AlphaIIbG236E causes Glanzmann thrombasthenia by impairing association with beta3

Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of the platelet fibrinogen receptor, integrin alphaIIbbeta3. The N-terminal domain of the alphaIIb subunit is folded in a beta-propeller that plays the role of binding fibrinogen and associating with the ligand-binding region of beta3. Analysing the mutations of Italian GT patients we found that a patient had a alphaIIb G236E missense substitution that substitutes a glycine from the highly conserved PhiPhiGPhi motif of blade 4 of the beta-propeller. To verify experimentally the effect of the substitution of glycine 236 human embryonic kidney (HEK) cells were transfected with normal or mutated alphaIIb in conjunction with normal beta3. Using flow cytometry analysis we found the percentage of HEK cells transfected with alphaIIbG236Ebeta3 that reacted with anti alphaIIbbeta3 was very low. In HEK cells transfected with either alphaIIbbeta3 or alphaIIbG236Ebeta3 and lysed, when immunoblotting was done in non-reducing conditions a band reacting with an antibody against alphaIIb was present in both lysates, although less intense in cells transfected with alphaIIbG236Ebeta3. In reducing condition alphaIIb from cells transfected with alphaIIbbeta3 was nearly all mature, while in cells transfected with alphaIIbG236Ebeta3 the ratio pro-alphaIIb: alphaIIb was 1 : 1, with signs of degradation of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against alphaIIb and immunoblotted with an antibody reacting with beta3. While in immunoblots from cells transfected with alphaIIbbeta3 a band corresponding to beta3 was strongly detectable, in immunoblots originating from cells transfected with alphaIIbG236Ebeta3 no band at the same level of normal beta3 was detected. Immunofluorescence studies showed accumulation of alphaIIbG236Ebeta3 in the endoplasmic reticulum and minimal transport to the Golgi. In conclusion we demonstrated that the alphaIIbG236E mutation causes GT by impairing the association with beta3 during biogenesis of the receptor.     

Mapping early conformational changes in alphaIIb and beta3 during biogenesis reveals a potential mechanism for alphaIIbbeta3 adopting its bent conformation

Current evidence supports a model in which the low-affinity state of the platelet integrin alphaIIbbeta3 results from alphaIIbbeta3 adopting a bent conformation. To assess alphaIIbbeta3 biogenesis and how alphaIIbbeta3 initially adopts the bent conformation, we mapped the conformational states occupied by alphaIIb and beta3 during biogenesis using conformation-specific monoclonal antibodies (mAbs). We found that alphaIIbbeta3 complex formation was not limited by the availability of either free pro-alphaIIb or free beta3, suggesting that other molecules, perhaps chaperones, control complex formation. Five beta3-specific, ligand-induced binding site (LIBS) mAbs reacted with much or all free beta3 but not with beta3 when in complex with mature alphaIIb, suggesting that beta3 adopts its mature conformation only after complex formation. Conversely, 2 alphaIIb-specific LIBS mAbs directed against the alphaIIb Calf-2 region adjacent to the membrane reacted with only minor fractions of free pro-alphaIIb, raising the possibility that pro-alphaIIb adopts a bent conformation early in biogenesis. Our data suggest a working model in which pro-alphaIIb adopts a bent conformation soon after synthesis, and then beta3 assumes its bent conformation by virtue of its interaction with the bent pro-alphaIIb.     

Two novel mutations in the alpha IIb calcium-binding domains identify hydrophobic regions essential for alpha IIbbeta 3 biogenesis

