Role for MKL1 in megakaryocytic maturation

Abstract

Megakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate, overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34(+) cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down, suggesting that MKL1 acts through SRF. Consistent with these findings in human cells, knockout of Mkl1 in mice leads to reduced platelet counts in peripheral blood, and reduced ploidy in bone marrow megakaryocytes. In conclusion, MKL1 promotes physiologic maturation of human and murine megakaryocytes.

Figure 1

Expression of Mkl1 during megakaryocyte differentiation. (A) Freshly isolated E12.5-E14.5 fetal liver cells were differentiated in the presence of TPO, IL-6, and IL-11. At day 4, different cell types (myelomonocytic cells vs megakaryocytes) were separated on a discontinuous BSA gradient. Analysis of cell ploidy validates the megakaryocyte fractionation. (B) Relative Mkl1 mRNA levels were assessed by quantitative RT-PCR on fetal liver subpopulations. The RNA from total fetal liver cells was used from day 0 to day 4 (left). At day 4, RNA from fractionated cells was used (right). The relative expression levels were normalized to 18S rRNA. (C) c-kit⁺CD41⁺ BM cells were sorted (day 1) and cultured with TPO for 4 more days. RNA at different time points was used to assess relative Mkl1 levels by quantitative RT-PCR. The Mkl1 expression was normalized to 18S rRNA or absolute cell number. Mean plus or minus SEM of duplicate experiments is represented.

Figure 2

MKL1 promotes human megakaryocytic differentiation. (A) Validation of 3 HEL cell clones (clone numbers 5, 8, and 9) using Western blot analysis of MKL1 expression in the absence (left) and presence (right) of Dox for 2 days. MKL1-His protein is detected by anti-His antibody. (B) Based on analysis of Wright-Giemsa–stained cytospins, MKL1 increases the percentage of mature megakaryocytes in response to TPA over 4 days (P < .05). (C) Representative data showing ploidy of TPA-stimulated HEL/MKL1 cells or control cells after 4 days of differentiation with (solid line) or without (tinted) Dox. MKL1 increases the ploidy of cells exposed to TPA. Note increased 8N and 16N peaks in HEL/MKL1 cells. (D) Morphology of HEL cells treated with TPA and/or Dox as indicated. MKL1 has no effect on cell morphology in the absence of TPA (compare untreated with Dox), and promotes adhesion and spreading when cells are exposed to TPA (compare TPA with TPA + Dox). (E,F) The effects of enforced expression of MKL1 in human CD34⁺ cells during megakaryocytic differentiation. (E) The figures shown are gated for GFP-positive cells infected with pCCL and pCCL-MKL1 virus. Average percentage-positive cells for CD61, CD41a, and CD42b expression plus or minus SD of 3 independent experiments. *P < .05 from pCCL versus pCCL-MKL1 in the respective immunophenotype group. (F) Ploidy distribution of CD41a⁺ fraction in pCCL and pCCL-MKL1–infected cells plus or minus SD of 3 independent experiments. *P < .05 from pCCL versus pCCL-MKL1 in the respective ploidy group.

Figure 3

Knockdown of SRF inhibits MKL1-induced increase in ploidy in response to TPA. HEL/MKL1 cells (A) and HEL/control cells (B) were treated with Dox and siRNA against SRF as indicated, and ploidy was measured over time (x-axis) after TPA addition. The y-axis represents the percentage of cells that had a ploidy of 8N. *P < .005, negative siRNA + Dox versus the rest of the groups.

Figure 4

Mkl1 deficiency does not affect LSK, pre-MegE cell numbers, and CFU-MK formation. (A) Two tibias and 1 femur from each mouse were collected. After BM harvest and red blood cell lysis, total cell numbers were counted (n = 6 per genotype). (B) Comparison of percentage of LSK cells in WT, HET, and Mkl1 KO BM (n = 9 per genotype). (C) Comparison of percentage of pre-MegE cells in WT, HET, and Mkl1 KO BM (n = 9 per genotype). (D) CFU-MK potential from total BM (n = 6 per genotype).

Figure 5

Mkl1 deficiency leads to an increased percentage of committed CD41⁺c-kit⁺ megakaryocytic progenitors and a decreased percentage of mature high-ploidy megakaryocytes. (A) Comparison of percentages of CD41⁺ cells in WT, HET, and Mkl1 KO BM (n = 6 per genotype). (B) Comparison of serum TPO concentration (n = 3 per genotype). (C) Comparison of percentages of CD41⁺ c-kit⁺ cells in WT, HET, and Mkl1 KO BM (n = 6 per genotype). (D) Comparison of numbers of megakaryocytes per high-power field in the femurs of WT, HET, and Mkl1 KO mice (n = 2 per genotype). (E,F) In vivo ploidy analysis of CD41-positive BM cells taken from WT, HET, and Mkl1 KO mice (n = 6 in WT and HET; n = 8 in Mkl1 KO mice). *P < .05 from both WT versus KO and HET versus KO.