Deletion of Gas2l3 in mice leads to specific defects in cardiomyocyte cytokinesis during development.

GAS2L3 is a recently identified cytoskeleton-associated protein that interacts with actin filaments and tubulin. The in vivo function of GAS2L3 in mammals remains unknown. Here, we show that mice deficient in GAS2L3 die shortly after birth because of heart failure. Mammalian cardiomyocytes lose the ability to proliferate shortly after birth, and further increase in cardiac mass is achieved by hypertrophy. The proliferation arrest of cardiomyocytes is accompanied by binucleation through incomplete cytokinesis. We observed that GAS2L3 deficiency leads to inhibition of cardiomyocyte proliferation and to cardiomyocyte hypertrophy during embryonic development. Cardiomyocyte-specific deletion of GAS2L3 confirmed that the phenotype results from the loss of GAS2L3 in cardiomyocytes. Cardiomyocytes from Gas2l3-deficient mice exhibit increased expression of a p53-transcriptional program including the cell cycle inhibitor p21. Furthermore, loss of GAS2L3 results in premature binucleation of cardiomyocytes accompanied by unresolved midbody structures. Together these results suggest that GAS2L3 plays a specific role in cardiomyocyte cytokinesis and proliferation during heart development.

Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells.

The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

Differential effects of mixed lymphocyte reaction supernatant on human mesenchymal stromal cells.

The concept that mesenchymal stromal cells (MSCs), a component of the hematopoietic microenvironment, can be a target for alloreactive effector cells in the context of graft-vs-host disease has not been investigated in detail. Mixed lymphocyte reaction (MLR) supernatant was used to mimic the inflammatory milieu induced by an allogeneic immune response in vitro. In addition to phenotype and proliferation, we monitored MSC differentiation, gene expression, and support of CD34(+) hematopoietic stem and progenitor cells after priming with MLR supernatant. Priming of MSCs with MLR supernatant led to an 11-fold decrease in cobblestone area-forming cells in the 4-week coculture (p < 0.05) and a threefold decrease of colony-forming unit macrophage in the colony-forming cell assay (p < 0.05). MSC proliferation over 8 days was increased 2.5-fold (p < 0.05). Osteogenic differentiation was enhanced, while adipogenesis was concurrently suppressed. In addition, the surface expression of HLA-DR and intercellular adhesion molecule-1 was increased 20-fold (p = 0.06) and 45-fold (p < 0.05), respectively. This was associated with increased adhesion of hematopoietic stem and progenitor cells to MLR-treated MSCs. In summary, our data shed light on the dysfunction of the stromal environment during graft-vs-host disease, possibly aggravating cytopenia and leading to an enhanced immunogenicity of MSCs.

Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ.

Signal transduction of FMS-like tyrosine kinase 3 (FLT3) is regulated by protein-tyrosine phosphatases (PTPs). We recently identified the PTP DEP-1/CD148/PTPRJ as a novel negative regulator of FLT3. This study addressed the role of DEP-1 for regulation of the acute myeloid leukemia (AML)-related mutant FLT3 internal tandem duplication (ITD) protein. Our experiments revealed that DEP-1 was expressed but dysfunctional in cells transformed by FLT3 ITD. This was caused by enzymatic inactivation of DEP-1 through oxidation of the DEP-1 catalytic cysteine. In intact cells, including primary AML cells, FLT3 ITD kinase inhibition reactivated DEP-1. DEP-1 reactivation was also achieved by counteracting the high levels of reactive oxygen species (ROS) production detected in FLT3 ITD-expressing cell lines by inhibition of reduced NAD phosphate (NADPH)-oxidases, or by overexpression of catalase or peroxiredoxin-1 (Prx-1). Interference with ROS production in 32D cells inhibited cell transformation by FLT3 ITD in a DEP-1-dependent manner, because RNAi-mediated depletion of DEP-1 partially abrogated the inhibitory effect of ROS quenching. Reactivation of DEP-1 by stable overexpression of Prx-1 extended survival of mice in the 32D cell/C3H/HeJ mouse model of FLT3 ITD-driven myeloproliferative disease. The study thus uncovered DEP-1 oxidation as a novel event contributing to cell transformation by FLT3 ITD.

Protein-tyrosine phosphatase DEP-1 controls receptor tyrosine kinase FLT3 signaling.

Fms-like tyrosine kinase 3 (FLT3) plays an important role in hematopoietic differentiation, and constitutively active FLT3 mutant proteins contribute to the development of acute myeloid leukemia. Little is known about the protein-tyrosine phosphatases (PTP) affecting the signaling activity of FLT3. To identify such PTP, myeloid cells expressing wild type FLT3 were infected with a panel of lentiviral pseudotypes carrying shRNA expression cassettes targeting different PTP. Out of 20 PTP tested, expressed in hematopoietic cells, or presumed to be involved in oncogenesis or tumor suppression, DEP-1 (PTPRJ) was identified as a PTP negatively regulating FLT3 phosphorylation and signaling. Stable 32D myeloid cell lines with strongly reduced DEP-1 levels showed site-selective hyperphosphorylation of FLT3. In particular, the sites pTyr-589, pTyr-591, and pTyr-842 involved in the FLT3 ligand (FL)-mediated activation of FLT3 were hyperphosphorylated the most. Similarly, acute depletion of DEP-1 in the human AML cell line THP-1 caused elevated FLT3 phosphorylation. Direct interaction of DEP-1 and FLT3 was demonstrated by "substrate trapping" experiments showing association of DEP-1 D1205A or C1239S mutant proteins with FLT3 by co-immunoprecipitation. Moreover, activated FLT3 could be dephosphorylated by recombinant DEP-1 in vitro. Enhanced FLT3 phosphorylation in DEP-1-depleted cells was accompanied by enhanced FLT3-dependent activation of ERK and cell proliferation. Stable overexpression of DEP-1 in 32D cells and transient overexpression with FLT3 in HEK293 cells resulted in reduction of FL-mediated FLT3 signaling activity. Furthermore, FL-stimulated colony formation of 32D cells expressing FLT3 in methylcellulose was induced in response to shRNA-mediated DEP-1 knockdown. This transforming effect of DEP-1 knockdown was consistent with a moderately increased activation of STAT5 upon FL stimulation but did not translate into myeloproliferative disease formation in the 32D-C3H/HeJ mouse model. The data indicate that DEP-1 is negatively regulating FLT3 signaling activity and that its loss may contribute to but is not sufficient for leukemogenic cell transformation.