HD3 Cells Research Articles

A novel cis-regulatory element regulates αD and αA-globin gene expression in chicken erythroid cells.

Cis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis. Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An α-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the α-globin domain in erythroid chicken cells. Erythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An α-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over αD and αA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that α-globin CRE-2 is required for the induction of αD and αA. Analysis of TF binding motifs at α-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding. Our findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells.

Hi-C analysis of genomic contacts revealed karyotype abnormalities in chicken HD3 cell line

BackgroundKaryotype abnormalities are frequent in immortalized continuous cell lines either transformed or derived from primary tumors. Chromosomal rearrangements can cause dramatic changes in gene expression and affect cellular phenotype and behavior during in vitro culture. Structural variations of chromosomes in many continuous mammalian cell lines are well documented, but chromosome aberrations in cell lines from other vertebrate models often remain understudied. The chicken LSCC-HD3 cell line (HD3), generated from erythroid precursors, was used as an avian model for erythroid differentiation and lineage-specific gene expression. However, karyotype abnormalities in the HD3 cell line were not assessed. In the present study, we applied high-throughput chromosome conformation capture to analyze 3D genome organization and to detect chromosome rearrangements in the HD3 cell line.ResultsWe obtained Hi-C maps of genomic interactions for the HD3 cell line and compared A/B compartments and topologically associating domains between HD3 and several other cell types. By analysis of contact patterns in the Hi-C maps of HD3 cells, we identified more than 25 interchromosomal translocations of regions ≥ 200 kb on both micro- and macrochromosomes. We classified most of the observed translocations as unbalanced, leading to the formation of heteromorphic chromosomes. In many cases of microchromosome rearrangements, an entire microchromosome together with other macro- and microchromosomes participated in the emergence of a derivative chromosome, resembling “chromosomal fusions'' between acrocentric microchromosomes. Intrachromosomal inversions, deletions and duplications were also detected in HD3 cells. Several of the identified simple and complex chromosomal rearrangements, such as between GGA2 and GGA1qter; GGA5, GGA4p and GGA7p; GGA4q, GGA6 and GGA19; and duplication of the sex chromosome GGAW, were confirmed by FISH.ConclusionsIn the erythroid progenitor HD3 cell line, in contrast to mature and immature erythrocytes, the genome is organized into distinct topologically associating domains. The HD3 cell line has a severely rearranged karyotype with most of the chromosomes engaged in translocations and can be used in studies of genome structure–function relationships. Hi-C proved to be a reliable tool for simultaneous assessment of the spatial genome organization and chromosomal aberrations in karyotypes of birds with a large number of microchromosomes.

Proteomic response of bacteria during the interaction with a host cell in a model of Mycoplasma gallisepticum

Parasitism, which is a way of the coexistence of bacteria with a host, is a very interesting phenomenon. Although the pathogenicity of many microorganisms has been studied in detail, many aspects of this phenomenon remain poorly understood for most bacteria. Among interesting objects for studying pathogen–host interactions are the members of the Mycoplasma genus. Despite a very broad geographical range of habitat of these bacteria, their pathogenicity has been poorly studied. Almost all living beings are the hosts of mycoplasmas, and the reduced genome of these microorganisms makes them a convenient object for model studies. Here, we have described for the first time an experimental label-free method for the analysis of the protein profile of a bacterium immediately during its interaction with a host cell, which made it possible to understand what changes the pathogen undergoes at this stage of infection. The method involves the sampling of eukaryotic cells at different times of cultivation with the pathogen, the sedimentation of infected cells, the preparation of a cell lysate, the hydrolytic cleavage of cell proteins, and their identification by mass spectrometry (HPLC–MS). Changes in the protein abundance were analyzed by the method of multiple reaction monitoring (MRM). In the present work, a model of the interaction of the bacterium Mycoplasma gallisepticum with chicken erythroblasts (HD3 cells) at the stage of acute infection was studied. A panel of 100 proteins was taken for the analysis, which were chosen based on their functions and literature data. To obtain the greatest number of identified proteins, several methods of their hydrolytic cleavage with subsequent preparation of the peptide extract were used: digestion in solution using the RapiGest SF surfactant, digestion using sodium deoxycholate and urea, and the digestion of proteins in gel. As a result, proteins were identified that, probably, are involved in the interaction of M. gallisepticum with the host cell at early infection stages; most of them were the proteins of adhesion, glycolysis, and ribosomal proteins.

