Showing papers on "Cellular differentiation published in 1999"

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis

Abstract: The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families.

Abstract: Osteoblasts/stromal cells are essentially involved in osteoclast differentiation and function through cell-to-cell contact (Fig. 8). Although many attempts have been made to elucidate the mechanism of the so-called "microenvironment provided by osteoblasts/stromal cells," (5-8) it has remained an open question until OPG and its binding molecule were cloned. The serial discovery of the new members of the TNF receptor-ligand family members has confirmed the idea that osteoclast differentiation and function are regulated by osteoblasts/stromal cells. RANKL, which has also been called ODF, TRANCE, or OPGL, is a member of the TNF ligand family. Expression of RANKL mRNA in osteoblasts/stromal cells is up-regulated by osteotropic factors such as 1 alpha, 25(OH)2D3, PTH, and IL-11. Osteoclast precursors express RANK, a TNF receptor family member, recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into pOCs in the presence of M-CSF. RANKL is also involved in the survival and fusion of pOCs and activation of mature osteoclasts. OPG, which has also been called OCIF or TR1, is a soluble receptor for RANKL and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.

Cardiomyocytes can be generated from marrow stromal cells in vitro

Abstract: We have isolated a cardiomyogenic cell line (CMG) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneously beating cells were repeatedly screened. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in approximately 30% of the cells; they connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. They expressed atrial natriuretic peptide and brain natriuretic peptide and were stained with anti-myosin, anti-desmin, and anti-actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials, such as sinus node-like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain, and alpha-actin, indicated that their muscle phenotype was similar to that of fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C mRNA before 5-azacytidine treatment and expressed MEF-2A and MEF-2D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation.

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand

Abstract: A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.

Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation

Abstract: The mechanisms that control cell proliferation and cell differentiation during morphogenesis of the endochondral skeleton of vertebrates are poorly understood. Indian hedgehog (Ihh) signaling from prehypertrophic chondrocytes has been implicated in the control of chondrocyte maturation by way of feedback control of a second secreted factor parathyroid hormone-related peptide (PTHrP) at the articular surfaces. Analysis of an Ihh null mutant suggests a more extensive role for Ihh in skeletal development. Mutants display markedly reduced chondrocyte proliferation, maturation of chondrocytes at inappropriate position, and a failure of osteoblast development in endochondral bones. Together, the results suggest a model in which Ihh coordinates diverse aspects of skeletal morphogenesis through PTHrP-dependent and independent processes.

Sox9 is required for cartilage formation.

Abstract: Chondrogenesis results in the formation of cartilages, initial skeletal elements that can serve as templates for endochondral bone formation Cartilage formation begins with the condensation of mesenchyme cells followed by their differentiation into chondrocytes Although much is known about the terminal differentiation products that are expressed by chondrocytes, little is known about the factors that specify the chondrocyte lineage SOX9 is a high-mobility-group (HMG) domain transcription factor that is expressed in chondrocytes and other tissues In humans, SOX9 haploinsufficiency results in campomelic dysplasia, a lethal skeletal malformation syndrome, and XY sex reversal During embryogenesis, Sox9 is expressed in all cartilage primordia and cartilages, coincident with the expression of the collagen alpha1(II) gene (Col2a1) Sox9 is also expressed in other tissues, including the central nervous and urogenital systems Sox9 binds to essential sequences in the Col2a1 and collagen alpha2(XI) gene (Col11a2) chondrocyte-specific enhancers and can activate these enhancers in non-chondrocytic cells Here, Sox9 is identified as a regulator of the chondrocyte lineage In mouse chimaeras, Sox9-/- cells are excluded from all cartilages but are present as a juxtaposed mesenchyme that does not express the chondrocyte-specific markers Col2a1, Col9a2, Col11a2 and Agc This exclusion occurred cell autonomously at the condensing mesenchyme stage of chondrogenesis Moreover, no cartilage developed in teratomas derived from Sox9-/- embryonic stem (ES) cells Our results identify Sox9 as the first transcription factor that is essential for chondrocyte differentiation and cartilage formation

