The myocardin-related transcription factor MKL co-regulates the cellular levels of two profilin isoforms

Abstract

Megakaryoblastic leukemia (MKL)/serum-response factor (SRF)-mediated gene transcription is a highly conserved mechanism that connects dynamic reorganization of the actin cytoskeleton to regulation of expression of a wide range of genes, including SRF itself and many important structural and regulatory components of the actin cytoskeleton. In this study, we examined the possible role of MKL/SRF in the context of regulation of profilin (Pfn), a major controller of actin dynamics and actin cytoskeletal remodeling in cells. We demonstrated that despite being located on different genomic loci, two major isoforms of Pfn (Pfn1 and Pfn2) are co-regulated by a common mechanism involving the action of MKL that is independent of its SRF-related activity. We found that MKL co-regulates the expression of Pfn isoforms indirectly by modulating signal transducer and activator of transcription 1 (STAT1) and utilizing its SAP-domain function. Unexpectedly, our studies revealed that cellular externalization, rather than transcription of Pfn1, is affected by the perturbations of MKL. We further demonstrated that MKL can influence cell migration by modulating Pfn1 expression, indicating a functional connection between MKL and Pfn1 in actin-dependent cellular processes. Finally, we provide initial evidence supporting the ability of Pfn to influence MKL and SRF expression. Collectively, these findings suggest that Pfn may play a role in a possible feedback loop of the actin/MKL/SRF signaling circuit.

Keywords: MKL; SAP-domain; actin; cell migration; cytoskeleton; profilin; serum-response factor (SRF).

Figure 1.

Effect of loss-of-function of MKL on the expression of Pfn isoforms in HEK-293 and MDA-231 cells. A, immunoblot analyses of MKL1, SRF, Pfn1, and Pfn2 expression in HEK-293 and MDA-231 cells 72 h after transfection with either MKL1 or control (cont) siRNA. The bar graphs show quantification (mean ± S.D.) of changes in Pfn1 and Pfn2 expression in response to MKL1 knockdown (data summarized from at least three experiments; *, p < 0.05; **, p < 0.01). B–D, immunoblot analyses of Pfn1 and Pfn2 expression in MDA-231 cells in response to either overnight (O/N ∼16 h) LatB (B) or 3 h LatB (C) or 24 h CCG-1423 treatment (D). LatB and CCG-1423 were used at 5 μm concentration. LatB experiments were performed with cultures in complete growth media. For CCG-1423 experiments, cells were serum-starved for 4 h before treating with CCG-1423 in complete growth media. The bar graphs show the mean ± S.D. values of the fold-changes in Pfn1 and Pfn2 expression with respect to the corresponding control conditions (data summarized from at least three experiments; **, p < 0.01). GAPDH blots serve as the loading control.

Figure 2.

Effect of MKL1 overexpression on Pfn expression. A, immunoblot analyses of MKL1, SRF, Pfn1, and Pfn2 expression 48 h after transfection with either empty vector (EV) or FLAG-tagged wild-type (WT) MKL1 in HEK-293 and MDA-231 cells (images of MKL1 bands were acquired at a very low exposure (0.05 s) to prevent saturation of the MKL1 overexpression lane signal, which prevented the endogenous MKL1 band from being detected). B, schematic of MKL structure (RPEL, three actin-binding regions that also has basic rich B2 region containing nuclear localization signal); B1, basic region that has a second nuclear localization signal and SRF-binding site; Q, glutamine-rich domain; SAP, DNA-binding domain; LZ, leucine zipper (dimerization) domain; TA, transcriptional activation domain). C, HEK-293 cells expressing various FLAG-tagged MKL1 constructs (WT, ΔN100 (lacks the first 100 amino acids), ΔB1 (internal deletion of amino acids 222–237)) were immunostained with anti-FLAG (green) antibody and DAPI (red) and scored for % cells for subcellular localization of FLAG-MKL1 as summarized in the graph below (N, exclusively nuclear; C, exclusively cytoplasmic; N/C, localized in both cytoplasmic and nuclear compartments; n indicates the number of cells analyzed in each group). D, anti-FLAG immunoblot analyses of nuclear (nucl) versus cytoplasmic (cyto) fractions prepared from HEK-293 cells 48 h after transfection with the indicated FLAG-tagged MKL constructs. Histone-H3 and tubulin blots serve as loading controls for nuclear and cytoplasmic fractions, respectively. E, total extracts of HEK-293 cells transfected with the above constructs were analyzed by immunoblotting for expression of Pfn1, Pfn2, and GAPDH (loading control); the anti-FLAG blot shows comparable expression levels of the various MKL1 constructs. The bar graph shows the average ± S.D. of the fold-changes in Pfn expression with respect to the corresponding control transfection condition (data summarized from three experiments; **, p < 0.01; *, p < 0.05). The electrophoretic mobility shift of ΔN100-MKL1 was not apparent in this blot as these samples were run on a high percentage (15%) gel.