NF-κB Signaling Is Increased in HD3 Cells Following Exposure to 1,4-Benzoquinone: Role of Reactive Oxygen Species and p38-MAPK a

In utero exposure to benzene, a known environmental contaminant, is associated with increased risk of leukemia. We have previously shown that in utero benzene exposure can alter the redox-sensitive transcription factor NF-κB, and we hypothesize that this is through benzene-induced reactive oxygen species (ROS) production interfering with the signaling pathway involving NF-κB and p38-Mitogen Activated Protein Kinase (MAPK). As benzoquinone (BQ) is one of benzene's most toxic metabolites, the objectives of this study were to determine whether ROS and p38-MAPK-mediated BQ-induced increased NF-κB activity. HD3 chicken erythroblast cells were transfected with an NF-κB luciferase-linked reporter plasmid and exposed to BQ (25 μM) for 2-24 h. NF-κB activities were determined through luciferase assays; Western blotting was conducted to assess changes in protein levels in nontransfected cells; and the presence of ROS was determined via 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) flow cytometric assays. Results demonstrated that NF-κB activity was significantly increased following exposure to BQ for 16 and 24 h and DCFDA assays and pretreatment with antioxidants indicated that BQ-mediated ROS production was responsible for this increase. Furthermore, decreased inhibitor of kappaB-alpha (IκB-α) expression suggests that nuclear factor-kappaB (NF-κB) translocates into the nucleus and that p38-MAPK activation through a ROS-dependent pathway mediates BQ-mediated increases in NF-κB activity. Future studies investigating the role of p38-MAPK in this pathway are warranted. Evaluating the effects of toxicant exposure on cell signaling pathways is vital for understanding mechanisms of xenobiotic-induced toxicity.

CTCF demarcates chicken embryonic α-globin gene autonomous silencing and contributes to adult stage-specific gene expression

Genomic loci composed of more than one gene are frequently subjected to differential gene expression, with the chicken α-globin domain being a clear example. In the present study we aim to understand the globin switching mechanisms responsible for the epigenetic silencing of the embryonic π gene and the transcriptional activation of the adult αD and αA genes at the genomic domain level. In early stages, we describe a physical contact between the embryonic π gene and the distal 3′ enhancer that is lost later during development. We show that such a level of regulation is achieved through the establishment of a DNA hypermethylation sub-domain that includes the embryonic gene and the adjacent genomic sequences. The multifunctional CCCTCC-binding factor (CTCF), which is located upstream of the αD gene promoter, delimits this sub-domain and creates a transition between the inactive sub-domain and the active sub-domain, which includes the adult αD gene. In avian-transformed erythroblast HD3 cells that are induced to differentiate, we found active DNA demethylation of the adult αD promoter, coincident with the incorporation of 5-hydroxymethylcytosine (5hmC) and concomitant with adult gene transcriptional activation. These results suggest that autonomous silencing of the embryonic π gene is needed to facilitate an optimal topological conformation of the domain. This model proposes that CTCF is contributing to a specific chromatin configuration that is necessary for differential α-globin gene expression during development.

Sialylation of the Fas Death Receptor by ST6Gal-I Provides Protection against Fas-mediated Apoptosis in Colon Carcinoma Cells

The glycosyltransferase, ST6Gal-I, adds sialic acid in an α2-6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that α2-6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that α2-6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that α2-6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, α2-6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis.

