ALDH1L1 inhibits cell motility via dephosphorylation of cofilin by PP1 and PP2A

Abstract

Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin toward strong preponderance of filamentous actin and formation of actin stress fibers. A549 cells expressing FDH showed a much slower recovery of green fluorescent protein-actin fluorescence in a fluorescence recovery after photobleaching assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases, but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis. Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can also be prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate specific and not a general response to nutrient starvation. Overall, this study shows the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.

Figure 1

Changes of motile characteristics in A549 cells upon FDH expression. (a) Migration and invasion of FDH deficient and FDH-expressing cells (inset shows levels of FDH with actin as a loading control) in the absence or in the presence zVAD-fmk. (b) Migration track of a single cell in the absence (-) and in the presence (+) FDH (top panel); bottom panel shows average track length calculated with NIH Image Software. (c) Adhesion potential of FDH-expressing and FDH-deficient cells. Experiments were performed in triplicate; average ± SD is shown.

Figure 2

FDH induces shift in G/F actin ratio and inhibits actin dynamics. (a) Levels of F-actin (green) and G-actin (red) in FDH deficient (-) and FDH expressing (+) A549 cells imaged by confocal microscopy. Yellow indicates co-localization. Bar, 20 μm. (b) Bar graph, G/F actin ratio in cytosol of -FDH and +FDH cells (quantified from three Western blots as described in Materials and Methods). Inset shows a representative Western blot of F, G and total actin in FDH-expressing and FDH-deficient cells (c) FRAP analysis of actin treadmilling rate in A549 cells. Representative microphotographs show re-distribution of GFP-actin fusion after photobleaching in control FDH-deficient (-FDH) and FDH-expressing (+FDH) A549 cells. Time (seconds) after photobleaching is indicated. The first panel (-20 s) shows cells before photobleaching. (d) Quantification of FRAP data from (c) for FDH-deficient cells (-FDH, closed circles) and FDH-expressing cells (+FDH, open circles). The rate of fluorescence recovery was analyzed using Leica Confocal Software; average of nine experiments was calculated for each time-point. (e) Actin polymerization rate by A549 cell lysates with (closed circles) or without (open circles) FDH expression evaluated by the increase of fluorescence intensity of pyrene conjugated to G-actin. Control experiment (open diamonds): pyrene G-actin was incubated with lysis buffer. (f) Actin depolymerization rate by the same lysates as in (e) evaluated by the decrease of fluorescence intensity of the pre-formed pyrene F-actin.

Figure 3

Decrease of phosphorylated cofilin upon FDH expression and effects of cofilin mutants/siRNA on G/F-actin and the FDH-induced phenotype. (a) Western blot analysis of cofilin and its phosphorylated form in A549/Tet-On cells induced for FDH expression with increasing concentrations of Dox (left panel) and at different time points after induction with 2.5 μg/ml of Dox (right panel). (b) Levels of cofilin (red) and its phosphorylated form (green) assessed by confocal microscopy in FDH deficient (-) and FDH expressing cells (+). Bar, 20 μm. (c) Confocal imaging of F-actin (green) and G-actin (red) in FDH-deficient or FDH-expressing (+FDH) A549/Tet-On cells with different cofilin status: S3A and S3D, cells transfected with corresponding mutant; siRNA, cells with knocked down cofilin. Yellow indicates co-localization. Bar, 20 μm. (d) Western blot analysis (top panel) of G-, F-, and the total actin in cells treated as indicated for panel (c); bar graph, ratio of G/F actin quantified from the the western blots using Quantity One software (Bio-Rad). Average (±SD) of two independent experiments is shown. Cells were analyzed 48 h or 72 h (siRNA) post-transfection. FDH induction was initiated 6 h after transfection.

Figure 4

FDH-induced cofilin dephosphorylation is not a part of antiproliferative response. (a) Levels of cofilin and p-cofilin (Western blot) in A549/Tet-On cells synchronized at different phases of the cell cycle, in the absence or in the presence of FDH. FDH was induced by the addition of 2.5 μg/ml doxycycline 48 h prior to the cell collection. Actin is shown as a loading control. (b) FACS analysis of the above cells. Apoptotic cells (sub-G0) were excluded from calculation of the percentage of cells for different phases, shown in the histogram. Synchronization was achieved as described in the Supplemental Materials. (c) Western blot analysis of cofilin and p-cofilin in the cells induced for FDH expression (2.5 μg/ml of Dox for 48 h) and treated with either 50 μM of zVAD (top panel) or 10 μM of SP600125 (bottom panel).

Figure 5

Folate supplementation controls cellular motility and related effects of FDH. (a) Reversal of FDH effects on motile characteristics by high folate supplementation (top panel, G/F actin ratio; middle panel, migration; bottom panel, invasion). Control, untreated cells/regular media; FDH, cells induced for FDH expression/regular media; FDH+LU, cells induced for FDH expression grown on regular media supplemented with 10 μM leucovorin. (b) G/F actin ratio (top panel), and migration (middle panel) and invasion (bottom panel) characteristics of A549 cells grown in normal folate (NM, control, 2.2 μM folic acid), folate-depleted (FD, folate free) and folate replete (FR, 2.2 μM folate added after folate depletion) medium. Insets show phosphorylated cofilin. In depletion experiments, prior to analysis cells were kept for 3 days in folate-free media supplemented with dialyzed FBS. In repletion experiments, cells were analyzed 24 h after the return to regular folate-containing media.

Figure 6

FDH activates dephosphorylation of cofilin by PP1 and PP2A. (a) Time dependent in vitro dephosphorylation of p-cofilin by lysate from FDH-expressing cells (top panel); the same assay in the presence of 1.0 μM of PP1/PP2A inhibitor calyculin (bottom panel); bar graph, levels of p-cofilin quantified from the panels (-Inh, in the absence of calyculin; +Inh, in the presence calyculin) using Quantity One software (Bio-Rad). Average (±SD) of three independent experiments is shown. (b) Western blot analysis of phosphatases pulled down with cofilin antibody from FDH expressing and FDH-deficient cells. (c) Motility of FDH-expressing A549 cells (wound healing assays) is restored by the addition of calyculin. (d) Calyculin prevents FDH-induced cofilin dephosphorylation in vivo. Time (h) after addition of calyculin to the cell culture is indicated. FDH expression was induced 24 h before addition of calyculin.