Natural angiogenesis inhibitor signals through Erk5 activation of peroxisome proliferator-activated receptor gamma (PPARgamma)

Abstract

Erk-5, a member of the MAPK superfamily, has a catalytic domain similar to Erk1/2 and a unique C-terminal domain enabling binding with transcription factors. Aberrant vascularization in the Erk5-null mice suggested a link to angiogenesis. Ectopic expression of constitutively active Erk5 blocks endothelial cell morphogenesis and causes HIF1-alpha destabilization/degradation. However the mechanisms by which endogenous Erk5 regulates angiogenesis remain unknown. We show that Erk5 and its activating kinase MEK5 are the upstream mediators of the anti-angiogenic signal by the natural angiogenesis inhibitor, pigment epithelial-derived factor (PEDF). We demonstrate that Erk5 phosphorylation allows activation of PPARgamma transcription factor by displacement of SMRT co-repressor. PPARgamma, in turn is critical for NFkappaB activation, PEDF-dependent apoptosis, and anti-angiogenesis. The dominant negative MEK5 mutant and Erk5 shRNA diminished PEDF-dependent apoptosis, inhibition of the endothelial cell chemotaxis, and angiogenesis. This is the first evidence of Erk5-dependent transduction of signals by endogenous angiogenesis inhibitors.

FIGURE 1.

Erk5 is activated by PEDF and is critical for PEDF angioinhibitory functions. A–D, Erk5 activation by PEDF. HMVECs were activated with VEGF at indicated doses (A) or bFGF (10 ng/ml, B) and treated with PEDF at an anti-angiogenic concentration of 10 nm. Total cell lysates were analyzed by Western blotting for phosphorylated Erk5 (p-Erk5). HMVECs expressing constitutively active MEK5 (CA) were used as a positive control. The membrane was stripped and probed for tubulin to assess loading. The results of densitometry analysis performed with ImageJ software are shown below. The results were normalized to the ratio between p-Erk5 and tubulin in untreated control samples. C and D, time-dependent induction of Erk5. The cells were treated with VEGF and PEDF (10 nm) for the indicated time periods, p-Erk5 was measured by Western blot (C), quantitative analysis was performed by densitometry and normalized against p-Erk/tubulin ratio in the untreated control (D). E, Erk5 activation by PEDF anti-angiogenic epitope. HMVECs were activated with VEGF (0.5 ng/ml) and treated fro 20 min with 20 nm PEDF or 20 nm 34-mer, as indicated. Densitometry analysis was performed as in A. F, Erk5 is required for PEDF blockade of the HMVEC chemotaxis. HMVECs were transfected with the dominant-negative and constitutively active MEK5 (DN and CA, respectively) or vector control (WT). Chemotaxis was induced with 20 ng/ml bFGF and blocked with 10 nm PEDF or with 10 nm 34-mer. The results were normalized to the WT background migration. *, p < 0.05; **, p < 0.01; ***, p < 0.0001. □, BSA control; ■, bFGF; gray, PEDF or the 34-mer; hatches, PEDF or the 34-mer + bFGF. G, role of Erk5 in PEDF-dependent apoptosis. HMVECs generated as in F were transferred in a serum-free medium, treated with protective bFGF (20 ng/ml) and 20 nm PEDF where indicated. *, p < 0.05; ***, p < 0.0001.

FIGURE 2.

Erk5 knock-down abrogates PEDF anti-angiogenic activity. A, Erk5 knock-down. We used two distinct shRNAmir cloned in pGIPZ lentiviral vectors. Western blot of the total cell lysates showed 70 and 90% knock-down (KO1 and KO2, respectively). KO2 population has been selected for further analysis. B and C, scratch wound assay was performed on confluent endothelial cell monolayer. B, representative images of the scratch wounds at 0 and 6 h are shown and the areas of the wound used for the quantitative measurements are marked. C, rate of the endothelial cell migration into the wound surface was measured as percent wound closure. □, control; ■, 20 nm PEDF. Asterisk indicates statistically significant difference (p < 0.05 by one-way ANOVA).

