Biomedical subjects

B A Miller

Publications and source records attributed to B A Miller.

At least 19 recordsLinked to original sources

PubMed2006

The role of TRP channels in oxidative stress-induced cell death.

The transient receptor potential (TRP) protein superfamily is a diverse group of voltage-independent calcium-permeable cation channels expressed in mammalian cells. These channels have been divided into six subfamilies, and two of them, TRPC and TRPM, have members that are widely expressed and activated by oxidative stress. TRPC3 and TRPC4 are activated by oxidants, which induce Na(+) and Ca(2+) entry into cells through mechanisms that are dependent on phospholipase C. TRPM2 is activated by oxidative stress or TNFalpha, and the mechanism involves production of ADP-ribose, which binds to an ADP-ribose binding cleft in the TRPM2 C-terminus. Treatment of HEK 293T cells expressing TRPM2 with H(2)O(2) resulted in Ca(2+) influx and increased susceptibility to cell death, whereas coexpression of the dominant negative isoform TRPM2-S suppressed H(2)O(2)-induced Ca(2+) influx, the increase in [Ca(2+)](i), and onset of apoptosis. U937-ecoR monocytic cells expressing increased levels of TRPM2 also exhibited significantly increased [Ca(2+)](i) and increased apoptosis after treatment with H(2)O(2) or TNFalpha. A dramatic increase in caspase 8, 9, 3, 7, and PARP cleavage was observed in TRPM2-expressing cells, demonstrating a downstream mechanism through which cell death is mediated. Inhibition of endogenous TRPM2 function through three approaches, depletion of TRPM2 by RNA interference, blockade of the increase in [Ca(2+)](i) through TRPM2 by calcium chelation, or expression of the dominant negative splice variant TRPM2-S protected cell viability. H(2)O(2) and amyloid beta-peptide also induced cell death in primary cultures of rat striatal cells, which endogenously express TRPM2. TRPM7 is activated by reactive oxygen species/nitrogen species, resulting in cation conductance and anoxic neuronal cell death, which is rescued by suppression of TRPM7 expression. TRPM2 and TRPM7 channels are physiologically important in oxidative stress-induced cell death.

Animals↗

PubMed2005

Amyloid beta-peptide(1-42) and hydrogen peroxide-induced toxicity are mediated by TRPM2 in rat primary striatal cultures.

Amyloid beta-peptide (Abeta) is the main component of senile plaques which characterize Alzheimer's disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin-like transient receptor potential 2 (TRPM2) is a Ca(2+)-permeant non-selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Abeta and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2-S) inhibited both hydrogen peroxide- and Abeta-induced increases in intracellular-free Ca(2+) and cell death. Functional inhibition of TRPM2 activation by the poly(ADP-ribose)polymerase inhibitor SB-750139, a modulator of intracellular pathways activating TRPM2, attenuated hydrogen peroxide- and Abeta-induced cell death. Furthermore, a small interfering RNA which targets TRPM2, reduced TRPM2 mRNA levels and the toxicity induced by hydrogen peroxide and Abeta. These data demonstrate that activation of TRPM2, functionally expressed in primary cultures of rat striatum, contributes to Abeta- and oxidative stress-induced striatal cell death.

Alzheimer Disease↗

PubMed2002

Co-localization of insulin-like growth factor binding protein 3 and fibronectin in human articular cartilage.