In utero and in vitro effects of benzene and its metabolites on erythroid differentiation and the role of reactive oxygen species

Benzene is a ubiquitous occupational and environmental toxicant. Exposures to benzene both prenatally and during adulthood are associated with the development of disorders such as aplastic anemia and leukemia. Mechanisms of benzene toxicity are unknown; however, generation of reactive oxygen species (ROS) by benzene metabolites may play a role. Little is known regarding the effects of benzene metabolites on erythropoiesis. Therefore, to determine the effects of in utero exposure to benzene on the growth and differentiation of fetal erythroid progenitor cells (CFU-E), pregnant CD-1 mice were exposed to benzene and CFU-E numbers were assessed in fetal liver (hematopoietic) tissue. In addition, to determine the effect of benzene metabolite-induced ROS generation on erythropoiesis, HD3 chicken erythroblast cells were exposed to benzene, phenol, or hydroquinone followed by stimulation of erythrocyte differentiation. Our results show that in utero exposure to benzene caused significant alterations in female offspring CFU-E numbers. In addition, exposure to hydroquinone, but not benzene or phenol, significantly reduced the percentage of differentiated HD3 cells, which was associated with an increase in ROS. Pretreatment of HD3 cells with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) prevented hydroquinone-induced inhibition of erythropoiesis, supporting the hypothesis that ROS generation is involved in the development of benzene erythrotoxicity. In conclusion, this study provided evidence that ROS generated as a result of benzene metabolism may significantly alter erythroid differentiation, potentially leading to the development of Blood Disorders.

The D3 dopamine receptor inhibits dopamine release in PC‐12/hD3 cells by autoreceptor signaling via PP‐2B, CK1, and Cdk‐5

The function of the D(3) dopamine (DA) receptor remains ambiguous largely because of the lack of selective D(3) receptor ligands. To investigate the function and intracellular signaling of D(3) receptors, we established a PC-12/hD3 clone, which expresses the human D(3) DA receptor in a DA producing cell line. In this model, we find that the D(3) receptor functions as an autoreceptor controlling neurotransmitter secretion. Pre-treatment with 3,6a,11, 14-tetrahydro-9-methoxy-2 methyl-(12H)-isoquino[1,2-b] pyrrolo[3,2-f][1,3] benzoxanzine-1-carboxylic acid, a D(3) receptor preferring agonist, dose-dependently suppressed K+-evoked [3H]DA release in PC-12/hD3 cells but not in the control cell line. This effect was prevented by D(3) receptor preferring antagonists GR103691 and SB277011-A. Furthermore, activation of D(3) receptors significantly inhibits forskolin-induced cAMP accumulation and leads to transient increases in phosphorylation of cyclin-dependent kinase 5 (Cdk5), dopamine and cAMP-regulated phosphoprotein of M(r) 32 000 and Akt. Because we observed differences in Cdk5 phosphorylation as well as Akt phosphorylation after DA stimulation, we probed the ability of Cdk5 and phosphatidylinositol-3 kinase (PI3K) to influence DA release. Cdk5 inhibitors, roscovitine, or olomoucine, but not the PI3K inhibitor wortmannin, blocked the D(3) receptor inhibition of DA release. In a complimentary experiment, over-expression of Cdk5 potentiated D(3) receptor suppression of DA release. Pertussis toxin, 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one and cyclosporine A also attenuated D(3) receptor-mediated inhibition of DA release indicating that this phenomenon acts through Gi/oalpha and casein kinase 1, and phosphatase protein phosphatase 2B (calcineurin), respectively. In support of previous data that D(3) DA receptors reduce transmitter release from nerve terminals, the current results demonstrate that D(3) DA receptors function as autoreceptors to inhibit DA release and that a signaling pathway involving Cdk5 is essential to this regulation.