Figure 3.

MKL regulates Pfn expression likely through an SRF-independent SAP domain-directed function. A, immunoblot analyses of MKL1, SRF, Pfn1, Pfn2, and GAPDH (loading control) expression in HEK-293 and MDA-231 cells 72 h after transient transfection with either control or SRF-siRNA (bar graph shows quantification (mean ± S.D.) of immunoblot data summarized from three independent experiments; NS, not significant). B, left, immunoblot analyses of SRF, Pfn1, and GAPDH (loading control) expression in control versus SRF-shRNA-expressing mouse 3T3 fibroblasts (two independent SRF shRNA stable clones (#1, #2) are shown); data are representative of two independent experiments; right, immunoblot analyses of MKL1 expression in control versus SRF-shRNA (#1) expressers of 3T3 cells. C, immunoblot analyses of SRF, MKL1, Pfn1, and Pfn2 expression in HEK-293 and MDA-231 cells 48 h after transfection with either SRF overexpression vector or empty vector (EV) as control. Images of SRF bands were acquired at a very low exposure (0.05 s) to prevent saturation of the SRF overexpression lane signal, which prevented the endogenous SRF band from being detected. The bar graph shows the mean ± S.D. values of the fold-changes in Pfn1 and Pfn2 expression with respect to the corresponding empty vector control transfection condition. D and E, immunoblot analyses of lysates of HEK-293 cells showing the effects of overexpression of either WT versus 3p(mut-B1) mutant form of MKL1 (D) or WT versus various deletion mutants form of MKL1 (E) on Pfn expression (3p(mut-B1), K237A/Y238A/H239A; ΔSAP = deletion of amino acids 343–378; ΔTA = a truncated form consisting of the first 630 amino acids); all MKL1 constructs are FLAG-tagged. The bar graphs show the mean ± S.D. values of the fold-changes in Pfn1 and Pfn2 expression with respect to the corresponding empty vector control transfection conditions. All data are summarized from three independent experiments; **, p < 0.01; *, p < 0.05; NS, not significant). GAPDH blots serve as the loading control.

Figure 4.

MKL promotes the expression of STAT isoforms through its SAP domain function. A and B, immunoblot analyses of HEK-293 and MDA-231 extracts showing the effects of either knockdown (A) or overexpression (B) of MKL1 on STAT1 and STAT3 expression (lysates were prepared 72 and 48 h after siRNA and plasmid transfections, respectively). The bar graphs show the mean ± S.D. values of fold-changes in STAT isoforms associated with MKL1 perturbations. C, STAT1 and STAT3 immunoblot analyses of HEK-293 extracts following overexpression of either WT form or the indicated deletion mutants of MKL1. The bar graphs show the mean ± S.D. values of the fold-changes in STAT1 and STAT3 expression with respect to the corresponding control transfection condition. All data are summarized from three experiments (**, p < 0.01; *, p < 0.05, NS, not significant; EV, empty vector). GAPDH blots serve as the loading control.

Figure 5.

MKL promotes Pfn expression through modulating STAT1 in HEK-293 cells. A and B, immunoblot analyses of Pfn expression in HEK-293 cells following either knockdown (A) or overexpression (B) of STAT variants (lysates were prepared 72 and 48 h after siRNA and plasmid transfections, respectively). C, immunoblot analyses of HEK-293 extracts showing the effect of STAT1 overexpression with or without STAT3 knockdown on Pfn expression. D, immunoblot analyses of HEK-293 extracts showing the effect of FLAG-MKL1 overexpression with or without STAT1 knockdown on Pfn expression (siRNA transfection was performed 24 h prior to plasmid transfection). All bar graphs accompanying the immunoblots show the mean ± S.D. values of the fold-changes in Pfn expression with respect to the corresponding control transfection condition (all data summarized from three experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant; EV, empty vector). GAPDH blots serve as the loading control.

Figure 6.