Mitogen-activated protein kinases: specific messages from ubiquitous messengers

Abstract: Signal transduction networks allow cells to perceive changes in the extracellular environment and to mount an appropriate response. Mitogen-activated protein kinase (MAPK) cascades are among the most thoroughly studied of signal transduction systems and have been shown to participate in a diverse array of cellular programs, including cell differentiation, cell movement, cell division, and cell death. A key question in studies of this cascade is, how does a ubiquitously activated regulatory enzume generate a specific and biologically appropriate cellular response? In this review we describe recent findings that provide insight into ways that the regulation, structure, and localization of MAPKs and the participation of adapters and scaffolds can help determine biological outcomes. MAPK cascades are evolutionarily conserved in all eucaryotes and play a key role in the regulation of gene expression as well as cytoplasmic activities. They typically are organized in a three-kinase architecture consisting of a MAPK, a MAPK activator (MEK, MKK, or MAPK kinase), and a MEK activator (MEK kinase [MEKK] or MAPK kinase kinase). Transmission of signals is achieved by sequential phosphorylation and activation of the components specific to a respective cascade. In the yeast Saccharomyces cerevisiae, five MAPK modules have been described; they regulate mating, filamentation, high-osmolarity responses, cell wall remodeling, and sporulation (Fig. ​(Fig.1A)1A) (reviewed in references 56 and 77). In mammalian systems five distinguishable MAPK modules have been identified so far (Fig. ​(Fig.1B).1B). These include the extracellular signal-regulated kinase 1 and 2 (ERK1/2) cascade, which preferentially regulates cell growth and differentiation, as well as the c-Jun N-terminal kinase (JNK) and p38 MAPK cascades, which function mainly in stress responses like inflammation and apoptosis (reviewed in references 57, 74, and 103). Moreover, MAPK pathways control several developmental programs, such as morphogenesis and spatial patterning in Dictyostelium amoebae (17, 45), eye development in Drosophila melanogaster (124), vulva induction in Caenorhabditis elegans (113), and T-cell development in mammals (31). FIG. 1 Schematic overview of MAPK modules. (A) In S. cerevisiae, five MAPK modules regulate mating, filamentation, high-osmolarity responses, cell wall remodeling, and sporulation. (B) Mammalian MAPK modules regulate cell growth, differentiation, stress responses, ... Individual MAPK modules generally can signal independently from each other, and this specificity is manifested in distinct physiologic responses. This is most obvious when studying MAPK signaling in S. cerevisiae. Here a particular extracellular event characteristically activates a specific MAPK module and initiates a unique cellular program (reviewed in references 56 and 77). For example, stimulation of cells with pheromone leads to the activation of the pheromone response pathway (STE11, STE7, and FUS3) (Fig. ​(Fig.2),2), which ultimately results in cell cycle arrest and the induction of mating-specific genes. However, related MAPKs whose modules share some components with the pheromone response pathway are not affected by pheromone stimulation but are activated only in response to the appropriate stimulus. For example, under conditions of high osmolarity Ste11 can lead to activation of Hog1 but does not induce mating-specific genes. Conversely, conditions that activate the filamentation pathway (which utilizes STE11 and STE7) induce only genes that regulate filamentous growth without triggering pheromone responses or responses to high osmolarity. These observations suggest that yeast cells have developed efficient mechanisms to generate pathway specificity and to successfully suppress cross talk, even when individual components participate in more than one signaling pathway. FIG. 2 Scaffold and adapter molecules in MAPK pathways. MAPK scaffolds and adapters (gray shading) are thought to promote the formation of oligomeric protein complexes with components that function in a specific MAPK module. Scaffolds have been identified in ... In metazoan cells the problem is more complex because each cell is simultaneously exposed to multiple extracellular signals and must integrate these inputs to choose an appropriate response. Thus, the biological context of a signal plays a determinative role in the way that MAPK activation is interpreted. For example, although ERKs generally regulate cell growth and cell differentiation and JNKs participate in a stress response, this is not always the case and in certain cell types activation of JNKs can induce proliferation (110). This indicates that in mammalian systems physiologic responses associated with a certain MAPK module can be cell type specific. Moreover, in PC12 cells, transient stimulation of the ERK cascade leads to proliferation whereas sustained stimulation leads to differentiation, as measured by neurite outgrowth (81). Thus, activation of the ERK cascade can lead to contrasting physiological responses in the same cellular context, suggesting that signal specificity is also determined by regulatory mechanisms other than the selective activation of a MAPK module. In this short review, we outline recent advances in understanding of this signaling system that help to explain how MAPK cascades are regulated and how specificity can be generated. Because of the power of yeast genetics, understanding of MAPK signaling in S. cerevisiae is at an advanced level, and thus many examples that utilize this organism are given. However, analogous mechanisms appear to be operative in metazoans as well. We discuss in turn the role of enzyme-substrate interactions, scaffolding proteins, subcellular targeting and localization, temporal regulation, and signal integration in determining the biological outcome of MAPK activation.