In embryonic chicken erythrocytes actively transcribed alpha globin genes are not associated with the nuclear matrix

The spatial organization of a 250 Kb region of chicken chromosome 14, which includes the alpha globin gene cluster, was studied using in situ hybridization of a corresponding BAC probe with nuclear halos. It was found that in non-erythroid cells (DT40) and cultured erythroid cells of definite lineage (HD3) the genomic region under study was partially (DT40 cells) or fully (HD3 cells) associated with the nuclear matrix. In contrast, in embryonic red blood cells (10-day RBC) the same area was located in the crown of DNA loops surrounding the nuclear matrix, although both globin genes and surrounding house-keeping genes were actively transcribed in these cells. This spatial organization was associated with the virtual absence of RNA polymerase II in nuclear matrices prepared from 10-day RBC. In contrast, in HD3 cells a significant portion of RNA polymerase II was present in nuclear matrices. Taken together, these observations suggest that in embryonic erythroid cells transcription does not occur in association with the nuclear matrix.

Transcription- and Apoptosis-Dependent Long-Range Distribution of Tight DNA–Protein Complexes in the Chicken α-Globin Gene

The proteins tightly bound to DNA (TBP) are a group of proteins that remain attached to DNA with covalent or noncovalent bonds after its deproteinization, and have been hypothesized to be involved in regulation of gene expression. To investigate this question further, oligonucleotide DNA arrays were used to determine the distribution of tightly bound proteins along a 100-kb DNA fragment surrounding the chicken alpha-globin gene domain in DNA from chicken erythrocytes, liver, and AEV-transformed HD3 (erythroblast) cells in different physiological conditions. DNA was fractionated into TBP-free (F) and TBP-enriched (R) fractions by separation on nitrocellulose, and these fractions were used as probes for hybridization with the microarray. In erythrocytes, the site 60 kb from the 5' end of the sequence and containing a LINE family CR1 repeat was TBP enriched, but in HD3 cells this sequence was devoid of TBPs. Thus cessation of transcription of the domain is followed by an F-R transition of this site. In apoptotic HD3 cells, TBPs remained attached to DNA only at a site situated 16 kb from the 5' end of the sequence. These data confirm and extend previous conclusions about the specificity of the DNA sequences that preferably form tight complexes with proteins and about the differentiation-specific distribution of the TBPs in different cell lineages. Binding of TBPs appears to be independent of primary DNA sequence.

Benzene’s metabolites alter c-MYB activity via reactive oxygen species in HD3 cells

Benzene is a known leukemogen that is metabolized to form reactive intermediates and reactive oxygen species (ROS). The c-Myb oncoprotein is a transcription factor that has a critical role in hematopoiesis. c-Myb transcript and protein have been overexpressed in a number of leukemias and cancers. Given c-Myb’s role in hematopoiesis and leukemias, it is hypothesized that benzene interferes with the c-Myb signaling pathway and that this involves ROS. To investigate our hypothesis, we evaluated whether benzene, 1,4-benzoquinone, hydroquinone, phenol, and catechol generated ROS in chicken erythroblast HD3 cells, as measured by 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) and dihydrorhodamine-123 (DHR-123), and whether the addition of 100 U/ml of the antioxidating enzyme superoxide dismutase (SOD) could prevent ROS generation. Reduced to oxidized glutathione ratios (GSH:GSSG) were also assessed as well as hydroquinone and benzoquinone’s effects on c-Myb protein levels and activation of a transiently transfected reporter construct. Finally we attempted to abrogate benzene metabolite mediated increases in c-Myb activity with the use of SOD. We found that benzoquinone, hydroquinone, and catechol increased DCFDA fluorescence, increased DHR-123 fluorescence, decreased GSH:GSSG ratios, and increased reporter construct expression after 24 h of exposure. SOD was able to prevent DCFDA fluorescence and c-Myb activity caused by benzoquinone and hydroquinone only. These results are consistent with other studies, which suggest metabolite differences in benzene-mediated toxicity. More importantly, this study supports the hypothesis that benzene may mediate its toxicity through ROS-mediated alterations in the c-Myb signaling pathway.

CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Serial" chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the beta-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

The role of c-MYB in benzene-initiated toxicity

Chronic exposure to benzene has been correlated with increased oxidative stress and leukemia. Oncogene activation, including c-Myb activation, is one of the earliest steps leading to the formation of leukemic cells, however the molecular mechanisms involved in these events are poorly understood. Given that oxidative stress can alter the activity and fate of cell signaling pathways we hypothesize that the bioactivation of benzene leads to the formation of reactive oxygen species (ROS), which if not detoxified can alter the c-Myb signaling pathway. Using chicken erythroblast HD3 cells we have shown that exposure to the benzene metabolites catechol, benzoquinone, and hydroquinone leads to increased c-Myb activity, increased phosphorylation of c-Myb and increased production of ROS supporting our hypothesis. Activation of the aryl hydrocarbon receptor (AhR) by environmental contaminants has also been associated with carcinogenesis and mice lacking this receptor are resistant to benzene-initiated hematotoxicity. Using wild type and AhR deficient cells we are investigating the role of this receptor in benzene-initiated alterations in the c-Myb signaling pathway. We have found that both wild type and AhR deficient cells are sensitive to catechol and hydroquinone-initiated increases in c-Myb activity while both cell types are resistant to benzene-initiated alterations leaving the role of the AhR still undetermined. Interestingly, protein expression of c-Myb is increased after catechol exposure in AhR deficient cells while decreased in wild-type cells. Further studies on the role of the AhR in benzene-initiated alterations on the c-Myb signaling pathway are on going.

The effects of benzene and the metabolites phenol and catechol on c-Myb and Pim-1 signaling in HD3 cells

Exposure to the environmental toxicant benzene has been proposed to lead to leukemogenesis. The transcription factor c-Myb plays a role in blood cell differentiation and can be regulated by the serine–threonine kinase Pim-1. Overexpressed versions of c-Myb and Pim-1 are believed to play a key role in the development of a wide variety of leukemias and tumors. In our study, we evaluated the effects of benzene and the metabolites catechol and phenol on c-Myb signaling to investigate our hypothesis that benzene exerts its toxicity by interfering with this pathway. To evaluate this hypothesis, HD3 chicken erythroblast cells were transiently transfected with a c-Myb responsive luciferase reporter plasmid and then exposed to benzene, catechol, or phenol (0–300 μM) for 1–24 h before nonproprietary dual luciferase activities were measured. Our results demonstrated that catechol exposure caused a time- and concentration-dependent increase in c-Myb activity with significance occurring at 100 and 300 μM after 24 h of exposure, which was independent of increased Pim-1 protein, but dependent on increased c-Myb phosphorylation. Benzene and phenol exposure resulted in small but significant decreases in c-Myb activity that were not dose- and time-dependent, nor was increased Pim-1 protein involved. These results are consistent with other studies, which suggest metabolite differences in benzene-mediated toxicity. More importantly, this study supports the hypothesis that benzene may mediate its toxicity through metabolite-mediated alterations in the c-Myb signaling pathway.

The Stress-activated Protein Kinases p38α and JNK1 Stabilize p21Cip1 by Phosphorylation

Stress signals activate the SAPK/JNK and p38 MAPK classes of protein kinases, which mediate cellular responses, including steps in apoptosis and the maturation of some cell types. We now show that stress signals initiated by transforming growth factor-beta 1 (TGF-beta 1) induce G(1) arrest through protein stabilization of the CDK inhibitor p21(Cip1). TGF-beta 1 was previously shown to increase p21 protein levels, which in turn mediated G(1) arrest through inactivation of the CDK2-cyclin E complex in HD3 cells (Yan, Z., Kim, G.-Y., Deng, X., and Friedman, E. (2002) J. Biol. Chem. 277, 9870-9879). We now demonstrate that the increase in p21 abundance is caused by a post-transcriptional, SMAD-independent mechanism. TGF-beta1 activated p38 alpha and JNK1, which initiated the phosphorylation of p21. TGF-beta1 treatment increased the half-life of p21 by 3-4-fold. The increase in p21 stability was detected following activation of p38 alpha and JNK1, and treatment of cells with the p38 inhibitor SB203580 prevented this increase in p21 stability. p38 alpha and JNK1 phosphorylated p21 in vivo, and both p38 alpha and JNK1 phosphorylated p21 at Ser(130) in vitro. Peptide mapping demonstrated that both TGF-beta 1 and p38 alpha induced phosphorylation of p21 at Ser(130) in vivo, and mutation of Ser(130) to alanine rendered p21 less stable than wild-type p21. TGF-beta 1 increased the stability of wild-type p21, but not the p21-S130A mutant. These findings demonstrate that SAPKs can mediate cell cycle arrest through post-translational modification of p21.

