Showing papers on "Cellular differentiation published in 1998"
TL;DR: It is reported that the activity of Oct4 is essential for the identity of the pluripotential founder cell population in the mammalian embryo and also determines paracrine growth factor signaling from stem cells to the trophectoderm.
3,461 citations
TL;DR: A culture system that facilitates the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal progenitor cells has been developed in this article, where cells obtained in bone marrow aspirates were first isolated by monolayer culture and then transferred into tubes and allowed to form three-dimensional aggregates in a chemically defined medium.
2,403 citations
TL;DR: It is reported that oscillations reduce the effective Ca2+ threshold for activating transcription factors, thereby increasing signal detection at low levels of stimulation and providing direct evidence that [Ca2+]i oscillations increase both the efficacy and the information content of Ca2+, leading to gene expression and cell differentiation.
Abstract: Cytosolic calcium ([Ca2+]i) oscillations are a nearly universal mode of signalling in excitable and non-excitable cells. Although Ca2+ is known to mediate a diverse array of cell functions, it is not known whether oscillations contribute to the efficiency or specificity of signalling or are merely an inevitable consequence of the feedback control of [Ca2+]i. We have developed a Ca2+ clamp technique to investigate the roles of oscillation amplitude and frequency in regulating gene expression driven by the proinflammatory transcription factors NF-AT, Oct/OAP and NF-kappaB. Here we report that oscillations reduce the effective Ca2+ threshold for activating transcription factors, thereby increasing signal detection at low levels of stimulation. In addition, specificity is encoded by the oscillation frequency: rapid oscillations stimulate all three transcription factors, whereas infrequent oscillations activate only NF-kappaB. The genes encoding the cytokines interleukin (IL)-2 and IL-8 are also frequency-sensitive in a way that reflects their degree of dependence on NF-AT versus NF-kappaB. Our results provide direct evidence that [Ca2+]i oscillations increase both the efficacy and the information content of Ca2+ signals that lead to gene expression and cell differentiation.
1,903 citations
TL;DR: It is demonstrated that the nuclear receptor PPARγ is induced in human monocytes following exposure to oxLDL and is expressed at high levels in the foam cells of atherosclerotic lesions, and it is suggested that endogenous PParγ ligands may be important regulators of gene expression during atherogenesis.
1,717 citations
TL;DR: Investigation of the role of STAT3 and inducible STAT3F in ES cells growing in the presence of LIF specifically abrogated self-renewal and promoted differentiation establish that STAT3 plays a central role in the maintenance of the pluripotential stem cell phenotype.
Abstract: The propagation of embryonic stem (ES) cells in an undifferentiated pluripotent state is dependent on leukemia inhibitory factor (LIF) or related cytokines. These factors act through receptor complexes containing the signal transducer gp130. The downstream mechanisms that lead to ES cell self-renewal have not been delineated, however. In this study, chimeric receptors were introduced into ES cells. Biochemical and functional studies of transfected cells demonstrated a requirement for engagement and activation of the latent trancription factor STAT3. Detailed mutational analyses unexpectedly revealed that the four STAT3 docking sites in gp130 are not functionally equivalent. The role of STAT3 was then investigated using the dominant interfering mutant, STAT3F. ES cells that expressed this molecule constitutively could not be isolated. An episomal supertransfection strategy was therefore used to enable the consequences of STAT3F expression to be examined. In addition, an inducible STAT3F transgene was generated. In both cases, expression of STAT3F in ES cells growing in the presence of LIF specifically abrogated self-renewal and promoted differentiation. These complementary approaches establish that STAT3 plays a central role in the maintenance of the pluripotential stem cell phenotype. This contrasts with the involvement of STAT3 in the induction of differentiation in somatic cell types. Cell type-specific interpretation of STAT3 activation thus appears to be pivotal to the diverse developmental effects of the LIF family of cytokines. Identification of STAT3 as a key transcriptional determinant of ES cell self-renewal represents a first step in the molecular characterization of pluripotency.
1,595 citations
TL;DR: The role of Tcf-4 in colon cancer was investigated in this paper, where the authors found that Tcf7/2//- mice die shortly after birth from colon cancer.
