FXII protects from stereotaxic brain lesion
Our first objective was to explore the potential role of FXII in brain lesions induced by experimental process independent of the circulatory system. For this, we used a classical model of brain injury induced by the stereotaxic injection of NMDA (Figure 1). We observed that the administration of FXII (1 µg) reduced the mean lesion volume by 46% (Figure 1B and corresponding quantification, Figure 1C). These data indicate that FXII could exert direct neuroprotective effects in the brain parenchyma.
FXII protects neurons from apoptosis by activating EGFR and subsequent signaling pathways
Our next step was to decipher whether FXII may also display neuroprotective effects in vitro. Considering that the in vivo paradigm of brain lesion induces neuronal apoptosis (Supplemental Figure 1), we tested the effect of single-chain FXII in cortical neurons subjected to serum deprivation (SD), a classical model of apoptosis. We observed that FXII exerted a dose-dependent anti-apoptotic effect on cortical neurons (Figure 2). Our hypothesis to explain this effect was that FXII could act, at least in part, via binding to EGFR and activation of this receptor, such as previously reported for tPA10, 11, a serine protease presenting homologous EGF-like domains. To address this question, we treated cortical neurons with biotinylated FXII, extracted the proteins and subjected them to immunoprecipitation (IP) using an anti-EGFR antibody (Figure 3A). We detected biotinylated FXII among the EGFR-immunoprecipitated proteins as a ~80kDa band revealed by peroxidase-coupled avidin (Figure 3A), at the same molecular weight as biotinylated FXII ran in parallel. FXII was absent in untreated cells (SD). This data show that FXII and EGFR are part of a same protein complex in FXII-treated neurons.
Then, we asked whether the interaction of FXII with EGFR, and its subsequent activation could be responsible for the anti-apoptotic effect of FXII on neurons. In line with this hypothesis, the inhibitor of EGF receptor kinase, AG1478 (5 µM), reversed the effect of FXII on neurons during SD, while it showed no effect when applied alone (Figure 3B).
EGFR activation can trigger several signaling cascades, including mitogen-activated protein kinase/extracellular regulated kinase (MAPK/Erk). Moreover, Tyr1068 in EGFR, the residue phosphorylated upon FXII treatment, is involved in the transduction of EGF signal through Erk pathway15. Here, we observed that single-chain FXII (125 nM) induced the rapid (within 5 minutes) and transient (<1h) phosphorylation of Erk1/2 (Figure 4A). Accordingly, the pre-treatment with MEK/Erk1/2 inhibitor (SCH7772984, 5 µM) reversed the anti-apoptotic effect of FXII (Figure 4B). Together, these data show that the activation of EGFR by FXII triggers Erk activation, and that this pathway actively participates in the anti-apoptotic effects of FXII in neurons.
Apoptosis is regulated by a balance between pro- and anti-apoptotic factors that control downstream protease activity of effector caspases and subsequent cell death. Here, we studied whether the pro-apoptotic factor Bax and the anti-apoptotic factor Bcl-2 are regulated by FXII during SD. We observed that after 24h of SD, as compared to 1h, treatment with FXII (125 nM) significantly decreased Bax expression, while it induced the up-regulation of Bcl-2 (Figure 4C), thus reducing the ratio between pro- and anti-apoptotic factors. These data show that single-chain FXII triggers anti-apoptotic pathways in neurons subjected to SD.
