Signalling crosstalk in FGF2-mediated protection of endothelial cells from HIV-gp120

Abstract

Background: The blood brain barrier (BBB) is the first line of defence of the central nervous system (CNS) against circulating pathogens, such as HIV. The cytotoxic HIV protein, gp120, damages endothelial cells of the BBB, thereby compromising its integrity, which may lead to migration of HIV-infected cells into the brain. Fibroblast growth factor 2 (FGF2), produced primarily by astrocytes, promotes endothelial cell fitness and angiogenesis. We hypothesized that treatment of human umbilical vein endothelial cells (HUVEC) with FGF2 would protect the cells from gp120-mediated toxicity via endothelial cell survival signalling.

Results: Exposure of HUVEC to gp120 resulted in dose- and time-dependent cell death; whereas, pre-treatment of endothelial cells with FGF2 protected cells from gp120 angiotoxicity. Treatment of HUVEC with FGF2 resulted in dose- and time-dependent activation of the extracellular regulated kinase (ERK), with moderate effects on phosphoinositol 3 kinase (PI3K) and protein kinase B (PKB), also known as AKT, but no effects on glycogen synthase kinase 3 (GSK3beta) activity. Using pharmacological approaches, gene transfer and kinase activity assays, we show that FGF2-mediated angioprotection against gp120 toxicity is regulated by crosstalk among the ERK, PI3K-AKT and PKC signalling pathways.

Conclusions: Taken together, these results suggest that FGF2 may play a significant role in maintaining the integrity of the BBB during the progress of HIV associated cerebral endothelial cell damage.

Figure 1

Cell viability assays for FGF2 protection of HUVEC against gp120-mediated toxicity (A-D) Phase contrast, (F-I) TUNEL staining, and (K-O) fluorescent staining of HUVEC. Panels A, F, and K show images of untreated HUVEC control; B, G, and L show HUVEC treated for 24 h with FGF2 (20 ng/ml); panels C, H, and M show HUVEC treated for 24 h with gp120 (25 ng/ml) and panels D, I, and N show HUVEC pre-treated with FGF2 for 24 h before a 24 h exposure to gp120. Panels E, J and O show the percentage of cell death as determined by Trypan blue exclusion, percentage of DNA fragmentation by TUNEL and FA/PI staining, respectively. Results shown in panels E, J and O represent the average of three separate experiments performed in triplicate. * = P < 0.05 by One-Way ANOVA with post-hoc Dunnett's when compared to control. Bar = 20 microns.

Figure 2

Effects of FGF2 and gp120 on cell proliferation and viability A) Density of proliferating HUVEC exposed to FGF2 and/or gp120. B) Cell viability of cells exposed to gp120 after pre-treating HUVEC with FGF2 for different lengths of time. Cell viability was measured 24 h after gp120 addition. * = P < 0.05 by One-Way ANOVA with post-hoc Dunnett's when compared to control.

Figure 3

Effects of inhibitors on FGF2-mediated ERK activation A) Western blot (WB) reacted with anti-phospho-ERK after treatment with FGF2 for 0, 5, 10 min (lanes 1–3) and after inhibitors LY294002 (to block PI3K), U0126 (to block MEK), and Bis I and Gö6983 (to block PKC) (lanes 4–7, respectively). B) WB reacted with anti-total ERK antibody. (C) WB reacted with anti-phospho-GSK3β. D) WB reacted with anti-GSK3β. E) WB reacted with anti-PI3K antibody. The same WB was used in each experiment after stripping, reblocking and incubating with new antibodies for the given protein.

Figure 4

Effects of inhibitors on FGF2-stimulated ERK and GSK3β activity Immunocomplex kinase assays for (A) ERK and (B) GSK3β activity without FGF2 treatment, or after inhibition with PD98059, U0126, LY29004, Bisindolymaleimide I or Gö6983 followed by FGF2 treatment. (C) Inhibitor treatment alone. (D) Summary of changes in phosphorylation (P) versus changes in activity (Act) of ERK and GSK3β. ⇑ Indicates an increase, ⇓ indicates a decrease, – indicates no change. * = P < 0.05 by One-Way ANOVA with post-hoc Dunnett's when compared to control.