Abstract: Mutations of the genes encoding APC or beta-catenin in colon carcinoma induce the constitutive formation of nuclear beta-catenin/Tcf-4 complexes, resulting in activated transcription of Tcf target genes. To study the physiological role of Tcf-4 (which is encoded by the Tcf7/2 gene), we disrupted Tcf7/2 by homologous recombination. Tcf7/2-/- mice die shortly after birth. A single histopathological abnormality was observed. An apparently normal transition of intestinal endoderm into epithelium occurred at approximately embryonic day (E) 14.5. However, no proliferative compartments were maintained in the prospective crypt regions between the villi. As a consequence, the neonatal epithelium was composed entirely of differentiated, non-dividing villus cells. We conclude that the genetic program controlled by Tcf-4 maintains the crypt stem cells of the small intestine. The constitutive activity of Tcf-4 in APC-deficient human epithelial cells may contribute to their malignant transformation by maintaining stem-cell characteristics.
1,570 citations
TL;DR: The notion that PTEN haploinsufficiency plays a causal role in CD, LDD and BZS pathogenesis is supported, and it is demonstrated that Pten is a tumour suppressor essential for embryonic development.
Abstract: The PTEN gene encodes a dual-specificity phosphatase mutated in a variety of human cancers. PTEN germline mutations are found in three related human autosomal dominant disorders, Cowden disease (CD), Lhermitte-Duclos disease (LDD) and Bannayan-Zonana syndrome (BZS), characterized by tumour susceptibility and developmental defects. To examine the role of PTEN in ontogenesis and tumour suppression, we disrupted mouse Pten by homologous recombination. Pten inactivation resulted in early embryonic lethality. Pten-/- ES cells formed aberrant embryoid bodies and displayed an altered ability to differentiate into endodermal, ectodermal and mesodermal derivatives. Pten+/- mice and chimaeric mice derived from Pten+/- ES cells showed hyperplastic-dysplastic changes in the prostate, skin and colon, which are characteristic of CD, LDD and BZS. They also spontaneously developed germ cell, gonadostromal, thyroid and colon tumours. In addition, Pten inactivation enhanced the ability of ES cells to generate tumours in nude and syngeneic mice, due to increased anchorage-independent growth and aberrant differentiation. These results support the notion that PTEN haploinsufficiency plays a causal role in CD, LDD and BZS pathogenesis, and demonstrate that Pten is a tumour suppressor essential for embryonic development.
1,503 citations
TL;DR: In this paper, the authors derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage, which can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties.
Abstract: We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase–PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of α-cardiac myosin heavy chain, α-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at ≫+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/− 60 nM and a Bmax of 118 fmol per 105 cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.
1,452 citations
TL;DR: A culture of mouse blastocysts or early postimplantation trophoblasts in the presence of fibroblast growth factor 4 (FGF4) permitted the isolation of permanent trophoblast stem cell lines that exclusively contributed to the trophoplast lineage in vivo in chimeras.
Abstract: The trophoblast cell lineage is essential for the survival of the mammalian embryo in utero. This lineage is specified before implantation into the uterus and is restricted to form the fetal portion of the placenta. A culture of mouse blastocysts or early postimplantation trophoblasts in the presence of fibroblast growth factor 4 (FGF4) permitted the isolation of permanent trophoblast stem cell lines. These cell lines differentiated to other trophoblast subtypes in vitro in the absence of FGF4 and exclusively contributed to the trophoblast lineage in vivo in chimeras.
1,294 citations
TL;DR: It is shown that PPARγ is expressed at high levels in both well- and poorly-differentiated adenocarcinomas, in normal colonic mucosa and in human colon cancer cell lines, indicating that the growth and differentiation of colon cancer cells can be modulated through PPARβ.
Abstract: PPARgamma is a nuclear receptor that has a dominant regulatory role in differentiation of cells of the adipose lineage, and has recently been shown to be expressed in the colon. We show here that PPARgamma is expressed at high levels in both well- and poorly-differentiated adenocarcinomas, in normal colonic mucosa and in human colon cancer cell lines. Ligand activation of this receptor in colon cancer cells causes a considerable reduction in linear and clonogenic growth, increased expression of carcinoembryonic antigen and the reversal of many gene expression events specifically associated with colon cancer. Transplantable tumors derived from human colon cancer cells show a significant reduction of growth when mice are treated with troglitazone, a PPARgamma ligand. These results indicate that the growth and differentiation of colon cancer cells can be modulated through PPARgamma.