The recently published crystal structure of the external domains of alphaVbeta3 confirms the prediction that the aminoterminal portion of alphaV, which shares 40% homology with alphaIIb, folds into a beta-propeller structure and that the 4 calcium-binding domains are positioned on the bottom of the propeller. To gain insight into the role of the calcium-binding domains in alphaIIb biogenesis, we characterized mutations in the second and third calcium-binding domains of alphaIIb in 2 patients with Glanzmann thrombasthenia. One patient inherited a Val298Phe mutation in the second domain, and the other patient inherited an Ile374Thr mutation in the third domain. Mammalian cell expression studies were performed with normal and mutant alphaIIb and beta3 cDNA constructs. By flow cytometry, expression of alphaIIb Val298Phe/beta3 in transfected cells was 28% of control, and expression of alphaIIbIle374Thr/beta3 was 11% of control. Pulse-chase analyses showed that both mutant pro-alphaIIb subunits are retained in the endoplasmic reticulum and degraded. Mutagenesis studies of the Val298 and Ile374 residues showed that these highly conserved, branch-chained hydrophobic residues are essential at these positions and that biogenesis and expression of alphaIIbbeta3 is dramatically affected by structural variations in these regions of the calcium-binding domains. Energy calculations derived from a new model of the alphaIIb beta-propeller indicate that these mutations interfere with calcium binding. These data suggest that the alphaIIb calcium-binding domains play a key structural role in the beta-propeller, and that the structural integrity of the calcium-binding domains is critical for integrin biogenesis.     

Identification of distal regulatory regions in the human alpha IIb gene locus necessary for consistent,high-level megakaryocyte expression

The alphaIIb/beta3-integrin receptor is present at high levels only in megakaryocytes and platelets. Its presence on platelets is critical for hemostasis. The tissue-specific nature of this receptor's expression is secondary to the restricted expression of alphaIIb, and studies of the alphaIIb proximal promoter have served as a model of a megakaryocyte-specific promoter. We have examined the alphaIIb gene locus for distal regulatory elements. Sequence comparison between the human (h) and murine (m) alphaIIb loci revealed high levels of conservation at intergenic regions both 5' and 3' to the alphaIIb gene. Additionally, deoxyribonuclease (DNase) I sensitivity mapping defined tissue-specific hypersensitive (HS) sites that coincide, in part, with these conserved regions. Transgenic mice containing various lengths of the h(alpha)IIb gene locus, which included or excluded the various conserved/HS regions, demonstrated that the proximal promoter was sufficient for tissue specificity, but that a region 2.5 to 7.1 kb upstream of the h(alpha)IIb gene was necessary for consistent expression. Another region 2.2 to 7.4 kb downstream of the gene enhanced expression 1000-fold and led to levels of h(alpha)IIb mRNA that were about 30% of the native m(alpha)IIb mRNA level. These constructs also resulted in detectable h(alpha)IIb/m(beta)3 on the platelet surface. This work not only confirms the importance of the proximal promoter of the alphaIIb gene for tissue specificity, but also characterizes the distal organization of the alphaIIb gene locus and provides an initial localization of 2 important regulatory regions needed for the expression of the alphaIIb gene at high levels during megakaryopoiesis.     

Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alphaIIbbeta(3). As a result, alphaIIbbeta(3) cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, -889Pl(A2)beta(3), was transduced into peripheral blood CD34(+) cells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alphaIIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta(3) subunit. Proviral DNA and alphaIIbbeta(3) biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34(+) cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed alphaIIbbeta(3) on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the alphaIIbbeta(3) complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34(+) cells as targets for alphaIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)     

Characterization of the murine platelet alphaIIb gene and encoded cDNA

The alphaIIb/beta3 receptor is central to platelet aggregation. Biological studies of this receptor have been limited by the inability to reproduce alphaIIb/beta3 function in a cell system. Increasingly, efforts are being directed at studies of this receptor in mice models. The structure of murine (m) beta3 has been reported. We now have sequenced the malphaIIb gene and found that it has the same size and organization as the human gene. The exon/intron borders are reported here, as are the distances between exons. malphaIIb protein is 1,033 amino acids (aa), 7 and 5 aa shorter than human (h) and rodent (r) alphaIIb, respectively, with 79% and 90% homology, respectively. As part of the comparative analysis of the 3 known alphaIIb chains included in this report, we found that a particular region of the alphaIIb N-terminal beta-propeller is highly conserved and speculate that it directly participates in ligand binding.     

