Motility induced by human immunodeficiency virus-1 Tat on Kaposi's sarcoma cells requires platelet-activating factor synthesis

Abstract

In the present study, we evaluated whether motility of Kaposi's sarcoma (KS) spindle cells induced by HIV-1 Tat protein is dependent on the synthesis of platelet-activating factor (PAF). The results obtained indicate that Tat induced a dose-dependent synthesis of PAF from KS cells at a concentration as low as 0.1 ng/ml. PAF production started rapidly after Tat stimulation, peaking at 30 minutes and declining thereafter. Tat-induced cell migration was also a rapid event starting at 30 minutes. The motility was abrogated by addition of a panel of chemically unrelated PAF receptor antagonists (WEB 2170, CV 3988, CV 6209, and BN 52021), suggesting that the synthesized PAF mediates the motogenic effect of Tat. This effect was also present on cells plated on a type-I collagen-, fibronectin-, or basement membrane extract-coated surface. Expression of PAF receptor-specific mRNA was detected in KS cells. In addition, examination of the cytoskeletal organization showed that Tat-mediated KS cell redistribution of actin filaments and shape change was also inhibited by a PAF receptor antagonist. Moreover, PAF receptor blockade prevented the up-regulation of beta1 integrin and the down-regulation of alphavbeta3 observed after stimulation of KS cells with Tat. In conclusion, the results of the present study indicate that Tat-induced PAF synthesis plays a critical role in triggering the events involved in motility of KS cells.

Figure 1.

PAF synthesis by KS cells. A shows the time course of PAF synthesis by KS cells stimulated with 1 ng/ml Tat (□, cell-associated PAF; ▪, released PAF) or with vehicle alone (▴, cell-associated PAF; ▵, released PAF). Numbers are the mean ± SD of three individual experiments. B shows the effect of different doses of Tat on PAF synthesis by KS cells and of thrombin (2 U/ml) used as positive control. The numbers are the mean ± SD of five individual experiments. C shows the incorporation of radiolabeled acetate in the newly synthesized PAF after stimulation of KS cells with Tat. Representative TLC analysis of radiolabeled lipids extracted from 5 × 10 KS cells stimulated with 1 ng/ml Tat (solid line). Dotted line shows the chromatographic behavior of radiolabeled lipids extracted from unstimulated cells. The chromatographic behavior of synthetic C16 PAF is indicated at the top of the figure. This figure is representative of four different experiments performed with similar results.

Figure 2.

Expression of mRNA for human PAF-receptor by B16 cells (lane 2) , PAF receptor-transfected B16 cells (lane 3) or KS cells (lane 4) detected by RT-PCR. Empty plasmid vector (lane 5) or plasmid vector containing PAF-receptor cDNA (lane 6) detected by PCR. Lane 1 shows DNA markers.

Figure 3.

Motility of KS cells measured as described in the Materials and Methods section. A shows incubation of KS cells with vehicle alone or with different doses of Tat or with PAF (10 ng/ml). B shows the inhibitory effect of specific PAF receptor antagonists WEB2170, CV 3988, CV 6209, or BN 52021, on Tat-induced KS motility. Cells were incubated with Tat (10 ng/ml) in the presence or absence of 3 μmol/L WEB2170, 3 μmol/L CV 3988, 0.17 μmol/L CV 6209, or 0.34 μmol/L BN 52021. Data are expressed as mean ± SD of three different experiments. ANOVA with Newmann Keul’s multicomparison test was performed: cells incubated with vehicle alone (control) versus cells incubated with Tat (*P < 0.05); cells incubated with Tat versus cells incubated with Tat + WEB2170, Tat + CV 3988, Tat + CV 6209, or Tat + BN 52021 (^§P < 0.05).

Figure 4.

Micrographs representative of time-lapse analysis of KS cell motility performed by digital saving at 30-minute intervals. Migration tracks were generated by marking the position of the nucleus of individual cells of each image (see Materials and Methods). KS cells were incubated with vehicle alone (A), with 10 ng/ml Tat (B), or with Tat (10 ng/ml) in the presence of 3 μmol/L WEB2170 (C). Original magnification, ×120.

Figure 5.

Evaluation of the role of different matrix substrates on Tat-induced KS cell motility. KS cells plated on type 1 collagen (A), fibronectin (B), and Matrigel (C) were stimulated with vehicle alone or with of Tat (10 ng/ml) or Tat plus PAF-receptor antagonists WEB2170 (3 μmol/L). Three experiments were performed with similar results.

Figure 6.

Micrographs representative of shape change of KS cells as observed by comparison of the time 0 (A, C, and E) and 1-hour frames (B, D, and F). A and B show unstimulated KS cells; C and D show KS cells stimulated with 10 ng/ml Tat; E and F show KS cells stimulated with 10 ng/ml Tat in the presence of 3 μmol/L WEB 2170. Three experiments were performed with similar results. Original magnification, ×120.

Figure 7.

Micrographs representative of F-actin distribution in fixed and permeabilized KS cells after incubation for 1 hour in the following experimental conditions: A, unstimulated KS cells (vehicle alone); B, KS cells stimulated with 10 ng/ml Tat; C, KS cells stimulated with 10 ng/ml Tat in the presence of 3 μmol/L WEB 2170; D, KS cells stimulated with Tat 10 ng/ml and then washed and cultured for 12 hours with RPMI supplemented with 10% FBS to evaluate reversal of cytoskeletal changes induced by Tat. Three experiments were performed with similar results. Original magnification, ×400.

Figure 8.

Detection of αvβ3 and β1 integrins on KS cells by cytofluorimetry after 12 hours stimulation with PAF (10 ng/ml) or Tat (10 ng/ml) in the presence or absence of PAF receptor antagonist CV3988 (3 μmol/L). PAF-stimulated (dark line, A) as well as Tat-stimulated (dark line, B) KS cells showed a down-regulation of αvβ3 in respect to the unstimulated KS cells (gray line, A and B). In contrast, PAF-stimulated (dark line, E) as well as Tat-stimulated (dark line, G) KS cells showed an up-regulation of β1 in respect to the unstimulated KS cells (gray line, E and G). Treatment with the PAF receptor antagonist CV3988 abrogated both the down-regulation of αvβ3 (B and D) and the up-regulation of β1 (F and H) integrins induced by PAF and Tat. Three experiments were carried out with similar results. The Kolmogorov-Smirnov statistical analysis performed between unstimulated cells and cells stimulated with Tat or PAF was significant for the changes in the expression of both αvβ3 and β1 integrins (P < 0.05). Moreover, the inhibitory effect of CV 3988 on Tat- and PAF-stimulated KS cells was statistically significant (P < 0.05).