1,008 citations
TL;DR: The results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.
TL;DR: The data suggest that VEGF, at pathologically relevant concentrations in vivo, may exert effects on pluripotent stem cells that result in blocked DC development as well as affect many other hematopoietic lineages.
TL;DR: These studies show that the second-generation HPCs, unlike HMBA, are potent inhibitors of HDAC activity, and appear to induce differentiation by different pathways.
Abstract: Hybrid polar compounds (HPCs) have been synthesized that induce terminal differentiation and/or apoptosis in various transformed cells. We have previously reported on the development of the second-generation HPCs suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bishydroxamide (CBHA) that are 2,000-fold more potent inducers on a molar basis than the prototype HPC hexamethylene bisacetamide (HMBA). Herein we report that CBHA and SAHA inhibit histone deacetylase 1 (HDAC1) and histone deacetylase 3 (HDAC3) activity in vitro. Treatment of cells in culture with SAHA results in a marked hyperacetylation of histone H4, but culture with HMBA does not. Murine erythroleukemia cells developed for resistance to SAHA are cross-resistant to trichostatin A, a known deacetylase inhibitor and differentiation inducer, but are not cross-resistant to HMBA. These studies show that the second-generation HPCs, unlike HMBA, are potent inhibitors of HDAC activity. In this sense, HMBA and the second-generation HPCs appear to induce differentiation by different pathways.
TL;DR: It is demonstrated that ES cells can differentiate to hemopoietic cells through the proximal lateral mesoderm, merely upon culturing in type IV collagen-coated dishes, and implicates Flk1+VE-cadherin+ cells as a diverging point of hemopOietic and endothelial cell lineages.
Abstract: Totipotent murine ES cells have an enormous potential for the study of cell specification. Here we demonstrate that ES cells can differentiate to hemopoietic cells through the proximal lateral mesoderm, merely upon culturing in type IV collagen-coated dishes. Separation of the Flk1+ mesoderm from other cell lineages was critical for hemopoietic cell differentiation, whereas formation of the embryoid body was not. Since the two-dimensionally spreading cells can be monitored easily in real time, this culture system will greatly facilitate the study of the mechanisms involved in the cell specification to mesoderm, endothelial, and hemopoietic cells. In the culture of ES cells, however, lineages and stages of differentiating cells can only be defined by their own characteristics. We showed that a combination of monoclonal antibodies against E-cadherin, Flk1/KDR, PDGF receptor(alpha), VE-cadherin, CD45 and Ter119 was sufficient to define most intermediate stages during differentiation of ES cells to blood cells. Using this culture system and surface markers, we determined the following order for blood cell differentiation: ES cell (E-cadherin+Flk1-PDGFRalpha-), proximal lateral mesoderm (E-cadherin-Flk1+VE-cadherin-), progenitor with hemoangiogenic potential (Flk1+VE-cadherin+CD45-), hemopoietic progenitor (CD45+c-Kit+) and mature blood cells (c-Kit-CD45+ or Ter119+), though direct differentiation of blood cells from the Flk1+VE-cadherin- stage cannot be ruled out. Not only the VE-cadherin+CD45- population generated from ES cells but also those directly sorted from the yolk sac of 9.5 dpc embryos have a potential to give rise to hemopoietic cells. Progenitors with hemoangiogenic potential were identified in both the Flk1+VE-cadherin- and Flk1+VE-cadherin+ populations by the single cell deposition experiment. This line of evidence implicates Flk1+VE-cadherin+ cells as a diverging point of hemopoietic and endothelial cell lineages.
TL;DR: In this paper, the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system was demonstrated.