Ubiquitination of inducible nitric oxide synthase is required for its degradation

Inducible nitric oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. We have previously shown that iNOS is degraded through the 26S proteasome. Targeting of proteins for proteasomal degradation may or may not require their covalent linkage to multiubiquitin chains (ubiquitination). In addition, ubiquitination of a protein can serve functions other than signaling proteolysis. In this context, it is not known whether iNOS is subject to ubiquitination or whether ubiquitination is required for its degradation. In this study, we show that iNOS, expressed in HEK293 cells or induced in primary bronchial epithelial cells, A549 cells, or murine macrophages, is subject to ubiquitination. To investigate whether iNOS ubiquitination is required for its degradation, HEK293T cells were cotransfected with plasmids containing cDNAs of human iNOS and of the dominant negative ubiquitin mutant K48R. Disruption of ubiquitination by K48R ubiquitin resulted in inhibition of iNOS degradation. ts20 is a mutant cell line that contains a thermolabile ubiquitin-activating enzyme (E1) that is inactivated at elevated temperature, preventing ubiquitination. Incubation of ts20 cells, stably expressing human iNOS, at the nonpermissive temperature (40 degrees C) resulted in inhibition of iNOS degradation and marked accumulation of iNOS. These studies indicate that iNOS is subject to ubiquitination and that ubiquitination is required for its degradation.     

Single point mutations affect fatty acid block of human myocardial sodium channel alpha subunit Na+ channels

Suppression of cardiac voltage-gated Na(+) currents is probably one of the important factors for the cardioprotective effects of the n-3 polyunsaturated fatty acids (PUFAs) against lethal arrhythmias. The alpha subunit of the human cardiac Na(+) channel (hH1(alpha)) and its mutants were expressed in human embryonic kidney (HEK293t) cells. The effects of single amino acid point mutations on fatty acid-induced inhibition of the hH1(alpha) Na(+) current (I(Na)) were assessed. Eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I(Na) in HEK293t cells expressing the wild type, Y1767K, and F1760K of hH1(alpha) Na(+) channels. The inhibition was voltage and concentration-dependent with a significant hyperpolarizing shift of the steady state of I(Na). In contrast, the mutant N406K was significantly less sensitive to the inhibitory effect of EPA. The values of the shift at 1, 5, and 10 microM EPA were significantly smaller for N406K than for the wild type. Coexpression of the beta(1) subunit and N406K further decreased the inhibitory effects of EPA on I(Na) in HEK293t cells. In addition, EPA produced a smaller hyperpolarizing shift of the V(1/2) of the steady-state inactivation in HEK293t cells coexpressing the beta(1) subunit and N406K. These results demonstrate that substitution of asparagine with lysine at the site of 406 in the domain-1-segment-6 region (D1-S6) significantly decreased the inhibitory effect of PUFAs on I(Na), and coexpression with beta(1) decreased this effect even more. Therefore, asparagine at the 406 site in hH1(alpha) may be important for the inhibition by the PUFAs of cardiac voltage-gated Na(+) currents, which play a significant role in the antiarrhythmic actions of PUFAs.     

Therapeutic expression of the platelet-specific integrin, alphaIIbbeta3, in a murine model for Glanzmann thrombasthenia

Integrins mediate the adhesion of cells to each other and to the extracellular matrix during development, immunity, metastasis, thrombosis, and wound healing. Molecular defects in either the alpha- or beta-subunit can disrupt integrin synthesis, assembly, and/or binding to adhesive ligands. This is exemplified by the bleeding disorder, Glanzmann thrombasthenia (GT), where abnormalities of the platelet-specific integrin, alphaIIbbeta3, prevent platelet aggregation following vascular injury. We previously used a retrovirus vector containing a cDNA cassette encoding human integrin beta3 to restore integrin alphaIIbbeta3 on the surface of megakaryocytes derived from peripheral blood stem cells of GT patients. In the present study, bone marrow from beta3-deficient (beta3-/-) mice was transduced with the ITGbeta3-cassette to investigate whether the platelet progeny could establish hemostasis in vivo. A lentivirus transfer vector equipped with the human ITGA2B gene promoter confined transgene expression to the platelet lineage. Human beta3 formed a stable complex with murine alphaIIb, effectively restoring platelet function. Mice expressing significant levels of alphaIIbbeta3 on circulating platelets exhibited improved bleeding times. Intravenous immunoglobulin effectively diminished platelet clearance in animals that developed an antibody response to alphaIIbbeta3. These results indicate the feasibility of targeting platelets with genetic therapies for better management of patients with inherited bleeding disorders.     

Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation

Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the alphaIIbbeta3 receptor. In this study we identified regions on integrin beta3 that control 7E3 binding. Murine/human amino acid substitutions were created in two regions of the betaA domain that previous studies found to influence 7E3 binding: the C177-C184 loop and K125-N133. The T182N substitution and a K125Q mutation reduced 7E3 binding to human beta3 in complex with alphaIIb. The introduction of both the human C177-C184 region and human W129 into murine beta3 was necessary and sufficient to permit 7E3 binding to the human alphaIIb/murine beta3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 A of each other in the crystal structure and close to the beta3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that alphaIIbbeta3 changes from a bent to an extended conformation upon activation, we hypothesized that 7E3 binds less well to the bent than the extended conformation. In support of this hypothesis we found that 7E3 bound less well to an alphaIIbbeta3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with alphaIIbbeta3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in alphaIIbbeta3 adopting a more extended conformation.     

The human platelet alphaIIb gene is not closely linked to its integrin partner beta3.

alphaIIbb3 integrin is a heterodimeric receptor facilitating platelet aggregation. Both genes are on chromosome 17q21.32. Intergenic distance between them has been reported to be 125 to 260 kilobasepairs (kb) by pulsed-field gel electrophoresis (PFGE) genomic analysis, suggesting that they may be regulated coordinately during megakaryopoiesis. In contrast, other studies suggest these genes are greater than 2.0 megabasepairs (mb) apart. Because of the potential biological implications of having these two megakaryocytic-specific genes contiguous, we attempted to resolve this discrepancy. Taking advantage of large kindreds with mutations in either alphaIIb or beta3, we have developed a genetic linkage map between the thyroid receptor hormone-1 gene (THRA1) and beta3 as follows: cen-THRA1-BRCA1-D17S579/alphaIIb-beta3-qte r, with a distance of 1.3 centiMorgans (cM) between alphaIIb and beta3 and the two genes being oriented in the same direction. PFGE genomic and YAC clone analysis showed that the beta3 gene is distal and >/=365 kb upstream of alphaIIb. Additional restriction mapping shows alphaIIb is linked to the erythrocyte band 3 (EPB3) gene, and beta3 to the homeobox HOX2b gene. Analysis of alphaIIb(+)-BAC and P1 clones confirm that the EPB3 gene is approximately 110 kb downstream of the alphaIIb gene. Sequencing the region surrounding the human alphaIIb locus showed the Granulin gene approximately 18 kb downstream to alphaIIb, and the KIAA0553 gene approximately 5.7 kb upstream. This organization is conserved in the murine sequence. These studies show that alphaIIb and beta3 are not closely linked, with alphaIIb flanked by nonmegakaryocytic genes, and imply that they are unlikely to share common regulatory domains during megakaryopoiesis.     

Role of p21(Cip1/Waf1) in cell-cycle exit of endomitotic megakaryocytes.

The cyclin-dependent kinase inhibitor p21(Waf-1/Cip-1) is expressed at high level during megakaryocyte differentiation, but its precise function remains unknown. In this study, it is confirmed that p21 was expressed at a high level in hypoploid (2N and 4N) and polyploid (at least 8N) human megakaryocytes derived from CD34(+) cells. A high expression of p27(Kip1), p16, cyclin E, and cyclin D3 was also found in both populations associated with a hypophosphorylated form of retinoblastoma protein, suggesting that the majority of hypoploid and polyploid megakaryocytes are G(1)-arrested cells. As human megakaryocytes grown in vitro present a defect in their polyploidization, the study switched to the murine model. The modal ploidy of megakaryocytes derived from lineage-negative cells was 32N, and an elevated expression of p21 was found in high-ploidy megakaryocytes. In addition, p21 and p27 were coexpressed in the majority of mature polyploid megakaryocytes. The p21 was detected by immunofluorescence in megakaryocytes derived from p53(-/-) mice, demonstrating a p53-independent regulation during megakaryocyte differentiation. Megakaryocytopoiesis of p21(-/-) mice was subsequently studied. No marked abnormality in the ploidy of primary or cultured megakaryocytes was detected. Overexpression of p21 in p21(-/-) or normal murine megakaryocytes and in human megakaryocytes showed in all these cases a marked inhibition in megakaryocyte polyploidization. In conclusion, while a reciprocal relation is observed between p21 levels in megakaryocytes and the cycling state of the cells, p21 is not essential for the determination of the ploidy profile in normal megakaryocytes in vivo. However, high levels of its expression in cultured megakaryocytes arrest the endomitotic cell cycle.     