Figure 5

Viability assays indicate that ERK activation is required for FGF2 protection against gp120 (A) Trypan Blue Exclusion assay for cell survival after 30 min of treatment with inhibitors LY294002, U0126, Bis I, or Gö6983 followed by treatment with FGF2 and/or gp120. (B) Trypan Blue Exclusion assay for cell survival after 30 min of treatment with the MEK inhibitor U0126 alone, and followed by exposure to gp120 and/or FGF2 for 24 h. In cells treated with FGF2 and anti-FGF2, HUVEC were exposed to anti-FGF2 antibody for 1 h followed by treatment with FGF2 alone or in combination with gp120. * Indicates a significant difference from control. * = P < 0.05 by One-Way ANOVA with post-hoc Dunnett's when compared to control.

Figure 6

Gene transfer of constitutively active ERK and AKT protect cells from gp120 toxicity (A) After gene transfer of constitutively active (ca) ERK or ca AKT, HUVEC were exposed to gp120, FGF2 or a combination of both and cell viability was assayed via Trypan Blue Exclusion. Controls consisted of ca ERK, ca AKT and GFP gene transfer without further treatment. * indicates a significant difference from control. ** indicates a significant difference from *. * = P < 0.05 by One-Way ANOVA with post-hoc Dunnett's when compared to control. ** = P < 0.05 by One-Way ANOVA with post-hoc Tukey-Kramer when compared between experimental groups.

Figure 7

Effects of gene transfer of constitutively active ERK, AKT or GFP phosphorylation (A-D) Western Blot of HUVEC after treatment with FGF2, infected with the GFP, ca ERK or ca AKT adenoviral constructs, or treated with FGF2 and infected with adenoviral constructs. The same Western blot was used for all antibodies after stripping and rehybridizing. A) Reacted with anti-phospho ERK antibody (B) reacted with anti-total ERK antibody. (C) Reacted with anti-phospho AKT antibody. (D) Reacted with anti-total AKT antibody. (E) Immuno-complex assay showing changes in ERK activity with or without FGF2 treatment in HUVEC with caERK, caAKT or GFP. (F) Quantification of ERK activity levels in HUVEC +/- FGF2 treatment with caERK, caAKT or GFP with the PhosphorImager as described in the Materials and Methods. * indicates a significant difference from control. * = P < 0.05 by One-Way ANOVA with post-hoc Dunnett's when compared to control.

Figure 8

Effects of FGF2, gp120, and inhibitors on ERK and GSK3β phosphorylation (A, B) Western blots showing ERK and GSK3β phosphorylation with (A) gp120 alone (lane 2), and with inhibitors (lanes 3–6), (B) FGF2 and gp120 (lane 2), and FGF2 with inhibitors and gp120 (lanes 3–6). (C) Table summarizing data from western blots (A and B) showing changes in phosphorylation of ERK and GSK3β. ⇑ Indicates and increase, ⇓ indicates a decrease, – indicates no change.

Figure 9

Diagrammatic representation of signalling pathways that may be involved in FGF2-mediated protection from gp120 As indicated by the diagram and as described by the data, it is possible that the crosstalk between the PI3K/AKT/GSK3β and ERK pathways is mediated in part by PKC. PKC signalling also is reported to occur directly with PI3K and upstream of Ras. Furthermore, direct interaction between MEK and PKC downstream of Ras is reported. Likewise, ERK2 (p42) is reported to signal to Raf-1 via a positive feedback mechanism. The points of action of the inhibitors LY290042, U0126, Bisindolymaleimide I, and Gö6983 are also shown.