OBJECTIVE: The anabolic cytokine insulin-like growth factor I (IGF-I) stimulates chondrocyte synthesis of matrix macromolecules and several lines of evidence suggest that it has a major role in maintaining articular cartilage and possibly in cartilage repair. Despite the apparent importance of IGF-I in articular cartilage metabolism and its potential importance in joint diseases, little is known about the regulation of IGF-I activity within the tissue. Insulin-like growth factor binding proteins (IGFBPs) bind IGF-I and can modify its activity. At least three IGFBPs are expressed by chondrocytes: IGFBP-3, -4 and -5. Localization of IGFPBs in the articular cartilage extracellular matrix (ECM) could create reservoirs of IGF-I within the articular cartilage ECM and thereby regulate local IGF-I levels. We hypothesized that ECM molecules bind and concentrate IGFPBs in the pericellular/territorial matrix. DESIGN: Semi-quantitative immunohistological measures of co-localization were used to compare the spatial distribution of IGFBP-3, -4, and -5 with the distributions of three peri-cellularly-enriched matrix molecules fibronectin, tenascin-C, and type VI collagen in osteoarthritic and non-osteoarthritic human articular cartilage. Purified proteins were used in an agarose diffusion assay to compare IGFBP-3 binding to the same three matrix proteins. RESULTS: IGFBP-3 associated with fibronectin in the pericellular/territorial matrix (approximately 40% co-localization) but not with tenascin-C, or type VI collagen (approximately 6% and approximately 15% co-localization respectively, P< 0.05). Neither IGFBP-4, nor IGFBP-5 were associated with any of the three ECM proteins (P< 0.05). In agarose diffusion assays IGFBP-3 interacted with fibronectin and heparan sulfate proteoglycan but not with type VI collagen or tenascin-C. CONCLUSIONS: Direct binding between purified IGFBP-3 and fibronectin and the strong co-localization the two proteins in the cartilage matrix support the hypothesis that IGFPB-3 and fibronectin help regulate local IGF-I levels.

Adult↗

PubMed2001

Cerebral protection by hypoxic preconditioning in a murine model of focal ischemia-reperfusion.

Sublethal periods of hypoxia or ischemia can induce adaptive mechanisms to protect against subsequent lethal ischemic insults in a process known as ischemic preconditioning. In the present study, we developed a murine model of cerebral preconditioning using several common strains of adult mice. Animals were exposed to sublethal hypoxia (11% oxygen for 2 h) 48 h prior to a 90 min period of transient focal middle cerebral artery occlusion, induced by an intraluminal filament; injury was assessed 24 h later by TTC staining. Infarct volume in hypoxia-preconditioned animals was reduced 46%, 58%, and 64% in C57Bl/6, 129SvEv, and Swiss-Webster ND4 mice relative to their respective untreated controls. This non-invasive murine model of ischemic tolerance should be useful for elucidating the molecular basis of this protection using transgenic and knockout mice.

Animals↗

PubMed2001

LUCA-15 suppresses CD95-mediated apoptosis in Jurkat T cells.

The candidate tumour suppressor gene, LUCA-15, maps to the lung cancer tumour suppressor locus 3p21.3. Overexpression of an alternative RNA splice variant of LUCA-15 has been shown to retard human Jurkat T cell proliferation and to accelerate CD95-mediated apoptosis. An antisense cDNA to the 3'-UTR of this splice variant was able to suppress CD95-mediated apoptosis. Here, we report that overexpression of LUCA-15 itself suppresses CD95-mediated apoptosis in Jurkat cells. This suppression occurs prior to the final execution stage of the CD95 signalling pathway, and is associated with up-regulation of the apoptosis inhibitory protein Bcl-2. LUCA-15 overexpression is also able to inhibit apoptosis induced by the protein kinase inhibitor staurosporine, but is not able to significantly suppress apoptosis mediated by the topoisomerase II inhibitor etoposide. These findings suggest that LUCA-15 is a selective inhibitor of cell death, and confirm the importance of the LUCA-15 genetic locus in the control of apoptosis.

Alternative Splicing↗

PubMed2001

A minimal cytoplasmic subdomain of the erythropoietin receptor mediates p70 S6 kinase phosphorylation.