Notch signalling controls pancreatic cell differentiation

Abstract: The pancreas contains both exocrine and endocrine cells, but the molecular mechanisms controlling the differentiation of these cell types are largely unknown. Despite their endodermal origin, pancreatic endocrine cells share several molecular characteristics with neurons, and, like neurons in the central nervous system, differentiating endocrine cells in the pancreas appear in a scattered fashion within a field of progenitor cells. This indicates that they may be generated by lateral specification through Notch signalling. Here, to test this idea, we analysed pancreas development in mice genetically altered at several steps in the Notch signalling pathway. Mice deficient for Delta-like gene 1 (Dll1) or the intracellular mediator RBP-JK showed accelerated differentiation of pancreatic endocrine cells. A similar phenotype was observed in mice over-expressing neurogenin 3(ngn 3) or the intracellular form of Notch3 (ref. 13) (a repressor of Notch signalling). These data provide evidence that ngn3 acts as pro-endocrine gene and that Notch signalling is critical for the decision between theendocrine and progenitor/exocrine fates in the developing pancreas.

Commitment to the B-lymphoid lineage depends on the transcription factor Pax5.

Abstract: The Pax5 gene encoding the B-cell-specific activator protein (BSAP) is expressed within the haematopoietic system exclusively in the B-lymphoid lineage, where it is required in vivo for progression beyond the pro-B-cell stage. However, Pax5 is not essential for in vitro propagation of pro-B cells in the presence of interleukin-7 and stromal cells. Here we show that pro-B cells lacking Pax5 are also incapable of in vitro B-cell differentiation unless Pax5 expression is restored by retroviral transduction. Pax5-/- pro-B cells are not restricted in their lineage fate, as stimulation with appropriate cytokines induces them to differentiate into functional macrophages, osteoclasts, dendritic cells, granulocytes and natural killer cells. As expected for a clonogenic haematopoietic progenitor with lymphomyeloid developmental potential, the Pax5-/- pro-B cell expresses genes of different lineage-affiliated programmes, and restoration of Pax5 activity represses this lineage-promiscuous transcription. Pax5 therefore plays an essential role in B-lineage commitment by suppressing alternative lineage choices.

Embryonic Stem Cell-Derived Glial Precursors: A Source of Myelinating Transplants

Abstract: Self-renewing, totipotent embryonic stem (ES) cells may provide a virtually unlimited donor source for transplantation. A protocol that permits the in vitro generation of precursors for oligodendrocytes and astrocytes from ES cells was devised. Transplantation in a rat model of a human myelin disease shows that these ES cell-derived precursors interact with host neurons and efficiently myelinate axons in brain and spinal cord. Thus, ES cells can serve as a valuable source of cell type-specific somatic precursors for neural transplantation.

Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation

Abstract: LEF/TCF DNA-binding proteins act in concert with activated beta -catenin, the product of Wnt signaling, to transactivate downstream target genes. To probe the role of activated LEF/TCF transcription factor complexes in hair follicle morphogenesis and differentiation, we engineered mice harboring TOPGAL, a beta -galactosidase gene under the control of a LEF/TCF and beta -catenin inducible promoter. In mice, TOPGAL expression was directly stimulated by a stabilized form of beta -catenin, but was also dependent upon LEF1/TCF3 in skin. During embryogenesis, TOPGAL activation occurred transiently in a subset of LEF1-positive cells of pluripotent ectoderm and underlying mesenchyme. Downgrowth of initiated follicles proceeded in the absence of detectable TOPGAL expression, even though LEF1 was still expressed. While proliferative matrix cells expressed the highest levels of Lef1 mRNAs, LEF1 concentrated in the precursor cells to the hair shaft, where TOPGAL expression was co-induced with hair-specific keratin genes containing LEF/TCF-binding motifs. LEF1 and TOPGAL expression ceased during catagen and telogen, but reappeared at the start of the postnatal hair cycle, concomitant with precortex formation. In contrast to hair shaft precursor cells, postnatal outer root sheath expressed TCF3, but not TOPGAL. TCF3 was also expressed in the putative follicle stem cells, and while TOPGAL was generally silent in this compartment, it was stimulated at the start of the hair cycle in a fashion that appeared to be dependent upon stabilization of beta -catenin. Taken together, our findings demonstrate that LEF1/TCF3 is necessary but not sufficient for TOPGAL activation, revealing the existence of positive and negative regulators of these factors in the skin. Furthermore, our findings unveil the importance of activated LEF/TCF complexes at distinct times in hair development and cycling when changes in cell fate and differentiation commitments take place.

STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells

Abstract: Embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). LIF acts through a receptor complex composed of a low affinity LIF receptor (LIFRbeta) and gp130. We reported that the intracellular domain of gp130 plays an important role in self-renewal of ES cells. In the present study, we examined the signaling pathway through which gp130 contributes to the self-renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 revealed that the tyrosine residue of gp130 responsible for STAT3 activation is necessary for self-renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we constructed a fusion protein composed of the entire coding region of STAT3 and the ligand binding domain of the estrogen receptor. This construction (STAT3ER) induced expression of junB (one of the targets of STAT3) in ES cells in the presence of the synthetic ligand 4-hydroxytamoxifen (4HT), thereby indicating that STAT3ER is a conditionally active form. ES cells transfected with STAT3ER cultured in the presence of 4HT maintained an undifferentiated state. Taken together, these results strongly suggest that STAT3 activation is required and sufficient to maintain the undifferentiated state of ES cells.

Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts

Abstract: Id proteins may control cell differentiation by interfering with DNA binding of transcription factors. Here we show that targeted disruption of the dominant negative helix–loop–helix proteins Id1 and Id3 in mice results in premature withdrawal of neuroblasts from the cell cycle and expression of neural-specific differentiation markers. The Id1–Id3 double knockout mice also display vascular malformations in the forebrain and an absence of branching and sprouting of blood vessels into the neuroectoderm. As angiogenesis both in the brain and in tumours requires invasion of avascular tissue by endothelial cells, we examined the Id knockout mice for their ability to support the growth of tumour xenografts. Three different tumours failed to grow and/or metastasize in Id1+/-Id3-/- mice, and any tumour growth present showed poor vascularization and extensive necrosis. Thus, the Id genes are required to maintain the timing of neuronal differentiation in the embryo and invasiveness of the vasculature. Because the Id genes are expressed at very low levels in adults, they make attractive new targets for anti-angiogenic drug design.

Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate.

Abstract: Marrow stromal cells (MSCs) were isolated from bone marrow obtained by aspirates of the iliac crest of normal volunteers. The cells were isolated by their adherence to plastic and then passed in culture. Some of the samples expanded through over 15 cell doublings from the time frozen stocks were prepared. Others ceased replicating after about four cell doublings. The replicative potential of the cells in culture was best predicted by a simple colony-forming assay in which samples from early passages were plated at low densities of about 10 cells per cm2. Samples with high colony-forming efficiency exhibited the greatest replicative potential. The colonies obtained by plating early passage cells at low density varied in size and morphology. The large colonies readily differentiated into osteoblasts and adipocytes when incubated in the appropriate medium. As samples were expanded in culture and approached senescence, they retained their ability to differentiate into osteoblasts. However, the cells failed to differentiate into adipocytes. The loss of multipotentiality following serial passage in culture may have important implications for the use of expanded MSCs for cell and gene therapy.

Direct activation of TERT transcription by c-MYC.

Abstract: The MYC proto-oncogene encodes a ubiquitous transcription factor (c-MYC) involved in the control of cell proliferation and differentiation. Deregulated expression of c-MYC caused by gene amplification, retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of c-MYC and its role in tumorigenesis are poorly understood because few c-MYC targets have been identified. Here we show that c-MYC has a direct role in induction of the activity of telomerase, the ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves chromosome integrity by maintaining telomere length. c-MYC activates telomerase by inducing expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c-MYC are expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally differentiated cells, and can both induce immortalization when constitutively expressed in transfected cells. Consistent with the recently reported association between MYC overexpression and induction of telomerase activity, we find here that the TERT promoter contains numerous c-MYC-binding sites that mediate TERT transcriptional activation. c-MYC-induced TERT expression is rapid and independent of cell proliferation and additional protein synthesis, consistent with direct transcriptional activation of TERT. Our results indicate that TERT is a target of c-MYC activity and identify a pathway linking cell proliferation and chromosome integrity in normal and neoplastic cells.