Regulation of glucose transport in differentiating HD3 cells.

The chicken erythroblast cell line, HD3, has high glucose transport activity which is lost upon differentiation to the red cell phenotype. HD3 cells, when incubated under conditions where maturation occurs, show substantial loss of GLUT1 and GLUT3 mRNAs. To assess whether cAMP or cellular protein phosphorylation affected GLUT mRNA and protein, the HD3 cells were incubated in the presence of different phosphatase inhibitors. Treatment of HD3 cells with the phosphatase inhibitors okadaic acid, vanadate or with 3-isobutyl-1-methyl-xanthine induced glucose transport and GLUT mRNAs. This suggests that phosphorylation events enhance glucose transport and that their reduction may be involved in the decrease in glucose transport that occurs upon HD3 cells differentiation.

Erythrocytic differentiation and glyceraldehyde-3-phosphate dehydrogenase expression are regulated by protein phosphorylation and cAMP in HD3 cells

Utilisation of glucose undergoes a marked decline during erythroblastic differentiation in the chicken. Concomitantly there is a reduction in the expression of glucose transporter proteins and in the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAD). GAD activity declines, after an initial rise, while the level of GAD mRNA decreases rapidly after induction of differentiation. We have employed the temperature-sensitive chicken erythroblast cell line HD3 that differentiates to the erythrocyte phenotype at 42°C in the presence of inducers (hemin and butyric acid). The role of tyrosine and serine/threonine phosphorylation pathways were evaluated with the phosphatase inhibitors sodium vanadate and okadaic acid, respectively. In the presence of phosphatase inhibitors, HD3 cells underwent differentiation and increased their synthesis of hemoglobin which is a marker protein for red blood cells differentiation. The levels of both GAD mRNA and enzymatic activity were increased by phosphatase inhibitors. The role of cAMP in differentiation was also assessed. Differentiation of HD3 cells was associated with an increase in cAMP. However the phosphodiesterase inhibitor IBMX was not a good inducer of hemoglobin synthesis but did induce GAD mRNA and enzymatic activity. Together these results suggest that multiple pathways (including serine/threonine phosphorylation, tyrosine phosphorylation and elevated cAMP) are involved in the regulation of erythroblastic differentiation, hemoglobin synthesis, GAD gene expression and GAD activity in HD3 cells.