Abstract: Mesenchymal progenitor cells provide a source of cells for the repair of musculoskeletal tissue. However, in vitro models are needed to study the mechanisms of differentiation of progenitor cells. This study demonstrated the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The cells were then passaged into an aggregate culture system in a serum-free medium. Initially, the cell aggregates contained type-I collagen and neither type-II nor type-X collagen was detected. Type-II collagen was typically detected in the matrix by the fifth day, with the immunoreactivity localized in the region of metachromatic staining. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for an outer region of flattened, perichondrial-like cells in a matrix rich in type-I collagen. Aggrecan and link protein were detected in extracts of the cell aggregates, providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the newly differentiating chondrocytic cells; the small proteoglycans, biglycan and decorin, were also detected in extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate demonstrated a uniform distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. When the bone-marrow-derived cell preparations were passaged in monolayer culture as many as twenty times, with cells allowed to grow to confluence at each passage, the chondrogenic potential of the cells was maintained after each passage. CLINICAL RELEVANCE: Chondrogenesis of progenitor cells is the foundation for the in vivo repair of fractures and damaged articular cartilage. In vitro chondrogenesis of human bone-marrow-derived osteochondral progenitor cells should provide a useful model for studying this cellular differentiation. Furthermore, the maintenance of chondrogenic potential after greater than a billion-fold expansion provides evidence for the clinical utility of these cells in the repair of bone and cartilage.
TL;DR: Monocytes cultured with endothelium differentiated into dendritic cells within 2 days, particularly after phagocytosing particles in subendothelial collagen, and migrated across the endot Helium in the ablumenal-to-lumenal direction, as would occur during entry into lymphatics.
Abstract: Essential to the dendritic cell system of antigen-presenting cells are the veiled dendritic cells that traverse afferent lymph to enter lymph nodes, where they initiate immune responses. The origin of veiled cells, which were discovered 20 years ago, is unclear. Monocytes cultured with endothelium differentiated into dendritic cells within 2 days, particularly after phagocytosing particles in subendothelial collagen. These nascent dendritic cells migrated across the endothelium in the ablumenal-to-lumenal direction, as would occur during entry into lymphatics. Monocytes that remained in the subendothelial matrix became macrophages. Therefore, monocytes have two potential fates associated with distinct patterns of migration.
TL;DR: Examining gene expression in discrete generations of dividing cells reveals a novel mechanism governing Th cell fate that initially integrates proliferative and differentiative signals and subsequently maintains stability of the differentiated state.
TL;DR: The combination of TGZ and ATRA synergistically and irreversibly inhibits growth and induces apoptosis of MCF7 breast cancer cells and the combination may provide a novel, nontoxic and selective therapy for human breast cancers.
Abstract: Induction of differentiation and apoptosis in cancer cells through ligands of nuclear hormone receptors (NHRs) is a novel and promising approach to cancer therapy. All-trans-retinoic acid (ATRA), an RA receptor-specific NHR ligand, is now used for selective cancers. The NHR, peroxisome proliferator-activated receptor γ (PPARγ) is expressed in breast cancer cells. Activation of PPARγ through a synthetic ligand, troglitazone (TGZ), and other PPARγ-activators cause inhibition of proliferation and lipid accumulation in cultured breast cancer cells. TGZ (10−5 M, 4 days) reversibly inhibits clonal growth of MCF7 breast cancer cells and the combination of TGZ (10−5 M) and ATRA (10−6 M, 4 days) synergistically and irreversibly inhibits growth and induces apoptosis of MCF7 cells, associated with a dramatic decrease of their bcl-2 protein levels. Similar effects are noted with in vitro cultured breast cancer tissues from patients, but not with normal breast epithelial cells. The observed apoptosis mediated by TGZ and ATRA may be related to the striking down-regulation of bcl-2, because forced over-expression of bcl-2 in MCF7 cells cultured with TGZ and ATRA blocks their cell death. TGZ significantly inhibits MCF7 tumor growth in triple immunodeficient mice. Combined administration of TGZ and ATRA causes prominent apoptosis and fibrosis of these tumors without toxic effects on the mice. Taken together, this combination may provide a novel, nontoxic and selective therapy for human breast cancers.
TL;DR: Cryopreservable human NSCs may be propagated by both epigenetic and genetic means that are comparably safe and effective, and may allow the development of NSC transplantation for a range of disorders.