FXII promotes HGF maturation, leading to HGFR-mediated anti-apoptotic effects
FXII can be activated from a single-chain form (FXII) to a more proteolytically active two-chain form (αFXIIa) (Figure 5A). Our next step was to investigate if αFXIIa showed the same anti-apoptotic effects than its single chain form, FXII. When applied to cortical neurons during SD, αFXIIa exerted an anti-apoptotic effect, although at slightly higher doses than FXII (Figure 5A). Noteworthy, in contrast to what we observed for FXII, the anti-apoptotic effect of αFXIIa was not completely reversed by the inhibitor of EGFR activation, AG1478 (5 nM, Figure 5B). Moreover, we observed that proteolytically inactive αFXIIa (αFXIIa-PPACK) still retained an antiapoptotic capacity (Supplemental Figure 2A). Thus, we wanted to study if αFXIIa also acts via binding to EGFR. We repeated the immunoprecipitation studies with biotinylated αFXIIa, confirming that we detected biotinylated αFXIIa among the EGFR-immunoprecipitated proteins as a ~50kDa band revealed by peroxidase-coupled avidin (Figure 5C), at the same molecular weight as biotinylated αFXIIa ran in parallel. In addition, the above immunoprecipitated material showed a band at approximately 175 kDa corresponding to EGFR, when revealed with anti-EGFR antibodies (Figure 5C). These data show that αFXIIa and EGFR are part of a same protein complex in αFXIIa-treated neurons. Interestingly, the blockade of EGFR by AG1478 is not sufficient to block αFXIIa. Because αFXIIa differs from FXII by its proteolytic activity, we wondered whether this activity could be involved in its anti-apoptotic function. To address this question, we co-treated neurons subjected to SD with αFXIIa and Corn Trypsin Inhibitor (CTI, at 10 µM) an inhibitor of its proteolytic activity. Interestingly enough, CTI reversed the antiapoptotic effect of αFXIIa (Figure 5D). Together, these results suggest that αFXIIa exerts anti-apoptotic effects on neurons by a combination of proteolytic and non-proteolytic (“growth factor-like”) effects.
The proteolytic activity of αFXIIa is known, among other actions, to induce the activation of hepatocyte growth factor (HGF) from its pro-form to its active form16, which in turn can activate its receptor, HGFR (also known as c-Met). We thus hypothesized that the effect of αFXIIa could be mediated by the proteolytic activation of HGF and a subsequent stimulation of HGFR. When we co-treated neurons subjected to SD with αFXIIa and sc1356 (100ng/mL), a blocking antibody of HGF17, the anti-apoptotic effect of αFXIIa was completely reversed (Figure 6A). The same result was obtained when using JNJ, an inhibitor of HGFR phosphorylation (Figure 6B). These data show that αFXIIa triggers the activation of HGF, which in turn acts on HGFR to provide anti-apoptotic effects in neurons.
FXII activation into αFXIIa can occur either by the action of proteases1, or by surface-mediated auto-activation in certain conditions1, 2. Thus, we wanted to study whether FXII could be auto-activated at the surface of neurons. By the use of a chromogenic substrate specific for αFXIIa activity, we observed that FXII underwent activation into its two-chain form at the surface of neurons (Figure 7A) and not in the absence of cells (DMEM + FXII condition). Considering the protective effect of αFXIIa described above (Figure 6), we hypothesized that protease-mediated mechanisms could also be involved in FXII anti-apoptotic effects. Indeed, the anti-apoptotic effect of FXII was also reversed by CTI (Figure 7B). Furthermore, in purified conditions, incubation of pro-HGF with either FXII or αFXIIa led to an increase in the amount of mature HGF (Supplemental Figure 3). That may account in our conditions for an increased activation of pro-HGF in mature HGF by the protease activity which comes up from the activation of FXII into its protease-active form (Figure 7A). As previously observed with αFXIIa, FXII effects were also reversed by JNJ (Figure 7C) and sc1356 (Figure 7D). Finally, incubation of neurons with FXII led to phosphorylation of HGFR (Figure 7E), which reflects its activation. These data indicate that FXII can protect from apoptosis by protease-mediated, HGF-mediated effects, in addition to its protease-independent, EGFR mediated effects (Figure 3-4). In accordance with this, we observed that proteolytically-inactive PPACK-αFXIIa showed a residual, protease-independent, anti-apoptotic effect (Supplemental Figure 2A) reversed by AG1478 (Supplemental Figure 2B). Interestingly enough, this anti-apoptotic effect was also blocked by JNJ, showing that HGFR pathway is activated (Supplemental Figure 2C). We hypothesized that EGFR and HGFR could be active via a co-receptor crosstalk. To test if EGFR could transphosphorylate and activate HGFR independently of mature HGF generation, as reported previously, we added sc1356 (100 ng/mL) to block a putative interaction between HGFR and an extracellular ligand. The EGFR-dependent anti-apoptotic effect of αFXIIa-PPACK was not reversed when blocking the binding of HGF to HGFR (Supplemental Figure 2D), whereas – as described above - it was reversed when the phosphorylation of HGFR was blocked using JNJ. These results support the transphosphorylation of HGFR by the activation of EGFR by FXII. Finally, since it has been shown FXII activates EGFR signaling through uPAR4 we also tested this hypothesis using a uPAR blocking antibody. As presented in Supplemental Figure 4, FXII antiapoptotic effect appears independent of uPAR activation in this context.