Erythropoietin (EPO) is a lineage-restricted growth factor that is required for erythroid proliferation and differentiation. EPO stimulates the phosphorylation and activation of p70 S6 kinase (p70 S6K), which is required for cell cycle progression. Here, the minimal cytoplasmic domains of the EPO receptor (EPO-R) required for p70 S6K activation were determined.Ba/F3 cells were stably transfected with wild-type (WT) EPO-R or EPO-R carboxyl-terminal deletion mutants, designated by the number of amino acids deleted from the cytoplasmic tail (-99, -131, -221). Transfected cells were growth factor deprived and then stimulated with EPO. p70 S6K, JAK2, IRS-2, and ERK1/2 phosphorylation/activation were examined. The ability of transfected 3-phosphoinositide-dependent protein kinase 1 (PDK1) to reconstitute p70 S6K phosphorylation in EPO-R mutants also was determined. Phosphorylation and activation of p70 S6K, JAK2, IRS-2, and ERK1/2 in Ba/F3 cells transfected with EPO-R-99 or EPO-R-99Y343F were similar to WT EPO-R. In contrast, EPO-dependent p70 S6K phosphorylation/activation, as well as IRS-2 and ERK1/2 phosphorylation, were minimal or absent in cells transfected with EPO-R-131 or EPO-R-221. JAK2 phosphorylation was reduced significantly in cells transfected with EPO-R-131 and abolished with EPO-R-221. To examine the role of PDK1, a kinase known to phosphorylate p70 S6K, Ba/F3 EPO-R-131 cells were transiently transfected with PDK1. WT constitutively active PDK1 restored p70 S6K phosphorylation in Ba/F3 EPO-R-131 cells but not in Ba/F3 EPO-R-221 cells. The results demonstrate that a minimal cytoplasmic subdomain of the EPO-R extending between -99 and -131 is required for p70 S6K phosphorylation and activation. The results also demonstrate that PDK1 is a critical component in this signaling pathway, which requires the presence of domains between -131 and -221 for its activation of p70 S6K.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2001

Molecular mechanisms of erythropoietin signaling.

Erythropoietin is an obligatory growth factor for red blood cell production. The receptor for erythropoietin contains a single membrane-spanning domain with no intrinsic tyrosine kinase motifs. On binding to erythropoietin, the receptor dimerizes and activates multiple intracellular signaling molecules, including but not limited to JAK2, STAT5, PI 3-kinase, IRS-2, RAS, and Ca2+ channels. This review focuses on cytoplasmic signaling cascades involved in erythropoietin action.

Animals↗

PubMed2000

Expression of angiotensin II type I receptor on erythroid progenitors of patients with post transplant erythrocytosis.

BACKGROUND: The pathogenesis of posttransplant erythrocytosis (PTE) has been elusive. Angiotensin converting enzyme inhibitors (ACEI) are efficacious in lowering the hematocrit of patients with PTE and angiotensin II (AII) type I receptors (AT1R) were recently detected on red blood cell precursors, burst-forming unit-erythroid- (BFU-E) derived cells. The purpose of this study was to determine whether there is increased expression of the AT1R on BFU-E-derived cells of patients with PTE, which might contribute to the pathogenesis of PTE. METHODS: Twelve healthy volunteers and 25 transplant recipients (13 patients with and 12 without PTE) were studied. BFU-E from peripheral blood were cultured in methylcellulose and BFU-E-derived colonies were harvested on day 10. Western blotting was used to detect AT1R and erythropoietin receptor (EpoR) expression. Intracellular free calcium in response to AII and erythropoietin (Epo) was measured with digital video imaging. RESULTS: There were no differences between transplant patients, with and without PTE, with respect to weight, age, sex, blood pressure, serum creatinine, circulating renin, angiotensin II, and Epo levels. Hematocrit, red blood cell number, BFU-E-derived colony number,and size were significantly increased in PTE compared with other two groups. AT1R expression was increased by 44% on the erythroid progenitors of PTE versus non posttransplant erythrocytosis patients and by 32% in PTE patients versus normal volunteers. AT1R expression correlated significantly with the hematocrit in PTE (Spearman r=0.68, P=0.01). In contrast, EpoR expression was equivalent in all groups. The AT1R was functional since a significant increase in [Ca(i)] was observed in Fura-2 loaded day 10 cells when stimulated with AII (182%, P<0.0001). CONCLUSION: An increase in AT1R density was observed in erythroid precursors of transplant patients with PTE compared to those without PTE and normal volunteers, and the level of AT1R expression in PTE correlated significantly with the hematocrit. In contrast, EpoR expression was not different in PTE compared with non posttransplant erythrocytosis or normal controls. This study supports a role for the AT1 receptor signaling pathway in the pathogenesis of PTE.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2000

