the rounded appearance seen when cultured on BSA (D). ^*P<0.05 vs untreated control.

Figure 2

GRGDSP inhibition experiments. Following 72 h of culture GRGDSP peptides inhibited fibronectin-induced IL-8 secretion (A) and proliferation (B). No significant effect was seen with control GRADSP peptides. When IL-8 was expressed per percentage change in cell numbers, fibronectin resulted in a small drop in secretion in these experiments. This was abolished by GRGDSP peptides but not by GRADSP, suggesting that the fibronectin RGD motif is critical to its effects. ^*P<0.05 vs untreated control.

Figure 3

Fibronectin time course. An increase in the rate of IL-8 secretion was observed in wells coated with 1 μg ml⁻¹ fibronectin before any rise in secretion in BSA-coated wells (A). Interleukin-8 levels continued to rise at the same rate after 72 h in fibronectin- and BSA-coated wells. Cell numbers rose only in fibronectin-coated wells and slowed after 72 h (B). However, when results were expressed as IL-8 per cell (C), it was apparent that at 96 and 120 h secretion was decreased in fibronectin-coated wells.

Figure 4

Treatment of Mia PaCa2 cells with 1 μg ml⁻¹ cytochalasin D. Culture of Mia PaCa2 cells for 72 h in the presence of cytochalasin D resulted in a 24-fold increase in IL-8 secretion on fibronectin-coated wells and a 31-fold increase in IL-8 secretion on BSA-coated control wells (A). Cytochalasin treatment also inhibited proliferation in fibronectin-coated wells in comparison with no treatment or treatment with the cytochalasin carrier vehicle DMSO (B). When IL-8 levels were expressed as a fraction of the percentage change in cell numbers, a large increase in secretion was still apparent and remained lower on fibronectin than on BSA. Fibronectin-induced cell spreading was prevented by cytochalasin (D). ^*P<0.05 vs untreated control.

Figure 5

Effect of soluble anti-integrin antibodies on fibronectin-induced IL-8 secretion. Cells were cultured on either fibronectin or BSA substratums for 72 h prior to assays. The effects of fibronectin on both IL-8 secretion (A) and cell numbers (B) were abolished by both lone anti-β1 integrin antibodies and by anti-α5 and -αV antibodies used in combination. Use of the anti-α5 antibody alone did not affect IL-8 levels but abolished fibronectin-induced cell proliferation. Single use of the anti-αV antibody resulted in increased IL-8 levels but did not affect proliferation. When IL-8 levels were expressed as a fraction of the percentage change in cell numbers, secretion was decreased by fibronectin in these experiments (C). This decrease was not affected by control IgG or anti-α3 or -αVβ5 antibodies. Anti-β1 and α5 antibodies resulted in an increase in IL-8 secreted per cell, which was greater on fibronectin than on BSA. Anti-αV antibodies both alone and in combination with anti-α5 abolished the difference between fibronectin and BSA, although in the case of anti-αV alone this was due to an identical increase in cell proliferation and IL-8 secretion, whereas anti-αV and -α5 in combination abolished both. ^*P<0.05 vs untreated control.