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. 2012 Mar 13;22(3):573-84.
doi: 10.1016/j.devcel.2011.12.019. Epub 2012 Mar 1.

Role of RhoA-specific guanine exchange factors in regulation of endomitosis in megakaryocytes

Yuan Gao  1 , Elenoe Smith, Elmer Ker, Phil Campbell, Ee-chun Cheng, Siying Zou, Sharon Lin, Lin Wang, Stephanie Halene, Diane S Krause
Affiliations

Role of RhoA-specific guanine exchange factors in regulation of endomitosis in megakaryocytes

Yuan Gao et al. Dev Cell. .

Abstract

Polyploidization can precede the development of aneuploidy in cancer. Polyploidization in megakaryocytes (Mks), in contrast, is a highly controlled developmental process critical for efficient platelet production via unknown mechanisms. Using primary cells, we demonstrate that the guanine exchange factors GEF-H1 and ECT2, which are often overexpressed in cancer and are essential for RhoA activation during cytokinesis, must be downregulated for Mk polyploidization. The first (2N-4N) endomitotic cycle requires GEF-H1 downregulation, whereas subsequent cycles (>4N) require ECT2 downregulation. Exogenous expression of both GEF-H1 and ECT2 prevents endomitosis, resulting in proliferation of 2N Mks. Furthermore, we have shown that the mechanism by which polyploidization is prevented in Mks lacking Mkl1, which is mutated in megakaryocytic leukemia, is via elevated GEF-H1 expression; shRNA-mediated GEF-H1 knockdown alone rescues this ploidy defect. These mechanistic insights enhance our understanding of normal versus malignant megakaryocytopoiesis, as well as aberrant mitosis in aneuploid cancers.

