Loss of RhoB Expression Enhances the Myelodysplastic Phenotype of Mammalian Diaphanous-Related Formin mDia1 Knockout Mice

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis and hyperplastic bone marrow. Complete loss or interstitial deletions of the long arm of chromosome 5 occur frequently in MDS. One candidate tumor suppressor on 5q is the mammalian Diaphanous (mDia)-related formin mDia1, encoded by DIAPH1 (5q31.3). mDia-family formins act as effectors for Rho-family small GTP-binding proteins including RhoB, which has also been shown to possess tumor suppressor activity. Mice lacking the Drf1 gene that encodes mDia1 develop age-dependent myelodysplastic features. We crossed mDia1 and RhoB knockout mice to test whether the additional loss of RhoB expression would compound the myelodysplastic phenotype. Drf1−/−RhoB−/− mice are fertile and develop normally. Relative to age-matched Drf1−/−RhoB+/− mice, the age of myelodysplasia onset was earlier in Drf1−/−RhoB−/− animals—including abnormally shaped erythrocytes, splenomegaly, and extramedullary hematopoiesis. In addition, we observed a statistically significant increase in the number of activated monocytes/macrophages in both the spleen and bone marrow of Drf1−/−RhoB−/− mice relative to Drf1−/−RhoB+/− mice. These data suggest a role for RhoB-regulated mDia1 in the regulation of hematopoietic progenitor cells

Flow cytometry analysis of erythroid precursors in mouse bone marrow and spleen.

<p>Each point on the scatter plots represents data from a single mouse (o = <i>Drf1</i>^−/−<i>RhoB</i>^+/−; ▪ = <i>Drf1</i>^−/−<i>RhoB</i>^−/−) <i>A</i>. Scatter plot showing the percentage of CD71⁺ cells from the bone marrow and spleen of mice (* denotes <i>P</i>≤0.05). <i>B</i>. Percentage of TER-119+ cells from the bone marrow and spleen of mice (* denotes <i>P</i>≤0.05). <i>C</i>. Percentage of cells undergoing S phase from the bone marrow and spleen of mice (** denotes <i>P</i>≤0.01).</p

Flow cytometry analysis of mouse bone marrow and splenic cells.

<p><i>A</i>. Scatter plot showing the percentage of lymphocytes, monocytes, and granulocytes from bone morrow. Open shapes represent <i>Drf1</i>^−/−<i>RhoB</i>^+/− mice and filled shapes represent <i>Drf1</i>^−/−<i>RhoB</i>^−/− mice (** denotes <i>P</i>≤0.01; *** denotes <i>P</i>≤0.001). <i>B</i>. Scatter plot showing the percentage of lymphocytes, monocytes, and granulocytes from mice splenic single-cell suspensions. Legend is the same as in <i>A</i> (** denotes <i>P</i>≤0.01). <i>C</i>. Percentage of F4/80⁺, CD11b⁺, and CD29⁺ cells from the bone marrow and spleen of mice (* denotes <i>P</i>≤0.05; ** denotes <i>P</i>≤0.01) (o = <i>Drf1</i>^−/−<i>RhoB</i>^+/−; ▪ = <i>Drf1</i>^−/−<i>RhoB</i>^−/−).</p

Peripheral blood from <i>Drf1</i>^−/−<i>RhoB</i>^−/− mice show age-dependent abnormalities.

<p><i>A</i>. Peripheral blood smears stained with Wright-Giemsa from 400-day-old mice. Left and center panels are images at 40x; right panels are 60x. <i>B</i>. Total WBC count from peripheral blood. <i>C</i>. Platelet numbers from peripheral blood CBC analysis. In both <i>B</i> and <i>C</i>, each point on scatter plot represents data from a single mouse (o = <i>Drf1</i>^−/−<i>RhoB</i>^+/−; ▪ = <i>Drf1</i>^−/−<i>RhoB</i>^−/−) (** denotes <i>P</i>≤0.01).</p

Ubiquitin-mediated Degradation of the Formin mDia2 upon Completion of Cell Division*

Formins assemble non-branched actin filaments and modulate microtubule dynamics during cell migration and cell division. At the end of mitosis formins contribute to the generation of actin filaments that form the contractile ring. Rho small GTP-binding proteins activate mammalian diaphanous-related (mDia) formins by directly binding and disrupting an intramolecular autoinhibitory mechanism. Although the Rho-regulated activation mechanism is well characterized, little is known about how formins are switched off. Here we reveal a novel mechanism of formin regulation during cytokinesis based on the following observations; 1) mDia2 is degraded at the end of mitosis, 2) mDia2 is targeted for disposal by post-translational ubiquitin modification, 3) forced expression of activated mDia2 yields binucleate cells due to failed cytokinesis, and 4) the cytokinesis block is dependent upon mDia2-mediated actin assembly as versions of mDia2 incapable of nucleating actin but that still stabilize microtubules have no effect on cytokinesis. We propose that the tight control of mDia2 expression and ubiquitin-mediated degradation is essential for the completion of cell division. Because of the many roles for formins in cell morphology, we discuss the relevance of mDia protein turnover in other processes where ubiquitin-mediated proteolysis is an essential component