To elucidate whether caspase activation is involved in megakaryopoiesis, we characterized megakaryocytes (MKs) in vav-bcl-2 transgenic (Tg) mice, in which Bcl-2 is overexpressed in hematopoietic cells. To exclude the effect of splenomegaly in Tg mice on megakaryopoiesis, splenectomy was performed. After splenectomy, basal platelet counts in peripheral blood were not significantly different between Tg and wild-type (WT) mice. However, when experimental thrombocytopenia was induced by injecting 5-fluorouracil into splenectomized mice, overshoot of platelet counts during the recovery phase was hardly observed in Tg mice. Analyses of MK ploidy during the recovery phase showed that MKs less than 16 N ploidy were significantly decreased in Tg mice, suggesting that MK supply from progenitors is impaired. Supporting this, differentiation of CD34À/c-kit þ /Sca-1 þ /LineageÀ stem cells into MKs was significantly hampered in Tg mice, whereas megakaryocyteerythroid progenitors (MEPs) normally differentiated into MKs. It suggests that differentiation into MKs is impaired in Tg mice before the stage of MEP. Furthermore, MK colony formation in WT cells was dose-dependently inhibited in the presence of a caspase inhibitor. Contrary, Bcl-2-overexpressing MKs showed normal ability for in vitro platelet production. We thus believe that caspase activation is involved in the differentiation of progenitors into megakaryocytic lineage but not in platelet production.
Summary. Background: One of the important biological activities of thrombopoietin (TPO) is to prevent the apoptosis of megakaryocytes. As the antiapoptotic protein Bcl‐xL, which has been proven to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl‐xL plays an important role in megakaryopoiesis. Objectives: We investigated the expression of Bcl‐xL during megakaryopoiesis and the underlying regulatory mechanism. Methods and Results: In stem cell‐derived megakaryocytes, expression of Bcl‐xL increased in the early and mid‐stages of the differentiation. Both in vitro in cell culture and in vivo in an animal model, expression of Bcl‐xL protein was maintained until the platelet‐producing stage. TPO depletion caused significant decrease in Bcl‐xL protein level without affecting its mRNA in both megakaryocytes and TPO‐dependent megakaryocytic UT‐7/TPO cells, suggesting that Bcl‐xL protein level in TPO‐dependent cells is post‐translationally regulated. In agreement with this finding, we recognized the appearance of a 12‐kD fragment of Bcl‐xL upon TPO depletion. This cleavage of Bcl‐xL was inhibited by a caspase‐3‐specific inhibitor. Furthermore, pretreatment of UT‐7/TPO with a phosphatidylinositol 3‐kinase (PI3 K) inhibitor resulted in the cleavage of Bcl‐xL even in the presence of TPO. We thus hypothesized that PI3 K inhibits the activation of caspase‐3 and consequent cleavage of Bcl‐xL. To prove this, we prepared UT‐7/TPO cells transfected with constitutively active Akt‐1. When TPO was depleted, the transfectant was significantly less liable to caspase‐3 activation and Bcl‐xL cleavage. Conclusions: Bcl‐xL protein is expressed throughout megakaryopoiesis until platelets are produced, and its expression level is at least in part post‐translationally regulated through TPO‐mediated Akt activation.