Development of peripheral lymphoid organs and natural killer cells depends on the helix–loop–helix inhibitor Id2

Abstract: Transcription factors with a basic helix-loop-helix (HLH) motif have been shown to be crucial for various cell differentiation processes during development of multicellular organisms. Id proteins inhibit the functions of these transcription factors in a dominant-negative manner by suppressing their heterodimerization partners through the HLH domains. Members of the Id family also promote cell proliferation, implying a role in the control of cell differentiation. Here we show that Id2 is indispensable for normal development of mice. Id2-/- mice lack lymph nodes and Peyer's patches. However, their splenic architecture is normal, exhibiting T-cell and B-cell compartments and distinct germinal centres. The cell population that produces lymphotoxins, essential factors for the development of secondary lymphoid organs, is barely detectable in the Id2-/- intestine. Furthermore, the null mutants show a greatly reduced population of natural killer (NK) cells, which is due to an intrinsic defect in NK-cell precursors. Our results indicate that Id2 has an essential role in the generation of peripheral lymphoid organs and NK cells.

Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation.

Abstract: While the transmembrane protein Notch plays an important role in various aspects of development, and diseases including tumors and neurological disorders, the intracellular pathway of mammalian Notch remains very elusive. To understand the intracellular pathway of mammalian Notch, the role of the bHLH genes Hes1 and Hes5 (mammalian hairy and Enhancer-of-split homologues) was examined by retrovirally misexpressing the constitutively active form of Notch (caNotch) in neural precursor cells prepared from wild-type, Hes1-null, Hes5-null and Hes1-Hes5 double-null mouse embryos. We found that caNotch, which induced the endogenous Hes1 and Hes5 expression, inhibited neuronal differentiation in the wild-type, Hes1-null and Hes5-null background, but not in the Hes1-Hes5 double-null background. These results demonstrate that Hes1 and Hes5 are essential Notch effectors in regulation of mammalian neuronal differentiation.

Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300.

Abstract: The cytokines LIF (leukemia inhibitory factor) and BMP2 (bone morphogenetic protein-2) signal through different receptors and transcription factors, namely STATs (signal transducers and activators of transcription) and Smads. LIF and BMP2 were found to act in synergy on primary fetal neural progenitor cells to induce astrocytes. The transcriptional coactivator p300 interacts physically with STAT3 at its amino terminus in a cytokine stimulation-independent manner, and with Smad1 at its carboxyl terminus in a cytokine stimulation-dependent manner. The formation of a complex between STAT3 and Smad1, bridged by p300, is involved in the cooperative signaling of LIF and BMP2 and the subsequent induction of astrocytes from neural progenitors.

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development

Abstract: The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (ΔCbfa1) in differentiated osteoblasts only postnatally. ΔCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. ΔCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that ΔCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally.

Leptin acts on human marrow stromal cells to enhance differentiation to osteoblasts and to inhibit differentiation to adipocytes.

Abstract: Both bone mass and serum leptin levels are increased in obesity. Because osteoblasts and adipocytes arise from a common precursor in bone marrow, we assessed the effects of human recombinant leptin on a conditionally immortalized human marrow stromal cell line, hMS2-12, with the potential to differentiate to either the osteoblast or adipocyte phenotypes. By RT-PCR and Western immunoblot analysis, the hMS2-12 cells expressed messenger RNA (mRNA) and protein for the leptin receptor. Leptin did not affect hMS2-12 cell proliferation, but resulted in dose- and time-dependent increases in mRNA and protein levels of alkaline phosphatase, type I collagen, and osteocalcin, and in a 59% increase in mineralized matrix. Leptin increased mRNA levels of lipoprotein lipase at 3 days, but decreased mRNA levels of adipsin and leptin at 9 days and decreased lipid droplet formation by 50%. Leptin did not affect the expression of Cbfa1 or peroxisome proliferator-activated receptor-gamma2, transcription factors involved in commitment to the osteoblast and adipocyte pathways, respectively. Thus, leptin acts on human marrow stromal cells to enhance osteoblast differentiation and to inhibit adipocyte differentiation. Our data support the hypothesis that leptin is a previously unrecognized, physiological regulator of these two differentiation pathways, acting primarily on maturation of stromal cells into both lineages.

The Ligand for Osteoprotegerin (OPGL) Directly Activates Mature Osteoclasts

Abstract: Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309–319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165–176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects.

Location and Clonal Analysis of Stem Cells and Their Differentiated Progeny in the Human Ocular Surface

Abstract: We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45–50 cell doublings and at ∼15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic “cell doubling clock.” These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium.

Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

Abstract: Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound alpha5 and beta1 integrin subunits but not alphav or beta3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of alpha5beta1 integrin bound to Fn, and differentiation was inhibited by anti-alpha5, but not anti-alphav, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications.