Abstract: Stable clones of neural stem cells (NSCs) have been isolated from the human fetal telencephalon. These self-renewing clones give rise to all fundamental neural lineages in vitro. Following transplantation into germinal zones of the newborn mouse brain they participate in aspects of normal development, including migration along established migratory pathways to disseminated central nervous system regions, differentiation into multiple developmentally and regionally appropriate cell types, and nondisruptive interspersion with host progenitors and their progeny. These human NSCs can be genetically engineered and are capable of expressing foreign transgenes in vivo. Supporting their gene therapy potential, secretory products from NSCs can correct a prototypical genetic metabolic defect in neurons and glia in vitro. The human NSCs can also replace specific deficient neuronal populations. Cryopreservable human NSCs may be propagated by both epigenetic and genetic means that are comparably safe and effective. By analogy to rodent NSCs, these observations may allow the development of NSC transplantation for a range of disorders.
TL;DR: It is concluded that the kinase cascade initiated by Raf can regulate the expression of p16(Ink4a) and the proliferative arrest and senescence that follows and may provide a defense against neoplastic transformation when the MAP kinase signaling cascade is inappropriately active.
Abstract: The oncogenes RAS and RAF came to view as agents of neoplastic transformation. However, in normal cells, these genes can have effects that run counter to oncogenic transformation, such as arrest of the cell division cycle, induction of cell differentiation, and apoptosis. Recent work has demonstrated that RAS elicits proliferative arrest and senescence in normal mouse and human fibroblasts. Because the Raf/MEK/MAP kinase signaling cascade is a key effector of signaling from Ras proteins, we examined the ability of conditionally active forms of Raf-1 to elicit cell cycle arrest and senescence in human cells. Activation of Raf-1 in nonimmortalized human lung fibroblasts (IMR-90) led to the prompt and irreversible arrest of cellular proliferation and the premature onset of senescence. Concomitant with the onset of cell cycle arrest, we observed the induction of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p16Ink4a. Ablation of p53 and p21Cip1 expression by use of the E6 oncoprotein of HPV16 demonstrated that expression of these proteins was not required for Raf-induced cell cycle arrest or senescence. Furthermore, cell cycle arrest and senescence were elicited in IMR-90 cells by the ectopic expression of p16Ink4a alone. Pharmacological inhibition of the Raf/MEK/MAP kinase cascade prevented Raf from inducing p16Ink4a and also prevented Raf-induced senescence. We conclude that the kinase cascade initiated by Raf can regulate the expression of p16Ink4a and the proliferative arrest and senescence that follows. Induction of senescence may provide a defense against neoplastic transformation when the MAP kinase signaling cascade is inappropriately active.
TL;DR: In vitro and in vivo observations shed light on the cell–cell interactions that occur during vessel development, as well as in pathologies in which developmental processes are recapitulated.
Abstract: We aimed to determine if and how endothelial cells (EC) recruit precursors of smooth muscle cells and pericytes and induce their differentiation during vessel formation. Multipotent embryonic 10T1/2 cells were used as presumptive mural cell precursors. In an under-agarose coculture, EC induced migration of 10T1/2 cells via platelet-derived growth factor BB. 10T1/2 cells in coculture with EC changed from polygonal to spindle-shaped, reminiscent of smooth muscle cells in culture. Immunohistochemical and Western blot analyses were used to examine the expression of smooth muscle (SM)-specific markers in 10T1/2 cells cultured in the absence and presence of EC. SM-myosin, SM22α, and calponin proteins were undetectable in 10T1/2 cells cultured alone; however, expression of all three SM-specific proteins was significantly induced in 10T1/2 cells cocultured with EC. Treatment of 10T1/2 cells with TGF-β induced phenotypic changes and changes in SM markers similar to those seen in the cocultures. Neutralization of TGF-β in the cocultures blocked expression of the SM markers and the shape change. To assess the ability of 10T1/2 cells to contribute to the developing vessel wall in vivo, prelabeled 10T1/2 cells were grown in a collagen matrix and implanted subcutaneously into mice. The fluorescently marked cells became incorporated into the medial layer of developing vessels where they expressed SM markers. These in vitro and in vivo observations shed light on the cell–cell interactions that occur during vessel development, as well as in pathologies in which developmental processes are recapitulated.
TL;DR: The data suggest that L‐Sox5/Sox6 and Sox9, which belong to two different classes of Sox transcription factors, cooperate with each other in expression of Col2a1 and possibly other genes of the chondrocytic program.
