Simultaneous Deletion of p21Cip1/Waf1 and Caspase-3 Accelerates Proliferation and Partially Rescues the Differentiation Defects of Caspase-3 Deficient Hematopoietic Stem Cells

Specialized blood cells are generated through the entire life of an organism by differentiation of a small number of hematopoietic stem cells (HSC). There are strictly regulated mechanisms assuring a constant and controlled production of mature blood cells. Although such mechanisms are not completely understood, some factors regulating cell cycle and differentiation have been identified. We have previously shown that Caspase-3 is an important regulator of HSC homeostasis and cytokine responsiveness. p21^cip1/waf1 is a known cell cycle regulator, however its role in stem cell homeostasis seems to be limited. Several reports indicate interactions between p21^cip1/waf1 and Caspase-3 in a cell type dependent manner. Here we studied the impact of simultaneous depletion of both factors on HSC homeostasis. Depletion of both Caspase-3 and p21^cip1/waf1 resulted in an even more pronounced increase in the frequency of hematopoietic stem and progenitor cells. In addition, simultaneous deletion of both genes revealed a further increase of cell proliferation compared to single knock-outs and WT control mice, while apoptosis or self-renewal ability were not affected in any of the genotypes. Upon transplantation, p21^cip1/waf1-/- bone marrow did not reveal significant alterations in engraftment of lethally irradiated mice, while Caspase-3 deficient HSPC displayed a significant reduction of blood cell production. However, when both p21^cip1/waf1 and Caspase-3 were eliminated this differentiation defect caused by Caspase-3 deficiency was abrogated.     

Caspase-3 is Involved in IFN-γ- and TNF-α-Mediated MIN6 Cells Apoptosis via NF-κB/Bcl-2 Pathway

TNF-α and IFN-γ are the major pro-inflammatory cytokines in the β-cell destruction. However, the underlying mechanism remains unclear. The present study used a murine insulinoma cell line MIN6 for further investigation of the effect of Caspase-3 on the cytokines-induced pancreatic β-cell apoptosis and analyzed the mechanisms involved in the activation of Caspase-3. It was showed that the combination of IFN-γ and TNF-α significantly reduced the viability of MIN6 cells and the observed cells growth inhibition was due to cell apoptosis as judged by the morphological changes under a confocal laser scanning microscopy and FACS assay of Annexin-V/7-AAD double staining. Accompanying with NF-κB activation and Bcl-2 downregulation, both the cleaved Caspase-3 and PARP, a known substrate of Caspase-3 in vivo, were observed at 24 and 12 h, respectively, after cells exposure to IFN-γ and TNF-α treatment. Pretreatment of Caspase-3 inhibitors remarkably attenuated IFN-γ- and TNF-α-induced cells apoptosis. Inhibition of NF-κB activation led to the increase in Bcl-2 expression, a significant attenuation in Caspase-3 activity, and an obvious amelioration in cells viability in IFN-γ- and TNF-α-treated MIN6 cells. Taken together, our results indicate that Caspase-3 is critical for the induction of MIN6 cells apoptosis and it’s activation is further confirmed to be related to the NF-κB-mediated Bcl-2 downregulation, which may be the underlying mechanism of IFN-γ- and TNF-α-mediated MIN6 cells apoptosis.     

Smad phosphoisoform signals in acute and chronic liver injury: similarities and differences between epithelial and mesenchymal cells

Hepatocellular carcinoma (HCC) usually arises from hepatic fibrosis caused by chronic inflammation. In chronic liver damage, hepatic stellate cells undergo progressive activation to myofibroblasts (MFB), which are important extracellular-matrix-producing mesenchymal cells. Concomitantly, perturbation of transforming growth factor (TGF)-β signaling by pro-inflammatory cytokines in the epithelial cells of the liver (hepatocytes) promotes both fibrogenesis and carcinogenesis (fibro-carcinogenesis). Insights into fibro-carcinogenic effects on chronically damaged hepatocytes have come from recent detailed analyses of the TGF-β signaling process. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad homology (MH) 1 and MH2 domains. TGF-β type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create phosphoisoforms phosphorylated at the COOH-terminal, linker, or both (L/C) regions. After acute liver injury, TGF-β-mediated pSmad3C signaling terminates hepatocytic proliferation induced by the pro-inflammatory cytokine-mediated mitogenic pSmad3L pathway; TGF-β and pro-inflammatory cytokines synergistically enhance collagen synthesis by activated hepatic stellate cells via pSmad2L/C and pSmad3L/C pathways. During chronic liver disease progression, pre-neoplastic hepatocytes persistently affected by TGF-β together with pro-inflammatory cytokines come to exhibit the same carcinogenic (mitogenic) pSmad3L and fibrogenic pSmad2L/C signaling as do MFB, thereby accelerating liver fibrosis while increasing risk of HCC. This review of Smad phosphoisoform-mediated signals examines similarities and differences between epithelial and mesenchymal cells in acute and chronic liver injuries and considers Smad linker phosphorylation as a potential target for the chemoprevention of fibro-carcinogenesis.     

