Induction of Megakaryocyte Differentiation Drives Nuclear Accumulation and Transcriptional Function of MKL1 Via Actin Polymerization and RhoA Activation

Abstract

3440 Background: How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryocyte Leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, plays critical roles in muscle, neuron, and megakaryocyte differentiation. Regulation of MKL1 subcellular localization is one mechanism by which a cell can control SRF activity with MKL1 localization to the nucleus being critical for its function as a transcriptional activator. MKL1 subcellular localization is cell-type specific; MKL1 is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types until it is sequestered in the nucleus following actin polymerization. In contrast, MKL1 is constitutively localized to the nucleus in neuronal cells. Objective: We tested the hypothesis that MKL1 subcellular localization is tightly regulated in megakaryocytic cells during induction of maturation. Methods and Results: Using a human erythroleukemia (HEL) cell line, we systematically dissected the events that occur after 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation to assess the relationships between RhoA activation, actin polymerization, MKL1 subcellular localization, and upregulation of SRF-target genes essential for megakaryocyte differentiation. In response to treatment with TPA, the percentage of HEL cells with predominantly nuclear localization went from <5% to over 50% in 2 hours. We found that TPA triggered RhoA activation and subsequent actin polymerization, each of which was necessary for MKL1 nuclear accumulation. In the absence of TPA, activation of RhoA and actin polymerization by calpeptin and Jasplakinolide, respectively, resulted in a statistically significant (p<.001) increase in MKL1 nuclear localization. Conversely, in cells exposed to TPA, Rho inhibition and actin depolymerization by a cell permeable C3 Transferase and Latrunculin B, respectively, caused a decrease in MKL1 nuclear localization (p<.01). Interference with MKL1 transcriptional activity using either dominant negative MKL1 (lacking the transcriptional activation domain) or the MKL1 chemical inhibitor CCG-1423 was sufficient to prevent TPA-induced expression of the SRF target genes MYL9, MYH9, and MMP9, all of which are necessary for proper megakaryocyte differentiation and maturation. Finally, we used timelapse microscopy to analyze the subcellular movement of a GFP tagged MKL1 in primary megakaryocytes. As predicted, Rho activation and actin polymerization were sufficient to drive MKL1 to the nucleus. Importantly, exposure of primary megakaryocytes to the physiological agonist thrombopoietin (TPO) stimulated MKL1 nuclear localization within minutes. Conclusions: Subcellular localization and regulation of MKL1 in megakaryocytes is dependent on RhoA activity and actin organization, similar to its regulation in fibroblasts and smooth muscle cells. Induction of megakaryocytic differentiation of HEL cells with TPA and primary megakaryocytes with TPO promotes MKL1 nuclear localization and downstream gene activation. This report broadens our knowledge of the mechanisms of action by which TPO promotes megakaryocyte differentiation. Disclosures: No relevant conflicts of interest to declare.

MRTFA augments megakaryocyte maturation by enhancing the SRF regulatory axis

“…Although some of these genes, such as CD41 and CD61, were already expressed by untreated HEL cells, TPA induction (both with and without MRTFA OE ) induced upregulation of ;200 Mk-associated genes, including VWF, KALRN, GFI1B, MYLK, FYN, GP1BB, GP5, GP9, SELP, and TBXA2R (genes upregulated in both TPA-induced HEL cells and in primary human cells are listed in supplemental Table 4). Promoter motif analysis 19 of the these genes indicated that many could be regulated by ETS factors, consistent with published data suggesting that TCFs, including ETS proteins, 26 play a role in TPA-induced gene activation in fibroblasts. 18 GO revealed enrichment for genes involved in integrin and receptor binding, which are important for Mk maturation and subsequent platelet formation.…”

“…In the MRTFA OE group without TPA treatment, the intensities of peaks for both binding categories (ie, increased and decreased SRF binding) were similar to those of the untreated control group, consistent with the cytoplasmic localization of MRTFA in the absence of TPA. 19 When comparing the peaks from cells treated with TPA, the peak for normalized reads for increased binding was much higher in the MRTFA OE TPA 1 group, indicating that MRTFA OE enhanced the TPA-induced association of SRF with these genomic regions. For those regions with decreased SRF binding in response to TPA, there was in contrast very little change between untreated and MRTFA OE TPA 1 groups, indicating that MRTFA OE may prevent SRF from being dislodged.…”

“…In this comparison, the percentage of upregulated genes with associated SRF binding (25%) is more than double that when comparing the TPA 1 only and untreated groups. Some up-/ downregulated genes had .1 SRF peak (for example, ACTA2) (supplemental the absence of TPA, 19 MRTFA OE had a weaker effect than the combined treatment. Of the 93 genes that were upregulated in response to MRTFA OE in the absence of TPA, 62 (66%) had associated SRF binding, and the SRF binding levels did not change significantly.…”

“…17 We previously showed that nuclear localization of MRTFA is induced by RhoA activation in primary Mks in response to thrombopoietin, and in HEL cells in response to the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate). 19 However, the direct and indirect targets of this activation in the context of Mk maturation are not known. Here, for the first time, we examine the genomic binding of the SRF/MRTFA complex and the consequent gene regulation changes during human Mk differentiation.…”

TIPE2 inhibits TNF-α-induced hepatocellular carcinoma cell metastasis via Erk1/2 downregulation and NF-κB activation

“…TIPE2 has been found to reduce F-actin polymerization and to inhibit cell migration (18). As actin polymerization is essential for the nuclear translocation of RhoA and protein trafficking (35,36), the effects of TIPE2 on MMP expression and cell migration might be due to the modulation of F-actin polymerization and epithelial-mesenchymal transition by TIPE2. In this study, we demonstrate that TIPE2 inhibits the effect of TNF-α on Erk1/2-NF-κB activation.…”