LUCA-15-encoded sequence variants regulate CD95-mediated apoptosis.

Using an expression cloning system to discover novel genes involved in apoptosis, we identified a 326 bp bone marrow cDNA fragment (termed Je2) that suppresses, upon transfection, CD95-mediated apoptosis in Jurkat T cells. Sequence homology revealed that Je2 maps to 3p21.3, to an intronic region of the candidate TSG LUCA-15 locus. It represents, in fact, an antisense transcript to the 3'-UTR of two novel splice variants of this gene. Overexpression of sequence representing one of these splice variants (a 2.6 kb cDNA termed Clone 26), inhibited proliferation of Jurkat cells and sensitized them to CD95-mediated apoptosis. This study therefore implicates the LUCA-15 gene locus in the control of apoptosis.

Alternative Splicing↗

PubMed2000

The human homologue of the Aspergillus nuclear migration gene nudC is preferentially expressed in dividing cells and ciliated epithelia.

We recently identified a novel human gene, HnudC, homologous to an Aspergillus nidulans gene coding for a protein crucial to nuclear migration, cell wall morphogenesis, and cell growth. While mRNA for this gene is expressed in most tissues, HNUDC protein expression is highly regulated. To provide insight into the function of this protein, we performed immunohistochemical analysis of the distribution of HNUDC in 19 different human tissues. Intense immunolabeling was observed in proliferating cells, including spermatocytes at all stages, early hematopoietic cells, cortical thymocytes, immunoblasts, and basal colonic and esophageal mucosa. Within a given tissue, cells with different proliferative capacities demonstrated different levels of HNUDC expression. HNUDC was also highly expressed in ciliated epithelia including those found in ependyma, bronchial mucosa, and fallopian tubes. Immunolabeling was moderate in several non-proliferating tissues, but little or no labeling was observed in most other tissues examined. We also demonstrated by western blotting that most cell lines express extremely high levels of HNUDC compared to their normal counterparts. While this supports a role for HnudC in cell proliferation, these data indicate that cell lines are not a reliable measure of HNUDC protein expression in normal tissues. We conclude that HNUDC is highly expressed in cell lines and the proliferating cells of normal tissues, consistent with our hypothesis that HNUDC is conserved throughout evolution for a crucial function in cell division. In addition, the high level in ciliated cells suggests an important role in ciliary motility or assembly, analogous to its role in A. nidulans nuclear movement.

Adult↗

PubMed2000

The nuclear migration gene NudC and human hematopoiesis.

The filamentous fungus Aspergillus nidulans nudC (nuclear distribution C) gene is required for movement of nuclei following mitosis and for normal colony growth. It is highly conserved, structurally and functionally, throughout most of evolution. The human homolog, called HnudC, has been cloned and has an important role in cell proliferation. In hematopoiesis, HNUDC is highly expressed in early hematopoietic precursors and declines during normal differentiation. Stimulation of proliferation of the erythroleukemia cell line TF-1 with GM-CSF enhances HnudC protein and mRNA expression and treatment with antisense (but not sense) oligonucleotides to HnudC mRNA significantly reduces cell division. A significant increase in HNUDC is present in bone marrow aspirates from patients with acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML) compared to the level in normal cellular counterparts, demonstrating dysregulated expression in leukemia. These data support the conclusion that HnudC plays a functional role in promoting hematopoietic cell growth and that it is involved in leukemogenesis.

