br Materials and methods br Results br Discussion Very
Materials and methods
Discussion Very few ARF inhibitors have been developed to study the key functions these small GTP-binding proteins play in pathophysiology. The natural compound Brefeldin A (BFA) has exhibited drastic effects in cells such as collapse of the Golgi because of its non-competitive inhibition mechanism . The small molecule Nav-2729 was identified in a screen as an ARF6 inhibitor . Recent data, however, has demonstrated that it can also block the activation of ARF1 . SecinH3 has the advantage of inhibiting the activation of both ARF1 and ARF6 to a similar extent, by cytohesins [21,30]. Although issues with autofluorescence have been noted in a fluorescence-based nucleotide exchange assay, and associated with insolubility at concentrations higher that 15 μM , this unique tool has allowed us to study the contribution of both ARF isoforms in the complex phenomenon of phenotypical VSMC switching. In our cell-response assays, we have not detected insolubility issues and observed dose-dependent responses. To confirm our results, we have assessed the contribution of each ARF independently by knocking down their expression and not limit ourselves to the specific inhibitory profiles of ARF/GEF pairs by other drugs. While depletion of ARF1 or ARF6 inhibited migration and proliferation, knockdown of ARF1 had a bigger effect than ARF6. The former is also the only ARF isoform that positively controlled sm22α and αSMA protein expression. Interestingly, ARF6 knockdown positively increased αSMA mRNA, without allowing us to distinguish a specific subset of events only regulated by one of the ARF. At last, overexpression of exogenous ARF6 was not able to rescue the defects caused by ARF1 knockdown, confirming the isoform selectivity of ARF1 in the control of the cellular functions that were observed in this study. In an attempt to further define the molecular mechanism regulated by ARF proteins, we observed that SecinH3 treatment or specific depletion of ARF1 by shRNA disrupted stress fibres and focal adhesions in VSMC. These effects, alongside with a drastic change into an elongated shape, were also seen in cells treated with an resazurin polymerization inhibitor, Latrunculin A. Consequently, we conclude that SecinH3 and ARF1 depletion inhibited the formation of actin filaments. We have previously shown, in another cell line, a link between ARF1 and Rac1 , a small GTPase that ultimately triggers actin polymerization through the Arp2/3 complex . In addition, we have demonstrated that ARF1 controls RhoA , a GTPase associated with stress fibre formation . While we could expect that these proteins could be regulated by ARF1 in our VSMC, ARF1 depletion had no effect on the activation of Rac1, RhoA and Cdc42 (data not shown). However, ARF1 inhibition prevented the formation of focal adhesions. Indeed, multiple studies have demonstrated that ARF1 or ARF GAP proteins can regulate paxillin recruitment to these complexes [33,34]. Our laboratory has previously shown that ARF1 can control focal adhesion assembly. However, this effect was only demonstrated in the context of agonist stimulation [35,36]. Here, we rather observed the role of ARF1 on the constitutive recruitment of paxillin to these sites. Similarly to the effects observed when we knocked down ARF1, Latrunculin A treatment also disrupted the formation of focal adhesions. These findings suggest that actin polymerization, controlled by ARF1, would be important for the establishment of these structures. Because focal adhesions were shown to be important for the promotion of VSMC differentiation , these data further suggest that ARF inhibition promotes a synthetic phenotype. Our data also revealed that ARF1 inhibition effectively controlled the localization of the myocardin-related protein Mkl1. We propose that this effect is mainly due to the ability of the GTPase ARF1 to control stress fibre formation and increased the ratio of G-actin to F-actin. Indeed, it has been previously reported that Mkl1 can be sequestered from the nucleus by binding to monomeric G-actin, limiting gene transcription . Here, we present evidence that SecinH3 treatment or ARF1 depletion leads to reduced levels of sm22α and αSMA mRNA suggesting regulation at the transcriptional level. In addition, inhibition of ARF1 increased G-actin levels in VSMC. Our observation that Mkl1 is not fully localized in the nucleus of control cells could be explained by the fact that the cellular localization of this protein also depends on its nuclear importation through importins, which expression has been reported to be reduced in dedifferentiated VSMC . Altogether, these findings provide a molecular mechanism for ARF1 in phenotypic VSMC regulation. As illustrated in Fig. 9, this GTPase controls first actin polymerization. Strategies effective in inhibiting the expression of this ARF or its activation results in actin depolymerization. G-actin filaments sequester Mkl1, preventing its translocation to the nucleus and contractile protein expression, necessary for VSMC contraction. In addition, limited actin polymerization leads to inhibition of cell migration, but also proliferation, which extensively requires the implication of the actin network. ARF6 knockdown also reduces VSMC migration and proliferation, but does so in a less drastic manner than ARF1 knockdown. We report that inhibition of expression of this GTPase does not affect contractile protein expression partly because it does not directly prevent actin filament formation, but rather controls the upstream signaling pathways that regulate migration and proliferation [19,20].