Background: The Matrigel-embedded epithelium of the mouse submandibular gland undergoes branching morphogenesis when cultured in medium supplemented with fibroblast growth factor 7 (FGF7) and lysophosphatidic acid (LPA), whereas it elongates a stalk with limited branching in medium with only FGF7. Because LPA is a well-known activator of epidermal growth factor (EGF) signaling, we hypothesized the involvement of autocrine EGF family growth factors in the branching morphogenesis. Results: Reverse transcriptase polymerase chain reaction studies showed that three members, Tgfa, Hbegf,and Nrg1 of the EGF family were expressed in the epithelium cultured with FGF7 1 LPA as well as in the epithelium freshly isolated from the rudiments. All the growth factors induced extensive branching morphogenesis in the Matrigel-embedded epithelium in the presence of LPA. Tyrphostin AG112, an inhibitor of EGF signaling, severely impaired branching morphogenesis induced by FGF7 1 LPA without exogenous addition of EGF family growth factors to the culture medium. The shaking cultures, which were expected to decrease the concentration of autocrine growth factors near the epithelium by promoting their diffusion, significantly reduced branching morphogenesis induced by FGF7 1 LPA. Conclusions: Autocrine EGF family growth factors are involved in epithelial branching morphogenesis induced by FGF7 1 LPA. Developmental Dynamics 243:552-559,
Megakaryocytes (Mgk) differentiate from hematopoietic stem cells through the complex process of endomitosis and cytoplasmic development, and finally form proplatelets in the terminal stage of the differentiation. To elucidate the role of a proapoptotic effecter protein, caspase-3, in megakaryopoiesis and thrombopiesis, we analyzed the phenotype of Mgk in vav-Bcl-2 transgenic (Tg) mice, which overexpress Bcl-2 exclusively in hematopoietic cells by the regulation of vav promotor. As was expected, Bcl-2-overexressing Mgk were remarkably resistant to caspase-3 activation induced by the depletion of thrombopoietin (TPO) from the culture. Percentages of proplatelet formation (PPF) assayed by culturing primary Mgk were slightly increased in the Tg mice. Platelet production from cultured Mgk, which was assayed by flowcytometry, was also slightly increased, indicating normal ability for Plt production from Bcl-2-overexpressing mature Mgk. To further analyze the ability for thrombopoiesis, we assayed the trends of Plt recovery following experimental thrombocytopenia. As Bcl-2-overexpressing mice had giant splenomegaly, we used splenectomized mice, which showed normal Plt counts. After the injection of 5-FU, we observed significant delay of the Plt recovery in the Tg mice, as well as the lack of the overshoot of Plt counts during the recovery phase. To gain insights about the mechanism underlying the delayed Plt recovery, we analyzeded the ploidy of DNA content. In the steady state, peak ploidy of Mgk obtained from the bone marrow of the Tg mice shifted to 2–4N. In a sharp contrast, in Plt recovery phase after 5-FU injection, peak ploidy shifted to 16–32N, and the percentages of Mgk possessing less than 16N ploidy were dramatically decreased. Consistent with this finding, we observed the delay of PPF in Bcl-2-overexpressing cells when Mgk were derived from c-kit+/lineage- progenitor cells in the presence of TPO. These data indicate that differentiation of immature Mgk into mature Plt-producing stage is significantly delayed in the Tg mice, but once the maturation proceeds, Mgk normally produce Plt. Taken together, we conclude that caspase-3 activation is involved in the early stage of the differentiation of Mgk, but not in Plt production.
Thrombopoietin (TPO) plays a relevant role for megakaryocyte differentiation from stem cells. One of the important biological activities of TPO is to prevent the apoptosis of megakaryocytic cells. As an anti-apoptotic protein Bcl-xL, which has been proved to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl-xL plays an important role for megakaryopoiesis. We thus investigated the expression of Bcl-xL during megakaryopoiesis and the underlying regulatory mechanism. In stem cell-derived megakaryocytes, expression of Bcl-xL increased in the early- and mid-stages of the differentiation. Both in vitro in stem cell-derived megakaryocyteic cell culture and in vivo in an animal model injected with anti-platelet antibody, expression of Bcl-xL protein was maintained until platelet-producing stage of the megakaryopoiesis. TPO-depletion caused significant decrease in Bcl-xL protein level without affecting its mRNA in both stem cell-derived megakaryocytes and TPO-dependent megakaryocytic UT7/TPO cells. As a 12-kD fragment of Bcl-xL appeared by the withdrawal of TPO, we considered that Bcl-xL was cleaved upon TPO-depletion. This cleavage was blocked by a caspase-3-specific inhibitor, suggesting that caspase cleaves Bcl-xL in TPO-depleted megakaryocytes. Furthermore, pretreatment of UT7/TPO cells with a phosphatidylinositol 3-kinase (PI3K) inhibitor resulted in the cleavage of Bcl-xL even in the presence of TPO. We thus hypothesized that PI3K or its downstream signaling molecule inhibits the activation of caspase-3 and consequent cleavage of Bcl-xL. To prove this possibility, we prepared UT7/TPO cells transfected with constitutively active Akt-1. When TPO was depleted, the transfectant was significantly less liable to caspase-3 activation and Bcl-xL cleavage. Concerning transcriptional regulation of Bcl-xL, suppression of GATA-1 in UT7/TPO using siRNA caused decreased expression of both c-Mpl and Bcl-xL. Taken together, we conclude that GATA-1 regulates the expression of both c-Mpl and Bcl-xL, and once Bcl-xL is expressed, its protein level is maintained by the TPO-mediated Akt activation.
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