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

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.

We tested the hypothesis that MKL1 subcellular localization is tightly regulated in megakaryocytic cells during induction of maturation.

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.

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.

No relevant conflicts of interest to declare.