Cell Cycle Proteins↗

PubMed2000

Family violence and victimization: treatment issues for women with alcohol problems.

A brief overview of the prevalence of violence in women's lives and psychological and physical consequences of these experiences is presented, with special attention to populations of women with alcohol and other drug problems. Barriers to identification of violent victimization within health care settings are discussed. Finally, potential areas for further consideration regarding policy, practice, and research are identified. The relationships between women's experiences of violent victimization and their use of alcohol and drugs are documented, although prior research has not fully explored or explained the etiologic nature of these relationships. Of additional importance is the recognition that various forms of violence occur throughout a single life span. Women's histories of violent victimization negatively affect their children, and thus, intergenerational issues are of concern. Understanding how the combined problems of alcohol and drugs interconnect with violence is relevant to the delivery of health services for women.

Adolescent↗

PubMed1999

Identification of the erythropoietin receptor domain required for calcium channel activation.

Erythropoietin (Epo) activates a voltage-independent Ca2+ channel that is dependent on tyrosine phosphorylation. To identify the domain(s) of the Epo receptor (Epo-R) required for Epo-induced Ca2+ influx, Chinese hamster ovary (CHO) cells were transfected with wild-type or mutant Epo receptors subcloned into pTracer-cytomegalovirus vector. This vector contains an SV40 early promoter, which drives expression of the green fluorescent protein (GFP) gene, and a cytomegalovirus immediate-early promoter driving expression of the Epo-R. Successful transfection was verified in single cells by detection of GFP, and intracellular Ca2+ ([Ca]i) changes were simultaneously monitored with rhod-2. Transfection of CHO cells with pTracer encoding wild-type Epo-R, but not pTracer alone, resulted in an Epo-induced [Ca]i increase that was abolished in cells transfected with Epo-R F8 (all eight cytoplasmic tyrosines substituted). Transfection with carboxyl-terminal deletion mutants indicated that removal of the terminal four tyrosine phosphorylation sites, but not the tyrosine at position 479, abolished Epo-induced [Ca]i increase, suggesting that tyrosines at positions 443, 460, and/or 464 are important. In CHO cells transfected with mutant Epo-R in which phenylalanine was substituted for individual tyrosines, a significant increase in [Ca]i was observed with mutants Epo-R Y443F and Epo-R Y464F. The rise in [Ca]i was abolished in cells transfected with Epo-R Y460F. Results were confirmed with CHO cells transfected with plasmids expressing Epo-R mutants in which individual tyrosines were added back to Epo-R F8 and in stably transfected Ba/F3 cells. These results demonstrate a critical role for the Epo-R cytoplasmic tyrosine 460 in Epo-stimulated Ca2+ influx.

Animals↗

PubMed1999

A homolog of the fungal nuclear migration gene nudC is involved in normal and malignant human hematopoiesis.

The filamentous fungus Aspergillus nidulans nudC gene has an essential function in movement of nuclei following mitosis and is required for normal colony growth. Here, the molecular cloning and role in hematopoiesis of a human gene (designated HnudC) homologous to A. nidulans nudC is reported. The amino terminus of the larger human protein (HNUDC = 45 kDa) does not overlap with A. nidulans NUDC (22 kDa). However, NUDC and the C-terminal 94 amino acids of HNUDC are 67% identical. The C-terminal region of the HnudC gene fully complements the A. nidulans temperature-sensitive nudC3 mutation, suggesting that nudC has an essential function in cell growth that is conserved from filamentous fungi to humans. In initial studies, HNUDC levels were much higher in erythroid precursors compared to most other human tissues. Therefore, the potential role of HnudC in hematopoiesis was explored. In normal human bone marrow, HNUDC protein and mRNA are highly expressed in early myeloid and erythroid precursors and decline as these cells terminally differentiate. To determine whether hematopoietic growth factors induce HnudC expression, TF-1 cells were stimulated by granulocyte-macrophage colony-stimulating factor. This induced a significant increase in HNUDC protein and HnudC mRNA, suggesting that enhancement of HnudC expression in response to growth factor stimulation may be mediated at the transcription level. Furthermore, HNUDC was significantly enhanced in lysates of bone marrow aspirates from patients with acute myelogenous and acute lymphoblastic leukemia compared to aspirates from normal controls, suggesting that HnudC is involved in malignant hematopoietic cell growth as well. These data demonstrate that HNUDC is highly expressed in normal and malignant human hematopoietic precursors and suggest it is of functional importance in the proliferation of these cells.