Defective Thymocyte Maturation in p44 MAP Kinase (Erk 1) Knockout Mice

Abstract: The p42 and p44 mitogen-activated protein kinases (MAPKs), also called Erk2 and Erk1, respectively, have been implicated in proliferation as well as in differentiation programs. The specific role of the p44 MAPK isoform in the whole animal was evaluated by generation of p44 MAPK-deficient mice by homologous recombination in embryonic stem cells. The p44 MAPK–/– mice were viable, fertile, and of normal size. Thus, p44 MAPK is apparently dispensable and p42 MAPK (Erk2) may compensate for its loss. However, in p44 MAPK−/− mice, thymocyte maturation beyond the CD4+CD8+ stage was reduced by half, with a similar diminution in the thymocyte subpopulation expressing high levels of T cell receptor (CD3high). In p44 MAPK−/− thymocytes, proliferation in response to activation with a monoclonal antibody to the T cell receptor in the presence of phorbol myristate acetate was severely reduced even though activation of p42 MAPK was more sustained in these cells. The p44 MAPK apparently has a specific role in thymocyte development.

Commitment and Differentiation of Osteoclast Precursor Cells by the Sequential Expression of C-Fms and Receptor Activator of Nuclear Factor κb (Rank) Receptors

Abstract: Osteoclasts are terminally differentiated cells derived from hematopoietic stem cells. However, how their precursor cells diverge from macrophagic lineages is not known. We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor κB (RANK), and have demonstrated that RANK expression in early-stage of precursor cells (c-Fms+RANK−) was stimulated by macrophage colony-stimulating factor (M-CSF). Although M-CSF and RANKL (ligand) induced commitment of late-stage precursor cells (c-Fms+RANK+) into osteoclasts, even late-stage precursors have the potential to differentiate into macrophages without RANKL. Pretreatment of precursors with M-CSF and delayed addition of RANKL showed that timing of RANK expression and subsequent binding of RANKL are critical for osteoclastogenesis. Thus, the RANK–RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation.

Impaired B Cell Development and Proliferation in Absence of Phosphoinositide 3-Kinase p85α

Abstract: Phosphoinositide 3-kinase (PI3K) activation has been implicated in many cellular responses, including fibroblast growth, transformation, survival, and chemotaxis. Although PI3K is activated by several agents that stimulate T and B cells, the role of PI3K in lymphocyte function is not clear. The mouse gene encoding the PI3K adapter subunit p85alpha and its splice variants p55alpha and p50alpha was disrupted. Most p85alpha-p55alpha-p50alpha-/- mice die within days after birth. Lymphocyte development and function was studied with the use of the RAG2-deficient blastocyst complementation system. Chimeric mice had reduced numbers of peripheral mature B cells and decreased serum immunoglobulin. The B cells that developed had diminished proliferative responses to antibody to immunoglobulin M, antibody to CD40, and lipopolysaccharide stimulation and decreased survival after incubation with interleukin-4. In contrast, T cell development and proliferation was normal. This phenotype is similar to defects observed in mice lacking the tyrosine kinase Btk.

Regulation of gene expression by dietary fat

Abstract: Dietary fat is an important macronutrient for the growth and development of all organisms. In addition to its role as an energy source and its effects on membrane lipid composition, dietary fat has profound effects on gene expression, leading to changes in metabolism, growth, and cell differentiation. The effects of dietary fat on gene expression reflect an adaptive response to changes in the quantity and type of fat ingested. Specific fatty acid-regulated transcription factors have been identified in bacteria, amphibians, and mammals. In mammals, these factors include peroxisome proliferator-activated receptors (PPAR alpha, -beta, and -gamma), HNF4 alpha, NF kappa B, and SREBP1c. These factors are regulated by (a) direct binding of fatty acids, fatty acyl-coenzyme A, or oxidized fatty acids; (b) oxidized fatty acid (eicosanoid) regulation of G-protein-linked cell surface receptors and activation of signaling cascades targeting the nucleus; or (c) oxidized fatty acid regulation of intracellular calcium levels, which affect cell signaling cascades targeting the nucleus. At the cellular level, the physiological response to fatty acids will depend on (a) the quantity, chemistry, and duration of the fat ingested; (b) cell-specific fatty acid metabolism (oxidative pathways, kinetics, and competing reactions); (c) cellular abundance of specific nuclear and membrane receptors; and (d) involvement of specific transcription factors in gene expression. These mechanisms are involved in the control of carbohydrate and lipid metabolism, cell differentiation and growth, and cytokine, adhesion molecule, and eicosanoid production. The effects of fatty acids on the genome provide new insight into how dietary fat might play a role in health and disease.