Impaired megakaryocytopoiesis in type 2B von Willebrand disease with severe thrombocytopenia

In type 2B von Willebrand disease, there is spontaneous binding of mutated von Willebrand factor (VWF) multimers to platelets. Here we report a family in which severe thrombocytopenia may also be linked to abnormal megakaryocytopoiesis. A heterozygous mutation in the VWF A1 domain gave a R1308P substitution in an interactive site for glycoprotein Ibalpha (GPIbalpha). Electron microscopy showed clusters of platelets in close contact. Binding of antibodies to the GPIbalpha N-terminal domain was decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB), GPIbalpha, alphaIIb, and beta3 were normally present. Proteins involved in Ca(2+) homeostasis were analyzed by quantitating platelet mRNA or by WB. Plasma membrane Ca(2+) ATPase (PMCA)-4b and type III inositol trisphosphate receptor (InsP(3)-R3) were selectively increased. The presence of degradation products of polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) suggested ongoing caspase-3 activity. These were findings typical of immature normal megakaryocytes cultured from peripheral blood CD34(+) cells with TPO. Significantly, megakaryocytes from the patients in culture produced self-associated and interwoven proplatelets. Immunolocalization showed VWF not only associated with platelets, but already on the megakaryocyte surface and within internal channels. In this family, type 2B VWD is clearly associated with abnormal platelet production.     

Thrombospondin-1 stimulates platelet aggregation by blocking the antithrombotic activity of nitric oxide/cGMP signaling

Platelet alpha-granules constitute the major rapidly releasable reservoir of thrombospondin-1 in higher animals. Although some fragments and peptides derived from thrombospondin-1 stimulate or inhibit platelet aggregation, its physiologic function in platelets has remained elusive. We now show that endogenous thrombospondin-1 is necessary for platelet aggregation in vitro in the presence of physiologic levels of nitric oxide (NO). Exogenous NO or elevation of cGMP delays thrombin-induced platelet aggregation under high shear and static conditions, and exogenous thrombospondin-1 reverses this delay. Thrombospondin-1-null murine platelets fail to aggregate in response to thrombin in the presence of exogenous NO or 8Br-cGMP. At physiologic concentrations of the NO synthase substrate arginine, thrombospondin-1-null platelets have elevated basal cGMP. Ligation of CD36 or CD47 is sufficient to block NO-induced cGMP accumulation and mimic the effect of thrombospondin-1 on aggregation. Exogenous thrombospondin-1 also reverses the suppression by NO of alphaIIb/beta3 integrin-mediated platelet adhesion on immobilized fibrinogen, mediated in part by increased GTP loading of Rap1. Thrombospondin-1 also inhibits cGMP-mediated activation of cGMP-dependent protein kinase and thereby prevents phosphorylation of VASP. Thus, release of thrombospondin-1 from alpha-granules during activation provides positive feedback to promote efficient platelet aggregation and adhesion by overcoming the antithrombotic activity of physiologic NO.     

Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation

The exocytic pathway provides a physical route through which newly synthesized secretory and membrane proteins are deployed to the eukaryote cell surface. For newly synthesized alpha1-antitrypsin (AAT), the modification of its asparagine-linked oligosaccharides by a slow-acting mannosidase partitions the misfolded monomer into the proteasomal degradation pathway. Herein, we asked whether, and how, modification by endoplasmic reticulum mannosidase I (ERManI) contributes to the preferential selection of the misfolded AAT monomer for proteasomal degradation. Transiently expressed mutant and WT AAT variants underwent rapid destabilization in response to an artificially elevated ERManI concentration in the murine hepatoma cell line, Hepa1a. Based on the mannosidase- and lactacystin-sensitive properties of intracellular turnover, a stochastic model is proposed in which the delayed onset of the glycan modification, relative to the duration of nonnative protein structure, coordinates the preferential degradation of the misfolded monomer and spares the native molecule from destruction. Newly synthesized endogenous transferrin underwent degradation in response to an elevated concentration of ERManI, whereas the nonglycosylated secretory glycoprotein albumin was not affected. Taken together, these findings indicate that efficient conformational maturation might function as the initial quality control standard for a broad population of glycoproteins.     