Antibody Specificity↗

PubMed1999

Mothers' alcohol and other drug problems and their punitiveness toward their children.

OBJECTIVE: The relationship between mothers' alcohol or other drug (AOD) problems and their punitiveness toward their children is examined in this study. METHOD: Women (N = 170) were recruited from five different sources, including clinical interventions and the community. Women's punitiveness toward their children was assessed by three different maternal self-report measures: Conflict Tactics Scale, Parental Punitiveness Scale and Child Abuse Potential Inventory. Women's histories of AOD problems, violent victimization and hostility were assessed using standardized assessment tools. RESULTS: Women were categorized by their AOD problems as follows: current problem (22.4%), past only problem (44.7%) or no problems (32.9%). Results indicate that mothers with current or past AOD problems are more punitive toward their children, controlling for potentially confounding demographic factors. Mothers' histories of partner violence and parental violence also predicted higher levels of mother-to-child punitiveness. However, histories of childhood sexual abuse predicted lower levels of mothers' punitiveness. Hostility served as a predictor of mothers' punitiveness and moderated some of the relationships between their AOD problems and their punitiveness. CONCLUSIONS: Findings suggest that women with AOD problems are more likely to be punitive toward their children, but other factors may also contribute to this risk. Treatment and prevention interventions need to be developed to decrease the risk of violent victimization among children whose mothers have alcohol and other drug problems.

Adolescent↗

PubMed1998

Duration of antiviral prophylaxis during nursing home outbreaks of influenza A: a comparison of 2 protocols.

BACKGROUND: We performed a randomized trial of 2 protocols guiding the duration of antiviral chemoprophylaxis during outbreaks of influenza A in a rural, 700-bed nursing home for veterans and their spouses with 14 nursing units in 4 buildings. METHODS: Half of all residents volunteered to participate. Nursing units were randomized, and the effectiveness of short-term (minimum, 14 days and 7 days without the onset of a case in the building) vs long-term (minimum, 21 days and 7 days without the onset of a case in the 4-building facility) prophylaxis was compared using amantadine hydrochloride in the influenza seasons of 1991-1992 and 1993-1994 and rimantadine hydrochloride in the influenza season of 1994-1995. A "case" is defined as an incident of a respiratory tract illness and the isolation of an influenza virus organism. We compared the number of cases after the discontinuation of short- vs long-term chemoprophylaxis. Prospective surveillance identified residents with new respiratory tract symptoms, and specimens for viral cultures were obtained even in the absence of temperature elevation. RESULTS: We documented influenza A virus activity during 3 seasons (32, 68, and 12 patients, respectively). During the 1991-1992, 1993-1994, and 1994-1995 influenza seasons, the patients on 11 floors were assigned to receive short-term chemoprophylaxis and those on 10 floors were assigned to long-term chemoprophylaxis. Only in 1993-1994 did chemoprophylaxis extend beyond 14 or 21 days when new cases continued beyond 14 days. Amantadine-resistant strains were circulating at that time. None of the participants in the prospective, controlled study had influenza develop after the termination of short- or long-term chemoprophylaxis. CONCLUSION: Antiviral chemoprophylaxis can be administered for the longer duration of 14 days or, in the absence of new culture-confirmed illness in the nursing building, for 7 days.

Aged↗

PubMed1998

Bcl-3 expression and nuclear translocation are induced by granulocyte-macrophage colony-stimulating factor and erythropoietin in proliferating human erythroid precursors.

