Platelets are critical for normal hemostasis. Their deregulation can lead to bleeding or to arterial thrombosis, a primary cause of heart attack and ischemic stroke. Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is a 5-phosphatase capable of dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate second messenger into phosphatidylinositol 3,4-bisphosphate. SHIP1 plays a critical role in regulating the level of these 2 lipids in platelets. Using SHIP1-deficient mice, we found that its loss affects platelet aggregation in response to several agonists with minor effects on fibrinogen binding and β 3 integrin tyrosine phosphorylation. Accordingly, SHIP1-null mice showed defects in arterial thrombus formation in response to a localized laser-induced injury. Moreover, these mice had a prolonged tail bleeding time. Upon stimulation, SHIP1-deficient platelets showed large membrane extensions, abnormalities in the open canalicular system, and a dramatic decrease in close cell-cell contacts. Interestingly, SHIP1 appeared to be required for platelet contractility, thrombus organization, and fibrin clot retraction. These data indicate that SHIP1 is an important element of the platelet signaling machinery to support normal hemostasis. To our knowledge, this is the first report unraveling an important function of SHIP1 in the activation of hematopoietic cells, in contrast to its well-documented role in the negative regulation of lymphocytes. IntroductionPlatelet activation is a highly regulated process and critical for mediating normal hemostasis. Vascular injury exposes subendothelial matrix proteins that allow platelet arrest and their subsequent spreading, activation, and secretion of soluble mediators. These soluble agonists recruit circulating platelets, allowing aggregation for the bleeding to stop. In pathological states, platelet activation can contribute to thrombosis and cerebrovascular and arterial occlusion and eventually to heart attacks and strokes. Several signaling mechanisms have been shown to contribute to platelet activation, including the phospholipase C (PLC)/Ca 2+ and the PI3K pathways. By generating phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P 2 ] second messengers, PI3K plays an important role in platelet activation and plug stability (1-3). Using a selective PI3K β inhibitor, Jackson et al. (3) recently suggested that this lipid kinase could be a potential target for antithrombotic therapy.It is now well established in several models that phosphoinositide phosphatases involved in the hydrolysis of PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 , such as the phosphatase and tensin homolog (PTEN), are essential for the negative regulation of PI3K pathways (4). PTEN is commonly deleted or inactivated in several cancers,
The specific mechanisms controlling the transition from proliferation to terminal differentiation in human intestinal epithelial cells (HIEC) remain largely undefined. Herein, we analyzed the expression and localization of Rb and E2F proteins in well-established normal intestinal epithelial cell models which allow for the re-enactment of the crypt-villus axis in vitro as well as in intact epithelium and in colon cancer cells. We report that (1) expression of E2F1 is down-regulated while E2F4 protein is sequestered in the cytoplasm during G(0) arrest associated with serum deprivation, confluency, and terminal differentiation of intestinal cells; (2) concurrently, there is an accumulation of the hypophosphorylated form of the pocket proteins into the nucleus with an increased association of E2F4 with pRb and p130; (3) cells which expressed high levels of nuclear E2F4 are all positive for Ki67 staining in human fetal intestine; (4) activation of HIEC crypt cells by growth factors leads to an increase in the nuclear localization of E2F4 which may be attributable to a decrease in the serine/threonine phosphorylation of this transcription factor; (5) inhibition of p38 MAP kinase with alpha/beta inhibitor SB203580 induces E2F4 translocation into the nucleus and its transcriptional activity. In conclusion, our data suggest a key role for E2F4 in proliferation of human intestinal crypt cells and that its cytoplasmic retention as well as its sequestration by Rb proteins may represent a critical step in initiating cell-cycle exit.
The high concentration of HCO(3)(-) ions (150 mM) in the human pancreatic ducts raises the question of the membrane proteins responsible for their secretion in addition to the Cl(-)/HCO(3)(-) exchanger. In this study, we investigated the expression of carbonic anhydrase IV (CA IV), a possible candidate. Experiments were carried out on specimens of normal human pancreas obtained from brain-dead donors ( n=9) as well as on isolated human ductal cells. Two antibodies were generated: CA IV NH(2) antibody directed against the NH(2) terminal of human glycosyl phosphatidylinositol (GPI)-anchored CA IV and CA IV COOH antibody directed against the COOH terminal of the same protein before its association with a GPI in the rough endoplasmic reticulum. A 35-kDa CA IV was detected in the homogenates of human pancreas. Immunocytochemistry demonstrated the expression of CA IV in centroacinar cells and in intercalated, intralobular, and interlobular ductal cells. The immunoreactivity observed with the CA IV COOH antibody was mainly localized on luminal membranes of ductal cells. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase C indicated that the CA IV expressed in pancreatic ducts was not GPI-anchored. Its detection in the same extracts by the CA IV COOH antibody indicated that it was anchored by a hydrophobic segment at the carboxy terminal. Taken together, these results suggest that normal human pancreatic ductal cells express a 35-kDa CA IV anchored in their luminal plasma membrane by a hydrophobic segment of the COOH terminus. In view of its localization and its mode of anchorage in luminal plasma membranes, this CA IV may participate in the maintenance of luminal pH.
E2F transcription factors control cell cycle progression. The localization of E2F4 in intestinal epithelial cells is cell cycle dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, whether nuclear translocation of E2F4 alone is sufficient to trigger intestinal epithelial cell proliferation remains to be established. Adenoviruses expressing fusion proteins between green fluorescent protein (GFP) and wild-type (wt)E2F4 or GFP and nuclear localization signal (NLS)-tagged E2F4 were used to infect normal human intestinal epithelial crypt cells (HIEC). In contrast to expression of wtE2F4, persistent expression of E2F4 into the nucleus of HIEC triggered phosphatidylserine exposure, cytoplasmic shrinkage, zeiosis, formation of apoptotic bodies, and activation of caspase 9 and caspase 3. Inhibition of caspase activities by zVAD-fmk partially inhibited cell death induced by E2F4-NLS. An induction of p53, phosphorylated Ser15-p53, PUMA, FAS, BAX, RIP, and phosphorylated JNK1 was also observed in HIEC expressing E2F4-NLS compared with wtE2F4-expressing cells. E2F1 and p14ARF expression remained unaltered. Downregulation of p53 expression by RNA interference attenuated cell death induced by E2F4-NLS. By contrast, the level of cell death was negligible in colon cancer cells despite the strong expression of E2F4 into the nucleus. In conclusion, deregulated nuclear E2F4 expression induces apoptosis via multiple pathways in normal intestinal epithelial cells but not in colon cancer cells. Hence, mutations that deregulate E2F4 localization may provide an initial proliferative advantage but at the same time accelerate cell death. However, intestinal cells acquiring mutations (e.g., p53, Bax loci, etc.) may escape apoptosis, thereby revealing the full mitogenic potential of the E2F4 transcription factor.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.