Loss-of-function of MKL1 down-regulates Pfn expression by promoting its extracellular release rather than its transcription. A, quantitative RT-PCR analyses show the mean ± S.D. values of the fold-changes in Pfn1 and Pfn2 mRNA levels after knockdown of MKL1 (data shown for two different MKL1 siRNAs) relative to control-siRNA transfectants of HEK-293 cells (data summarized from three independent experiments for each siRNA with three technical replicates/group; *, p < 0.05; NS, not significant). B, immunoblot analyses of MKL1, Pfn1, Pfn2, p27^Kip1, and GAPDH (loading control) expressions in HEK-293 cells 72 h after transfection with the indicated siRNAs and following treatment with either 5 μm MG-132 or DMSO (vehicle (veh) control) for 12 h. p27^Kip1, a cell cycle protein that is rapidly turned over by proteasomal degradation, shows elevation upon MG-132 treatment serving as a positive control in these experiments (data representative of three experiments). C, immunoblot analyses of MKL1, Pfn1, LC3, and GAPDH (loading control) expressions in HEK-293 cells 72 h after transfection with the indicated siRNAs and being treated with either 10 mm NH₄Cl (blocks lysosomal degradation pathway) or vehicle control for 12 h. LC3, an autophagy-related protein that is subjected to lysosomal degradation, shows elevation upon NH₄Cl treatment serving as a positive control in these experiments (data representative of three experiments). The bar graphs in B and C show the mean ± S.D. values of the fold-changes in Pfn isoforms (relative to control) associated with the indicated conditions. D–F, representative immunoblot analysis of Pfn1 level in the conditioned media prepared from HmVEC (D) and MDA-231 (E) cells following overnight treatment with either vehicle (veh) or LatB or CCG-1423. CD63, a well-known marker of extracellular vesicles, including exosomes, was used as a loading control for conditioned media samples in HmVEC-1 experiments. Coomassie staining of the SDS-PAGE of conditioned media derived from MDA-231 cells confirmed equal loading. F summarizes the quantification of fold-changes of Pfn1 (n = 3 experiments; **, p < 0.01). G, representative immunoblot analyses of Pfn1 along with the other indicated proteins in cellular extracts versus the conditioned media prepared from MDA-231 cells following transfection with either control or MKL1 siRNA (note that MKL1, STAT1, and SRF were not detected in the conditioned media). The numbers in parentheses represent the individual fold-changes in the released Pfn1 content in each of the two experiments with the mean fold-change equaling ∼4-fold.

Figure 7.

Initial evidence of sensitivity of MKL and SRF expression to perturbations of Pfn. A, immunoblot analyses of MDA-231 extracts showing the effect of co-depletion of Pfn isoforms (via transfection of pooled siRNAs targeting Pfn1 and Pfn2) on MKL1 and SRF levels. The bar graph summarizes the quantification (mean ± S.D.) of the fold-changes of MKL and SRF (n = 3 experiments; *, p < 0.05). B, immunoblot analyses of MDA-231 extracts showing the effect of transient overexpression of Pfn1 (cloned into GFP-IRES backbone vector) on MKL1, SRF, and Pfn2 levels (cells transfected with the GFP-IRES backbone vector served as a control group). The bar graphs show the mean ± S.D. values of the fold-changes in MKL1, SRF, and Pfn2 expressions with respect to the corresponding control transfection condition (n = 3 experiments; *, p < 0.05). GAPDH blots serve as the loading control. EV, empty vector. C, hypothetical model of actin/MKL/Pfn/SRF signaling circuit. This model integrates the current findings of Pfn being regulated downstream of MKL in an SRF-independent manner through STAT and our initial evidence supporting Pfn's ability to also modulate MKL and in turn SRF expression, thus possibly enabling a positive feedback loop. SRF activation can either elicit a feedforward (through promoting actin polymerization, MKL expression) action amplifying the response or a negative feedback action (through elevating G-actin level) thus dampening the response beyond a certain limit (dashed lines with question marks indicates mechanisms unknown).

Figure 8.

Loss-of-function of MKL1 promotes MDA-231 cell motility through down-regulating Pfn1 expression. A, immunoblot analyses of MKL1, SRF, Pfn1, and GADPH (loading control) expression in MDA-231 cells co-transfected with the indicated siRNAs (control versus MKL1) and overexpression vectors (GFP-IRES backbone (EV) versus GFP-IRES-Pfn1). B, box-whisker plot summarizing the average speed of migration of these four groups of cells (transfected cells were identified by GFP fluorescence) in random-motility assays. In the plot, the middle line, the upper and lower hinges of the box represent the median, 75th and 25th percentile of data, and the whiskers represent the maximum and minimum values (data summarized from three experiments; n, number of cells analyzed in each group pooled from all experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant).