Critical role of miR-9 in myelopoiesis and EVI1-induced leukemogenesis.

MicroRNA-9 (miR-9) is emerging as a critical regulator of organ development and neurogenesis. It is also deregulated in several types of solid tumors; however, its role in hematopoiesis and leukemogenesis is not yet known. Here we show that miR-9 is detected in hematopoietic stem cells and hematopoietic progenitor cells, and that its expression increases during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis and promotes apoptosis in vitro and in vivo. Conversely, in hematopoietic progenitor cells, the inhibition of miR-9 with a miRNA sponge blocks myelopoiesis. Ecotropic viral integration site 1 (EVI1), required for normal embryogenesis, is considered an oncogene because its inappropriate up-regulation induces malignant transformation in solid and hematopoietic cancers. Here we show that EVI1 binds to the promoter of miR-9-3, leading to DNA hypermethylation of the promoter and repression of miR-9. Moreover, miR-9 expression reverses a myeloid differentiation block that is induced by EVI1. Our findings indicate that EVI1, when inappropriately expressed, delays or blocks myeloid differentiation at least in part by DNA hypermethylation and down-regulation of miR-9. It was reported that Forkhead box class O genes (FoxOs) inhibit myeloid differentiation and prevent differentiation of leukemia-initiating cells. Here we identify both FoxO1 and FoxO3 as direct targets of miR-9 in hematopoietic cells and find that up-regulation of FoxO3 inhibits miR-9-induced myelopoiesis. These results reveal a unique role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms and provide insights into the epigenetic regulation of miR9 in tumorigenesis.

Activated mesenchymal stem cells increase wound tensile strength in aged mouse model via macrophages.

BACKGROUND: Wound healing is impaired in the aged. Mesenchymal stem cells (MSCs) can exert beneficial effects in wounds; however, promoting healing in the challenging setting of aged skin may require additional potency. MSCs can enhance the production of pro-regenerative cytokines and growth factors when activated with interferon gamma. We hypothesized that the increased potency of activated MSC could be used to facilitate wound healing in the aged mice. METHODS: Young and old C57BL6 mice underwent incisional wounds and were treated with naive MSCs, activated MSCs, or vehicle to examine MSC effects on tensile strength in the aged skin. To test whether the benefits of MSC treatment could be attributed to the participation of host macrophages, liposomal clodronate was used to deplete host macrophages. RESULTS: In older mice, tensile strength of healing wounds was significantly lower than that in younger mice. Older mice treated with activated MSCs showed significant increases in tensile strength restoring the strength to that observed in younger mice. Macrophage depletion abrogated the beneficial effect of MSC. CONCLUSIONS: Activated MSCs restored wound tensile strength in the aged mice, and this effect was dependent on host macrophage activity. These data provide encouraging support for the development of activated MSC therapies for enhanced tissue regeneration, especially for older population groups.

Methylation and silencing of miRNA-124 by EVI1 and self-renewal exhaustion of hematopoietic stem cells in murine myelodysplastic syndrome.

By expressing EVI1 in murine bone marrow (BM), we previously described a myelodysplastic syndrome (MDS) model characterized by pancytopenia, dysmegakaryopoiesis, dyserythropoiesis, and BM failure. The mice invariably died 11-14 months after BM transplantation (BMT). Here, we show that a double point mutant EVI1-(1+6Mut), unable to bind Gata1, abrogates the onset of MDS in the mouse and re-establishes normal megakaryopoiesis, erythropoiesis, BM function, and peripheral blood profiles. These normal features were maintained in the reconstituted mice until the study was ended at 21 months after BMT. We also report that EVI1 deregulates several genes that control cell division and cell self-renewal. In striking contrast, these genes are normalized in the presence of the EVI1 mutant. Moreover, EVI1, but not the EVI1 mutant, seemingly deregulates these cellular processes by altering miRNA expression. In particular, the silencing of miRNA-124 by DNA methylation is associated with EVI1 expression, but not that of the EVI1 mutant, and appears to play a key role in the up-regulation of cell division in murine BM cells and in the hematopoietic cell line 32Dcl3. The results presented here demonstrate that EVI1 induces MDS in the mouse through two major pathways, both of which require the interaction of EVI1 with other factors: one, results from EVI1-Gata1 interaction, which deregulates erythropoiesis and leads to fatal anemia, whereas the other occurs by interaction of EVI1 with unidentified factors causing perturbation of the cell cycle and self-renewal, as a consequence of silencing miRNA-124 by EVI1 and, ultimately, ensues in BM failure.

Mesenchymal Stem Cells Synergize with 635, 532, and 405 nm Laser Wavelengths in Renal Fibrosis: A Pilot Study.

OBJECTIVE: To address whether a single treatment of one of three visible light wavelengths, 635, 532, and 405 nm (constant wave, energy density 2.9 J/m2), could affect the hallmarks of established renal fibrosis and whether these wavelengths could facilitate mesenchymal stem cell (MSC) beneficence. BACKGROUND DATA: Chronic kidney disease is a global health problem with only 20% receiving care worldwide. Kidneys with compromised function have ongoing inflammation, including increased oxidative stress and apoptosis, peritubular capillary loss, tubular atrophy, and tubulointerstitial fibrosis. Promising studies have highlighted the significant potential of MSC-based strategies to mitigate fibrosis; however, reversal of established fibrosis has been problematic, suggesting that methods to potentiate MSC effects require further development. Laser treatments at visible wavelengths have been reported to enhance mitochondrial potential and available cellular ATP, facilitate proliferation, and inhibit apoptosis. We hypothesized that laser-delivered energy might provide wavelength-specific effects in the fibrotic kidney and enhance MSC responses. MATERIALS AND METHODS: Renal fibrosis, established in C57BL6 mice following 21 days of unilateral ureter obstruction (UUO), was treated with one of three wavelengths alone or with autologous MSC. Mitochondrial activity, cell proliferation, apoptosis, and cytokines were measured 24 h later. RESULTS: Wavelengths 405, 532, and 635 nm all significantly synergized with MSC to enhance mitochondrial activity and reduce apoptosis. Proliferative activity was observed in the renal cortices following combined treatment with the 532 nm laser and MSC; endothelial proliferation increased in response to the 635 nm laser alone and to the combined effects of MSC and the 405 nm wavelength. Reductions of transforming growth factor-ß were observed with 532 nm alone and when combined with MSC. CONCLUSIONS: Specific wavelengths of laser energy appear to induce different responses in renal fibrotic tissue. These findings support further study in the development of a customized laser therapy program of combined wavelengths to optimize MSC effects in the treatment of renal fibrosis.

The oncoprotein EVI1 and the DNA methyltransferase Dnmt3 co-operate in binding and de novo methylation of target DNA.

EVI1 has pleiotropic functions during murine embryogenesis and its targeted disruption leads to prenatal death by severely affecting the development of virtually all embryonic organs. However, its functions in adult tissues are still unclear. When inappropriately expressed, EVI1 becomes one of the most aggressive oncogenes associated with human hematopoietic and solid cancers. The mechanisms by which EVI1 transforms normal cells are unknown, but we showed recently that EVI1 indirectly upregulates self-renewal and cell-cycling genes by inappropriate methylation of CpG dinucleotides in the regulatory regions of microRNA-124-3 (miR-124-3), leading to the repression of this small gene that controls normal differentiation and cell cycling of somatic cells. We used the regulatory regions of miR-124-3 as a read-out system to investigate how EVI1 induces de novo methylation of DNA. Here we show that EVI1 physically interacts with DNA methyltransferases 3a and 3b (Dnmt3a/b), which are the only de novo DNA methyltransferases identified to date in mouse and man, and that it forms an enzymatically active protein complex that induces de novo DNA methylation in vitro. This protein complex targets and binds to a precise region of miR-124-3 that is necessary for repression of a reporter gene by EVI1. Based on our findings, we propose that in cooperation with Dnmt3a/b EVI1 regulates the methylation of DNA as a sequence-specific mediator of de novo DNA methylation and that inappropriate EVI1 expression contributes to carcinogenesis through improper DNA methylation.

EVI1 Impairs myelopoiesis by deregulation of PU.1 function.

EVI1 is an oncogene inappropriately expressed in the bone marrow (BM) of approximately 10% of myelodysplastic syndrome (MDS) patients. This disease is characterized by severe anemia and multilineage myeloid dysplasia that are thought to be a major cause of mortality in MDS patients. We earlier reported on a mouse model that constitutive expression of EVI1 in the BM led to fatal anemia and myeloid dysplasia, as observed in MDS patients, and we subsequently showed that EVI1 interaction with GATA1 blocks proper erythropoiesis. Whereas this interaction could provide the basis for the erythroid defects in EVI1-positive MDS, it does not explain the alteration of myeloid differentiation. Here, we have examined the expression of several genes activated during terminal myelopoiesis in BM cells and identified a group of them that are altered by EVI1. A common feature of these genes is their regulation by the transcription factor PU.1. We report here that EVI1 interacts with PU.1 and represses the PU.1-dependent activation of a myeloid promoter. EVI1 does not seem to inhibit PU.1 binding to DNA, but rather to block its association with the coactivator c-Jun. After mapping the PU.1-EVI1 interaction sites, we show that an EVI1 point mutant, unable to bind PU.1, restores the activation of PU.1-regulated genes and allows a normal differentiation of BM progenitors in vitro.