Abstract: Transcripts for a new form of Sox5, called L-Sox5, and Sox6 are coexpressed with Sox9 in all chondrogenic sites of mouse embryos. A coiled-coil domain located in the N-terminal part of L-Sox5, and absent in Sox5, showed >90% identity with a similar domain in Sox6 and mediated homodimerization and heterodimerization with Sox6. Dimerization of L-Sox5/Sox6 greatly increased efficiency of binding of the two Sox proteins to DNA containing adjacent HMG sites. L-Sox5, Sox6 and Sox9 cooperatively activated expression of the chondrocyte differentiation marker Col2a1 in 10T1/2 and MC615 cells. A 48 bp chondrocyte-specific enhancer in this gene, which contains several HMG-like sites that are necessary for enhancer activity, bound the three Sox proteins and was cooperatively activated by the three Sox proteins in non-chondrogenic cells. Our data suggest that L-Sox5/Sox6 and Sox9, which belong to two different classes of Sox transcription factors, cooperate with each other in expression of Col2a1 and possibly other genes of the chondrocytic program.
TL;DR: It is demonstrated that Nurr1 functions at the later stages of dopamine cell development to drive differentiation of ventral mesencephalic late dopaminergic precursor neurons, and is essential for specifying commitment of mesENCEphalic precursors to the full dopamine phenotype.
Abstract: Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is expressed predominantly in the central nervous system, including developing and mature dopaminergic neurons. Recent studies have demonstrated that Nurr1 is essential for the induction of phenotypic markers of ventral mid-brain dopaminergic neurons whose generation is specified by the floor plate-derived morphogenic signal sonic hedgehog (SHH), but the precise role of Nurr1 in this differentiative pathway has not been established. To provide further insights into the role of Nurr1 in the final differentiation pathway, we have examined the fate of dopamine cell precursors in Nurr1 null mutant mice. Here we demonstrate that Nurr1 functions at the later stages of dopamine cell development to drive differentiation of ventral mesencephalic late dopaminergic precursor neurons. In the absence of Nurr1, neuroepithelial cells that give rise to dopaminergic neurons adopt a normal ventral localization and neuronal phenotype characterized by expression of the homeodomain transcription factor and mesencephalic marker, Ptx-3, at embryonic day 11.5. However, these late precursors fail to induce a dopaminergic phenotype, indicating that Nurr1 is essential for specifying commitment of mesencephalic precursors to the full dopaminergic phenotype. Further, as development progresses, these mid-brain dopamine precursor cells degenerate in the absence of Nurr1, resulting in loss of Ptx-3 expression and a concomitant increase in apoptosis of ventral midbrain neurons in newborn null mutant mice. Taken together, these data indicate that Nurr1 is essential for both survival and final differentiation of ventral mesencephalic late dopaminergic precursor neurons into a complete dopaminergic phenotype.
TL;DR: The role of one family of transcription factors, the CCAAT/enhancer-binding proteins (C/EBPs), in inducing preadipocyte differentiation and in modulating gene expression in the fully differentiated adipocyte is explored.
TL;DR: It is demonstrated that PPARgamma is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes and raised the possibility that PPargamma ligands may influence the progression of atherosclerosis.
Abstract: The peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARgamma is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARgamma is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARgamma expression in primary macrophages and monocytic cell lines. PPARgamma mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARgamma expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARgamma expression in macrophages. TPA induced the expression of PPARgamma in RAW 264.7 macrophages by increasing transcription from the PPARgamma1 and PPARgamma3 promoters. In concert, these observations provide insights into the regulation of PPARgamma expression in activated macrophages and raise the possibility that PPARgamma ligands may influence the progression of atherosclerosis.
TL;DR: It is shown that DC activation induced by lipopolysaccharide can be separated into two distinct processes: first, maturation, leading to upregulation of MHC and costimulatory molecules, and second, rescue from immediate apoptosis after withdrawal of growth factors (survival).