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Figures

Figure 1
Figure 1
Endomitosis of MkP induced by Tpo. (A) 2N to 4N Mk endomitosis shows cleavage furrow ingression with subsequent regression, resulting in one 4N cell. (B) 4N to 8N Mk endomitosis showing no apparent cleavage furrow. The figures show overlays of DIC (gray) with green fluorescent H2B-GFP (green) images taken every 5 minutes. The time of each image relative to the first is indicated in each frame.
Figure 2
Figure 2
Active RhoA is absent from the cleavage furrow during the first endomitotic cleavage. Mouse primary PreMegE cells were transduced with the RhoA biosensor virus for 48 h before switching to fresh growth medium (A) or differentiation (B) medium. After 8 hours, the RhoA activation pattern throughout mitosis (A) or endomitosis (B) was assessed. The CFP channel indicates biosensor (and RhoA) localization (i, iii, v). The RhoA activation pattern was assessed by the RhoA biosensor’s FRET/CFP ratio (FRET). The red color in FRET images indicates high RhoA activation (ii, iv, vi). All images were processed identically. Elapsed time (minutes) with starting time set to 0 (8h post-Tpo administration) is indicated in each picture. Images are representative of at least 4 similar images for each condition.
Figure 3
Figure 3
GEF-H1 and ECT2 are down-regulated during Mk differentiation. Data shown for freshly sorted (d0) PreMegE and MkP cells from WT mice, as well as PreMegE cultured in DM for the time indicated. (A) Relative levels of GEF-H1 mRNA are reduced during Mk differentiation. ***, P<0.005, versus value of PreMegE day 0. (B) Relative levels of ECT2 mRNA were also decreased. ***, P<0.005, versus value of PreMegE day 0. (C) The protein levels of GEF-H1 and ECT2 are reduced with different kinetics as shown by Western-blotting. Anti-α-tubulin was used as the loading control. Relative protein level of each sample after normalization to tubulin and setting PreMegE level as 1 are indicated above each band.
Figure 4
Figure 4
GEF-H1 and ECT2 are reduced in endomitotic Mk. PreMegE were grown in GM and MkP in DM. (A) Cells were stained with anti-GEF-H1 (red), anti-α-tubulin (green) and DAPI. Examples of normal mitosis (i–iii, PreMegE cultured in growth medium), 2N to 4N endomitosis (iv–vi), and ≥ 4N endomitosis (vii–ix) are shown. GEF-H1 protein level is reduced at the 2N to 4N stage of endomitosis and increases at later stages (2 days) of endomitosis. (B) ECT2 protein, stained with anti-ECT2 antibody (red), is clearly detected in PreMegE undergoing mitosis (i – iii), and at the 2N to 4N stage of endomitosis (iv–vi), but ECT2 levels are reduced at later stages (2 days) of endomitosis (vii–ix).
Figure 5
Figure 5
Differential effects of overexpressing GEF-H1 or ECT2 on polyploidization of Mk. (A–C) PreMegE cells in growth medium were transduced with retroviral vectors (control and expressing individual GEFs) and cultured for two days, then transferred to differentiation medium for 3 days before the ploidy of each sample was assessed. (A) Shown is the effect of overexpressing control (GFP) virus, GEF-H1 or ECT2 encoding virus. Ploidy of GFP+ cells in each condition is shown. The percentages of Mk in 2N, 4N, and ≥ 8N ploidy are indicated. (B) The average ploidy of Mk expressing GFP only (control), GEF-H1 or ECT2 is compared. ***, P<0.005, versus control. (C) In this experiment, cells were transduced with 2 retroviral vectors (one encoding RFP and the other GFP). Controls received vectors encoding only RFP and GFP. GEF-H1 (upper second panel) received GFP control plus GEF-H1-ires-RFP vectors, ECT2 (upper third panel) received ECT2-ires-GFP plus control RFP vectors, the upper fourth panel shows cells transduced with ECT2-ires-GFP plus GEF-H1-ires-RFP. Samples in lower panel were transduced with RhoAN19-ires-GFP together with RFP control (left), GEF-H1-ires-RFP (middle), or ECT2-ires-RFP (right). Ploidy is shown for GFP+RFP+ (double positive) cells. (D) Western blot of HEL cells transduced with the indicated virus validate expression vectors. (E) Overexpression of GEF-H1 also decreases polyploidization of Mk in vivo. CD45.1 BM cells were transduced with control (GFP only) or GEF-H1–ires-GFP virus, and transplanted into lethally irradiated CD45.2 mice. After 6 weeks, the ploidy of GFP positive Mk was analyzed. Representative ploidy profiles from GFP+ Mk expressing empty virus or GEF-H1 are shown. (F) Average ploidy of GFP positive Mk recovered 6 weeks post-transplant. *, P<0.05, versus the value of control.
Figure 6
Figure 6
Down-regulation of GEF-H1 is MKL1- dependent. (A) The relative mRNA level of GEF-H1 is significantly increased in Mkl1−/− Mk, and shows very little decrease with Mk differentiation. WT or Mkl1−/− PreMegE or MkP were cultured in DM as indicated. ***, P<0.005, versus the corresponding WT control. (B) Relative ECT2 mRNA levels are the same for WT and Mkl1−/− cells. In both, ECT2 mRNA decreases during Mk differentiation. P>0.1 for the values of Mkl1−/− versus the corresponding WT control, (C) PreMegE cultured in growth medium (mitotic controls, i–iii and vii–ix) and MkP cultured in Tpo-only medium, which induces endomitosis, were immunostained as indicated. Unlike WT MkP (iv–vi), Mkl1−/− MkP do not show a loss of GEF-H1 protein level in response to TPO induction (x–xii). (D and E) 293FT cells were transduced with empty vector (−); WT Mkl1 (WT); constitutively active (CA) Mkl1, which lacks the actin binding domain; or dominant negative (DN) Mkl1, which lacks the transcriptional activation domain, but can still heterodimerize with endogenous Mkl1. Overexpression of CA MKL1 significantly reduces endogenous GEF-H1 mRNA (D) and protein (E) levels compared to cells transduced with empty vector (−), wild type Mkl1 (WT) and dominant negative (DN) Mkl1. GAPDH was used as a loading control in (E). ***, P<0.005, and **, P<0.01, versus the value of empty vector (−). (F) Quantitative RT-PCR reveals much higher levels of GEF-H1 and ECT2 mRNA in the 6133 cell line compared to WT PreMegE with PreMegE value set to 1. Values normalized to 18S RNA. ***, P<0.005.
Figure 7
Figure 7
Knockdown of GEF-H1 restores polyploidy in Mkl1−/− Mk in vitro. (A) WT or Mkl1−/− PreMegE were transduced with retrovirus encoding either shRNA targeting luciferase or GEF-H1, as indicated. After Tpo induced differentiation, ploidy was assessed. A representative ploidy plot for each condition is shown. The percentages of Mk in 2N, 4N, 8N and ≥16N ploidy are indicated. (B) Average ploidy of these samples. *** P <0.005. (C) Validation of shRNA mediated knockdown of GEF-H1 protein in NIH3T3 cells transduced with the indicated constructs. GFP positive shRNA expressing cells were sorted, and analyzed by Western Blot. GAPDH was used as the loading control.
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