FIGURE 3.

Erk5 is critical for PEDF anti-angiogenic action in vivo. A, cryogenic sections of the Matrigel plugs containing control HMVECs (WT), or HMVECs expressing dominant negative or constitutively active MEK5 (DN and CA, respectively). Angiogenesis was induced with 200 ng/ml VEGF and blocked with 100 μm 34-mer, where indicated. The sections were stained for the endothelial cell marker, CD31 (PECAM1). B, morphometric analysis of the digital images of the sections shown in A with MetaMorph software package. Microvascular density was quantified as relative CD31-positive area. **, p < 0.01; **, p < 0.001.

FIGURE 4.

Erk5 in the PPARγ transcription complexes is phosphorylated in response to PEDF. A, HMVECs were treated for the indicated time periods with the combinations of bFGF (10 ng/ml) and PEDF (10 nm). PPARγ and P53 levels were measured by Western blot. B, total RNA was extracted and PPARγ mRNA measured by real-time RT-PCR after 6 h of treatment. C, PPARγ mobility shift assay. HMVECs were treated with VEGF ± PEDF or the 34-mer, nuclear extracts were collected and subjected to EMSA with biotinylated PPRE probe. Non-biotinylated wild-type and non-biotinylated mutant PPRE probes were used as specific and nonspecific competitors (PPRE and NSP, respectively). C indicates positive control. D, PPARγ is necessary for PEDF inhibitory activity. HMVECs chemotaxis up the bFGF gradient (20 ng/ml across the membrane) was blocked by PEDF (20 nm) alone or in the presence of T0070907 (100 nm) Asterisks indicate statistically significant differences (p < 0.05, calculated by one-tailed Student's t test). E, wild-type HMVECs (MEK5-WT) were treated with PEDF, VEGF, or VEGF+PEDF. Untreated cells served as a negative control. Cell lysates were collected and subjected to immunoprecipitation with PPARγ antibody followed by Western blot with antibodies for p-Erk5, total Erk5, and SMRT. The input was controlled by Western blot with PPARγ antibody. F, HMVECs expressing MEK5-DN or MEK5-CA, were treated with VEGF (1 ng/ml) and PEDF (10 nm). Protein extracts were precipitated with PPARγ antibody and analyzed by Western blot for p-Erk5 and total Erk5. The input was controlled by Western blot with PPARγ antibody.

FIGURE 5.

PPARγ is critical for PEDF-dependent NFκB activation. PPARγ antagonist interferes with IκB-α phosphorylation in the presence of PEDF. HMVECs were pretreated 30 min with proteasome inhibitor MG132 (10 μm) and then treated 25 min with the indicated combinations of bFGF (10 ng/ml), PEDF (10 nm), and T0070907 (1 μm). A, whole cell extracts were subjected to Western blot analysis for IκB-α. B, HMVECs plated on coverslips were treated as in A and stained for p65/RelA. NFκB nuclear localization is indicated with white arrows. C, cells were activated with VEGF (0.5 ng/ml) and treated with PEDF or the 34-mer (10 nm). NFκB p65/RelA was measured in the whole cell extracts by Western blot.

FIGURE 6.

The results of PEDF-driven Erk5 activation. A, PEDF exposure causes sequential activation of MEK5 and Erk5, which leads to PPARγ activation followed by NFκB activation, endothelial cell apoptosis, and anti-angiogenesis. B, proposed mechanism of PPARγ-dependent induction of NFκB expression. PPARγ/RXR heterodimers recruit SMRT to the p65/RelA promoter: SMRT, in turn, recruits HDAC1, which causes transcriptional repression. When phosphorylated by PEDF, Erk5 displaces SMRT, and thus releases HDAC1 and alleviates trans-repression by PPARγ.