Bcl-3 is a proto-oncogene involved in the chromosomal translocation t(14;19) found in some patients with chronic lymphocytic leukemia. It shares structural similarities with and is a member of the IkappaB family of proteins. In this report, involvement of Bcl-3 in hematopoietic growth factor-stimulated erythroid proliferation and differentiation was examined. In TF-1 cells, an erythroleukemia cell line, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) greatly enhanced Bcl-3 expression at both the protein and mRNA levels in association with stimulation of proliferation. Bcl-3 protein was also highly expressed in early burst-forming unit-erythroid (BFU-E)-derived erythroid precursors (day 7) and decreased during maturation (days 10 and 14), suggesting that Bcl-3 is involved in normal erythroid proliferation. In these hematopoietic cells, Bcl-3 was hyperphosphorylated. GM-CSF and Epo modulated the subcellular localization of Bcl-3. Upon stimulation of TF-1 cells with GM-CSF or Epo, the nuclear translocation of Bcl-3 was dramatically enhanced. Overexpression of Bcl-3 in TF-1 cells by transient transfection along with the NF-kappaB factors p50 or p52 resulted in significant induction of an human immunodeficiency virus-type 1 (HIV-1) kappaB-TATA-luceriferase reporter plasmid, demonstrating that Bcl-3 has a positive role in transactivation of kappaB-containing genes in erythroid cells. Stimulation with GM-CSF enhanced c-myb mRNA expression in these cells. Bcl-3 in nuclear extracts of TF-1 cells bound to a kappaB enhancer in the c-myb promoter together with NF-kappaB2/p52 and this binding activity was enhanced by GM-CSF stimulation. Furthermore, cotransfection of Bcl-3 with p52 or p50 in TF-1 cells resulted in significant activation of a c-myb kappaB-TATA-luceriferase reporter plasmid. These findings suggest that Bcl-3 may participate in the transcriptional regulation of certain kappaB-containing genes involved in hematopoiesis, including c-myb.

B-Cell Lymphoma 3 Protein↗

PubMed1998

NF-kappaB transcription factors are involved in normal erythropoiesis.

NF-kappaB/Rel designates a widely distributed family of transcription factors involved in immune and acute phase responses. Here, the expression and function of NF-kappaB factors in erythroid proliferation and differentiation were explored. In an erythroleukemia cell line, TF-1, high levels of p105/p50, p100/p52, p65, and IkappaBalpha were detected 24 hours after growth factor deprivation. In response to granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation, significant induction of p52 expression was observed. GM-CSF also induced nuclear translocation of both p52 and p65. No induction of NF-kappaB factors was observed with erythropoietin stimulation of TF-1 cells. Overexpression of p52 and p65 in TF-1 cells by transient transfection resulted in significant induction of a kappaB-TATA-luciferase reporter plasmid, showing that these factors are functional in vivo in erythroid cells. To determine whether NF-kappaB factors may play a role in normal erythropoiesis, levels of these factors were determined in burst-forming unit-erythroid (BFU-E)-derived cells at different stages of differentiation. The NF-kappaB factors p105/p50, p100/p52, and p65 were highly expressed in early BFU-E-derived precursors, which are rapidly proliferating, and declined during maturation. Furthermore, nuclear levels of NF-kappaB factors p50, p52, and p65 were higher in less mature precursors (day 10 BFU-E-derived cells) compared with more differentiated (day 14) erythroblasts. In nuclear extracts from day 10 BFU-E-derived cells, p50, p52, and p65 were able to form complexes, which bound to kappaB sites in the promoters of both the c-myb and c-myc genes, suggesting that c-myb and c-myc may be among the kappaB-containing genes regulated by NF-kappaB factors in normal erythroid cells. Taken together, these data show that NF-kappaB factors are modulated by GM-CSF and suggest they function to regulate specific kappaB containing genes involved in erythropoiesis.

Cell Division↗