Unusual phenotypic alteration of beta amyloid precursor protein (betaAPP) maturation by a new Val-715 --> Met betaAPP-770 mutation responsible for probable early-onset Alzheimer's disease

We have identified a novel beta amyloid precursor protein (betaAPP) mutation (V715M-betaAPP770) that cosegregates with early-onset Alzheimer's disease (AD) in a pedigree. Unlike other familial AD-linked betaAPP mutations reported to date, overexpression of V715M-betaAPP in human HEK293 cells and murine neurons reduces total Abeta production and increases the recovery of the physiologically secreted product, APPalpha. V715M-betaAPP significantly reduces Abeta40 secretion without affecting Abeta42 production in HEK293 cells. However, a marked increase in N-terminally truncated Abeta ending at position 42 (x-42Abeta) is observed, whereas its counterpart x-40Abeta is not affected. These results suggest that, in some cases, familial AD may be associated with a reduction in the overall production of Abeta but may be caused by increased production of truncated forms of Abeta ending at the 42 position.     

Modulation of megakaryocytopoiesis by human basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) may act to modulate hematopoiesis in addition to its effects on mesenchymal cells. We studied the effects of bFGF on human and murine primary marrow megakaryocytes. bFGF modestly enhanced the size of the human megakaryocyte colony-forming unit (CFU-MK) and cell numbers per colony, in combination with interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF). Adhesion of human megakaryocytes to bone marrow (BM) stromal fibroblasts was enhanced when either stromal fibroblasts or megakaryocytes were treated with bFGF. This resulted in significantly increased proliferation of megakaryocytes. In addition, bFGF augmented secretion of the cytokines tumor necrosis factor alpha and IL-6 by human primary BM megakaryocytes. Immature murine megakaryocytes showed a significant growth response to bFGF as measured by the single cell growth assay. This effect was abrogated by specific antibodies for bFGF and combination of anti-IL-6 and anti-IL-1 beta antibodies. bFGF has no effect on murine CFU-MK formation, but significantly potentiated CFU-MK formation in the presence of IL-3 or GM-CSF. These results indicate that the effect of bFGF on various megakaryocyte populations is different and that bFGF may affect megakaryocytopoiesis via modulation of megakaryocyte-stromal interactions and via augmentation of cytokine secretion from megakaryocytes.     

A single viral protein HCMV US2 affects antigen presentation and intracellular iron homeostasis by degradation of classical HLA class I and HFE molecules

HFE is a nonclassical class I molecule that associates with beta 2-microglobulin (beta 2m) and with the transferrin receptor. HFE accumulates in transferrin-containing endosomes, and its overexpression in human cell lines correlates with decreased transferrin receptor (TFR)-mediated iron uptake and decreased intracellular iron pools. A mutation that interferes with proper folding and assembly of HFE complexes results in a severe iron-overload disease hereditary hemochromatosis. We previously suggested that viruses could also interfere with iron metabolism through the production of proteins that inactivate HFE, similarly to classical class I proteins. In particular, we demonstrated in a transient expression system that human cytomegalovirus (HCMV) US2 targeted HFE for proteasomal degradation. Here we demonstrate that the stable expression of HCMV US2 in HEK 293 cells constitutively expressing HFE leads to loss of HFE expression both intracellularly and on the cell surface, and the significant reduction of classical class I expression. Both HFE and classical class I molecules are targeted to degradation via a similar pathway. This HCMV US2-mediated degradation of HFE leads to increased intracellular iron pools as indicated by reduced synthesis of TfR and increased ferritin synthesis. Whether this interference with regulation of iron metabolism potentiates viral replication and/or promotes damage of HCMV-infected tissues remains to be determined. Nevertheless, the deleterious effect of US2 on the expression of HFE and classical class I major histo-compatibility complexes (MHC) provides HCMV with an efficient tool for altering cellular metabolic functions, as well as supporting the escape of virus-infected cells from cytotoxic T lymphocyte (CTL)-mediated immune responses.