Steps in integrin ?1-chain glycosylation mediated by TGF?1 signaling through ras

Ras is activated by transforming growth factor beta (TGFbeta) in several cell types, but the biological consequences of this activation are largely unknown. We now show that ras mediates two stages in integrin beta1-chain maturation: 1) glycosylation of the 86-kD core peptide, which is a TGFbeta1-independent process, and 2) TGFbeta1-mediated conversion of the 115-kD beta1 integrin precursor into the mature 130-kD form. HD3 colon epithelial cells maintain elevated levels of integrin alpha2beta1 heterodimers, strong binding to collagen I, and autocrine regulation by TGFbeta1, which converts beta1 integrin into the mature cell surface form. Each of three HD3 cell clones that stably express dominant negative ras (N17ras) exhibited abnormal glycosylation of the integrin beta1-chain, decreased cell surface expression of the mature integrin beta1, and impaired binding to collagen and laminin. Autocrine levels of TGFbeta were not altered by expression of N17ras. The aberrant glycosylation of the integrin beta1-chain was reversed by antisense oligonucleotides specific to the DNA sequence encoding the rasS17N mutation. Glycosylation of the 86-kD core peptide was delayed in the N17ras transfectants, but was not altered by either the addition of TGFbeta1 or inhibition of autocrine TGFbeta1. In contrast, conversion of the partially glycosylated beta1 integrin precursor into the mature 130-kD isoform was accelerated by exogenous TGFbeta1 and blocked by neutralizing antibody to autocrine TGFbeta1 in control cell lines. Neither effect was seen in the N17ras transfectants, indicating that TGFbeta1 modulates integrin beta1-chain maturation by activating ras proteins. Cell fractionation studies demonstrated that this conversion takes place within the Golgi.

Steps in integrin beta1-chain glycosylation mediated by TGFbeta1 signaling through Ras.

Ras is activated by transforming growth factor beta (TGFbeta) in several cell types, but the biological consequences of this activation are largely unknown. We now show that ras mediates two stages in integrin beta1-chain maturation: 1) glycosylation of the 86-kD core peptide, which is a TGFbeta1-independent process, and 2) TGFbeta1-mediated conversion of the 115-kD beta1 integrin precursor into the mature 130-kD form. HD3 colon epithelial cells maintain elevated levels of integrin alpha2beta1 heterodimers, strong binding to collagen I, and autocrine regulation by TGFbeta1, which converts beta1 integrin into the mature cell surface form. Each of three HD3 cell clones that stably express dominant negative ras (N17ras) exhibited abnormal glycosylation of the integrin beta1-chain, decreased cell surface expression of the mature integrin beta1, and impaired binding to collagen and laminin. Autocrine levels of TGFbeta were not altered by expression of N17ras. The aberrant glycosylation of the integrin beta1-chain was reversed by antisense oligonucleotides specific to the DNA sequence encoding the rasS17N mutation. Glycosylation of the 86-kD core peptide was delayed in the N17ras transfectants, but was not altered by either the addition of TGFbeta1 or inhibition of autocrine TGFbeta1. In contrast, conversion of the partially glycosylated beta1 integrin precursor into the mature 130-kD isoform was accelerated by exogenous TGFbeta1 and blocked by neutralizing antibody to autocrine TGFbeta1 in control cell lines. Neither effect was seen in the N17ras transfectants, indicating that TGFbeta1 modulates integrin beta1-chain maturation by activating ras proteins. Cell fractionation studies demonstrated that this conversion takes place within the Golgi.

Regulation of glyceraldehyde-3-phosphate dehydrogenase in differentiating HD3 cells

Red blood cells usually replenish their ATP pools by glycolysis, fueled by glucose imported via the cell membrane. Mature red cells of some species (e.g. pig, chicken) have, however, been reported to show very low glucose transport. The subject of this study was the possible dependency of the level of a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAD) on glucose transporter activity during the maturation of chicken red cells. The chicken pronormoblast cell line, HD3, was used as a model system. These cells contain higher levels of GAD and glucose transporter activities than normal chicken bone marrow cells, but reduce their levels during maturation. In an attempt to assess whether the decrease in GAD activity is regulated by the glucose transport, the chicken GLUT3 expressed under the control of viral promoter was introduced into HD3 cells by retroviral infection (pDOL-cGT3). Upon cell differentiation and maturation, both cells with and without the exogenous transporter decreased GAD activity. Butyric acid did not affect the regulation of GAD activity upon differentiation. These results show that the development of chicken red cells is manifested by reduction of their GAD activity and that this is not affected by their sugar transporter activity. The very low GAD activity in embryonic chicken red cells is thus due to a loss of this activity at an early stage in their development. Because of the very low glucose transport and GAD activities in mature chicken red cells, rates of glycolysis are likely to be low and suggesting an alternative pathway for ATP production in these cells.