Staying alive

Quiescence is a state of reversible cell cycle arrest that can grant protection against many environmental insults. In some systems, cellular quiescence is associated with a low metabolic state characterized by a decrease in glucose uptake and glycolysis, reduced translation rates and activation of autophagy as a means to provide nutrients for survival. For cells in multiple different quiescence model systems, including Saccharomyces cerevisiae, mammalian lymphocytes and hematopoietic stem cells, the PI3Kinase/TOR signaling pathway helps to integrate information about nutrient availability with cell growth rates. Quiescence signals often inactivate the TOR kinase, resulting in reduced cell growth and biosynthesis. However, quiescence is not always associated with reduced metabolism; it is also possible to achieve a state of cellular quiescence in which glucose uptake, glycolysis and flux through central carbon metabolism are not reduced. In this review, we compare and contrast the metabolic changes that occur with quiescence in different model systems.     

Staying alive: Metabolic adaptations to quiescence

Quiescence is a state of reversible cell cycle arrest that can grant protection against many environmental insults. In some systems, cellular quiescence is associated with a low metabolic state characterized by a decrease in glucose uptake and glycolysis, reduced translation rates and activation of autophagy as a means to provide nutrients for survival. For cells in multiple different quiescence model systems, including Saccharomyces cerevisiae, mammalian lymphocytes and hematopoietic stem cells, the PI3Kinase/TOR signaling pathway helps to integrate information about nutrient availability with cell growth rates. Quiescence signals often inactivate the TOR kinase, resulting in reduced cell growth and biosynthesis. However, quiescence is not always associated with reduced metabolism; it is also possible to achieve a state of cellular quiescence in which glucose uptake, glycolysis and flux through central carbon metabolism are not reduced. In this review, we compare and contrast the metabolic changes that occur with quiescence in different model systems.     

Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis

Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.     

Regulating quiescence: new insights into hematopoietic stem cell biology

Blood-forming hematopoietic stem cells (HSCs) ensure production of all mature blood cells during homeostatic and regenerative hematopoiesis. Proliferation, cell cycle regulation, and quiescence are key processes involved in this function, and in a recent issue of Cancer Cell, show that HSC quiescence is actively regulated by specific molecular mechanisms that appear to distinguish normal HSC maintenance from HSC responses to hematologic injury.     

Hematopoietic stem cell quiescence is maintained by compound contributions of the retinoblastoma gene family

Individual members of the retinoblastoma (Rb) tumor suppressor gene family serve critical roles in the control of cellular proliferation and differentiation, but the extent of their contributions is masked by redundant and compensatory mechanisms. Here we employed a conditional knockout strategy to simultaneously inactivate all three members, Rb, p107, and p130, in adult hematopoietic stem cells (HSCs). Rb family triple knockout (TKO) mice develop a cell-intrinsic myeloproliferation that originates from hyperproliferative early hematopoietic progenitors and is accompanied by increased apoptosis in lymphoid progenitor populations. Loss of quiescence in the TKO HSC pool is associated with an expansion of these mutant stem cells but also with an enhanced mobilization and an impaired reconstitution potential upon transplantation. The presence of a single p107 allele is sufficient to largely rescue these defects. Thus, Rb family members collectively maintain HSC quiescence and the balance between lymphoid and myeloid cell fates in the hematopoietic system.     

Regulation of myogenic stem cell behaviour by vessel cells: The "ménage à trois" of satellite cells,periendothelial cells and endothelial cells

In skeletal muscle, satellite cells, that are responsible of muscle repair, are localized close to capillaries. Although angiogenesis is known for a long time to be crucial for muscle repair and satellite cell survival, cellular interplays between vessel cells and satellite/myogenic cells have been poorly explored. We analyzed the interrelationships between myogenic cells, endothelial cells, and periendothelial cells that includes smooth muscle cells and endomysial fibroblasts. We found that endothelial cells strongly stimulate myogenic cell growth and, inversely, myogenic cells increase angiogenesis. VEGF plays a essential role in this bidirectional interaction. On the contrary, periendothelial cells promote the return to quiescence of a subset of muscle precursor cells to quiescence that ensures self-renewal of adult muscle stem cells. We have shown that Angiopoietin-1/Tie-2 signalling controls the entry into quiescence. We propose that during muscle regeneration, i.e. while vessels are not stabilized, endothelial cells and myogenic cells interact with each other to promote both myogenesis and angiogenesis, that have been shown to be concomitant processes in several models. On the other hand, once homeostasis of muscle is reached, the proximity of satellite cells and periendothelial cells allows the responsiveness of satellite cells, that bear Tie-2 receptor, to the secretion of Angiopoietin-1 by periendothelial cells, that, in the same time, stabilize vessels by promoting quiescence of endothelial cells.