Abstract: Although dendritic cell (DC) activation is a critical event for the induction of immune responses, the signaling pathways involved in this process have not been characterized. In this report, we show that DC activation induced by lipopolysaccharide (LPS) can be separated into two distinct processes: first, maturation, leading to upregulation of MHC and costimulatory molecules, and second, rescue from immediate apoptosis after withdrawal of growth factors (survival). Using a DC culture system that allowed us to propagate immature growth factor–dependent DCs, we have investigated the signaling pathways activated by LPS. We found that LPS induced nuclear translocation of the nuclear factor (NF)-κB transcription factor. Inhibition of NF-κB activation blocked maturation of DCs in terms of upregulation of major histocompatibility complex and costimulatory molecules. In addition, we found that LPS activated the extracellular signal–regulated kinase (ERK), and that specific inhibition of MEK1, the kinase which activates ERK, abrogated the ability of LPS to prevent apoptosis but did not inhibit DC maturation or NF-κB nuclear translocation. These results indicate that ERK and NF-κB regulate different aspects of LPS-induced DC activation: ERK regulates DC survival whereas NF-κB is responsible for DC maturation.
TL;DR: It is demonstrated that T cell differentiation results in long-range changes in the chromatin structure of effector cytokine genes, which persist in resting Th1 and Th2 cells in the absence of further stimulation.
TL;DR: A model in which GATA6 lies upstream of HNF4 in a transcriptional cascade that regulates differentiation of the visceral endoderm is suggested and it is demonstrated that GATA 6 is required for establishment of the endODermally derived bronchial epithelium.
Abstract: GATA6 belongs to a family of zinc finger transcription factors that play important roles in transducing nuclear events that regulate cellular differentiation and embryonic morphogenesis in vertebrate species. To examine the function of GATA6 during embryonic development, gene targeting was used to generate GATA6-deficient (GATA6−/−) ES cells and mice harboring a null mutation in GATA6. Differentiated embryoid bodies derived from GATA6−/− ES cells lack a covering layer of visceral endoderm and severely attenuate, or fail to express, genes encoding early and late endodermal markers, including HNF4, GATA4, α-fetoprotein (AFP), and HNF3β. Homozygous GATA6−/− mice died between embryonic day (E) 6.5 and E7.5 and exhibited a specific defect in endoderm differentiation including severely down-regulated expression of GATA4 and absence of HNF4 gene expression. Moreover, widespread programmed cell death was observed within the embryonic ectoderm of GATA6-deficient embryos, a finding also observed in HNF4-deficient embryos. Consistent with these data, forced expression of GATA6 activated the HNF4 promoter in nonendodermal cells. Finally, to examine the function of GATA6 during later embryonic development, GATA6−/−-C57BL/6 chimeric mice were generated. lacZ-tagged GATA6−/− ES cells contributed to all embryonic tissues with the exception of the endodermally derived bronchial epithelium. Taken together, these data suggest a model in which GATA6 lies upstream of HNF4 in a transcriptional cascade that regulates differentiation of the visceral endoderm. In addition, these data demonstrate that GATA6 is required for establishment of the endodermally derived bronchial epithelium.
TL;DR: Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that were first identified as mediators of cellular responses to interferons as mentioned in this paper.
Abstract: Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that were first identified as mediators of cellular responses to interferons (reviewed in references 12, 16 and 35). Signaling induced by the interaction of interferons and other cytokines with their cognate receptors is initiated by a cascade of events, including receptor aggregation and activation of Janus protein tyrosine kinases (JAKs) associated with the receptors. Subsequently, STAT proteins are recruited to the receptor-JAK complexes and activated by tyrosine phosphorylation, which promotes the formation of homodimers or heterodimers of STAT family members. Activated STATs, in turn, translocate to the nucleus and bind to specific DNA response elements that regulate gene expression. There are at least seven genes in the mammalian genome known to encode different STAT family members, which are activated in various combinations in response to stimulation by numerous cytokines (12, 16, 35).
It has become evident that, in addition to cytokines, mitogenic growth factors, such as platelet-derived growth factor and epidermal growth factor, also induce STAT signaling, particularly Stat1, Stat3, and Stat5 (21, 35). An emerging concept is that normal signaling by STAT proteins is involved in control of diverse biological processes regulated by cytokines and growth factors, including cell differentiation, proliferation, development, and apoptosis (2, 4, 10, 13, 19, 20, 22, 23, 26, 29, 31, 37, 39, 40, 50). Increasingly, evidence that indicates an association between abnormal activation of STAT signaling and oncogenesis has also been accumulating. For example, we and other investigators have demonstrated constitutive activation of Stat1, Stat3, Stat5, and Stat6 in cells transformed by Src, Abl, and various other oncoproteins and tumor viruses (7, 8, 11, 14, 24, 25, 47, 51). In the context of human cancer, there is a high frequency of activation of Stat1, Stat3, and Stat5 in breast carcinoma cells (14, 32, 42) and in lymphoid malignancies, including lymphomas and leukemias (15, 43, 49). Although these findings suggest a role for STAT signaling in cell transformation as well as in human cancer, direct evidence for the obligatory requirement of STAT signaling in oncogenesis has not been reported previously.
Because transformation of mammalian fibroblasts by viral Src (v-Src) specifically induces constitutive activation of one STAT family member, Stat3, this system is ideal for investigating the role of Stat3 signaling in oncogenesis (7, 47). The embryonic lethality of targeted disruption of the Stat3 gene (39), however, precludes generation of viable Stat3-deficient mouse models for these studies. An alternative approach for disrupting Stat3 function is to make use of Stat3 dominant negative proteins that interfere with Stat3 signaling. One such variant of Stat3, known as Stat3β, has been shown to block Stat3 function in response to interleukin 5 (IL-5) stimulation (5). Stat3β is a naturally occurring splice variant with a deletion in the C-terminal portion that harbors the transcriptional activation domain and the Ser-727 residue, phosphorylation of which is required for efficient transcriptional activation (44, 45). As a result of this C-terminal deletion, dimers formed with human Stat3β lack transcriptional activity (5). In some studies, however, mouse Stat3β has been shown to activate the promoters of certain genes in a cell type-dependent manner (33, 34), suggesting that the dominant negative effect of Stat3β may be cell type specific.
To investigate Stat3-mediated gene regulation by v-Src and the role of Stat3 in oncogenesis, we disrupted Stat3 signaling by using human Stat3β. We report that in NIH 3T3 fibroblasts transiently expressing luciferase reporter constructs, v-Src induced Stat3-specific transcriptional activation. As a potent dominant negative modulator of Stat3-mediated signaling, Stat3β effectively blocked Stat3-specific gene expression induced by v-Src in these cells. Furthermore, cotransfection of expression vectors encoding Stat3β and v-Src suppressed cell transformation of NIH 3T3 fibroblasts, and stable cell lines overexpressing Stat3β were found to be resistant to transformation by v-Src. Our findings establish that activation of Stat3 by v-Src induces specific gene regulation and provide evidence for the requirement of Stat3 signaling in cell transformation by the Src oncoprotein.
Journal Article•
TL;DR: DC activation by type I IFNs represents a novel mechanism of immunomodulation by these cytokines, which could be important during antiviral responses and autoimmune reactions.
Abstract: This study identifies type I IFNs as activating cytokines in a serum-free system in which human dendritic cells (DC) were generated from CD34+ progenitor cells. After 14 days of culture in GM-CSF, TNF-alpha, and IL-4, CD34+ progenitors gave rise to a population of large, immature DC expressing CD1a and CD11b but lacking CD14, CD80, CD83, CD86, and CMRF44. During the next 2 wk, this population spontaneously matured into nonadherent, CD1a(low/-), CD11b(low/-), CD14-, CD80+, CD83+, CD86+, CMRF44+ DC with high allostimulatory activity in the MLR. To examine which factors influenced this maturation, 25 different cytokines or factors were added to the immature DC culture. Only type I IFNs (alpha or beta) accelerated this maturation in a dose-dependent manner, so that after only 3 days the majority of large cells acquired the morphology, phenotype, and function characteristics of mature DC. Furthermore, supernatants from cultures containing spontaneously maturing DC revealed low levels of endogenous IFN production. Because of the similarity of the activation of DC in our culture system with the phenotypic and functional changes observed during Langerhans cells activation and migration in vivo, we investigated the effect of IFN-alpha on human Langerhans cell migration. IFN-alpha also activated the migration of human split skin-derived DC, demonstrating that this effect was not limited to DC derived in vitro from hemopoietic progenitor cells. DC activation by type I IFNs represents a novel mechanism of immunomodulation by these cytokines, which could be important during antiviral responses and autoimmune reactions.