Glycophorin A dimerization and band 3 interaction during erythroid membrane biogenesis: in vivo studies in human glycophorin A transgenic mice.

Band 3 and glycophorin A (GPA) are the 2 most abundant integral proteins in the human erythrocyte membrane. Earlier studies suggested that the 2 proteins may associate not only in the mature erythrocyte membrane, but also during their posttranslational processing and intracellular trafficking. The purpose of this study was to directly examine the GPA-band 3 interaction in vivo and determine the nature of this association during erythroid membrane biogenesis. Transgenic mice were generated expressing the human glycophorin A gene and were used to examine how the induction of human GPA expression affected the levels of murine GPA and band 3 expression in the red cell membrane. Murine GPA expression was reduced in erythrocytes expressing human GPA, whereas the level of band 3 expression remained constant, implying a tight coupling of band 3 and GPA expression in the membrane of mature red cells. In vivo GPA dimerization was not modulated solely by the GPA transmembrane motif, but the distance between this motif and the basic residues on the cytoplasmic side of the transmembrane domain may also be important. In addition, GPA monomers with varying degrees of glycosylation dimerized, providing clear evidence that carbohydrate structures on the extracellular domain do not affect dimerization. The association between the multiple transmembrane-spanning protein, band 3, and the single transmembrane-spanning sialoglycoprotein, GPA, may serve as a model for interactions of other multi-pass and single-pass polypeptides during membrane biogenesis.

Characterization of multiple isoforms of protein 4.1R expressed during erythroid terminal differentiation.

In erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4. 1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5' end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2' and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2' and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.

Expression and function of integrins on hematopoietic progenitor cells.

The growth and differentiation of hematopoietic stem cells are highly dependent on regulatory molecules produced by stromal cells of the marrow environment. Evidence has accumulated over the past years which shows that adhesive receptors on hematopoietic cells and their ligands on stromal cells and extracellular matrix play a crucial role in these interactions. Integrins of the beta 1 family, mostly VLA-4 and VLA-5, are the best characterized and have been identified on committed progenitor cells of the hematopoietic hierarchy as well as on more primitive stem cells defined by their long-term repopulating capacity assayed in vitro as well as in vivo. Functional assays demonstrate that most progenitor cells efficiently bind to ECM components through beta 1 integrins and lineage- and maturation stage-specific differences have been described. Evidence exists on the direct control of late erythroid differentiation by VLA-4, but whether or not the triggering of beta 1 integrins is critically required for hematopoietic stem cell functioning at more immature steps is unclear. Many other integrin and non-integrin receptors involved in adhesive interactions are expressed on hematopoietic progenitor cells and tightly regulated during differentiation but their function is still controversial. Our main purpose in this review is to describe recent advances in the knowledge of integrin expression on hematopoietic progenitor cells in both mouse and man. The emerging importance of the synergy between integrins and cytokine signalling pathways in the regulation of hematopoietic differentiation will also be discussed.

Nerve growth factor is involved in the supportive effect by bone marrow--derived stromal cells of the factor-dependent human cell line UT-7.

We previously demonstrated that murine MS-5 and SI/SI4 cell lines induce the proliferation of human factor-dependent UT-7 cells in the absence of normally required human cytokines and also stimulate the differentiation of CD34+/CD38-LTC-ICs. We report in this study that the effect of MS-5 cells on UT-7 cells can be completely explained by the synergistic action of nerve growth factor (NGF) and stem cell factor (SCF) produced by these murine stromal cells. Purified murine NGF was able to support short-term clone formation and long-term growth of UT-7 cells in suspension cultures as efficiently as rhu-granulocyte-macrophage colony-stimulating factor. NGF action was mediated through the TrkA receptor, in which messenger RNA (mRNA) was easily detected in UT-7 cells by Northern blot. MS-5 cells strongly expressed NGF mRNA in Northern blot, and direct implication of MS-5-derived NGF in the induction of UT-7 cells proliferation was demonstrated in inhibition assays with an anti-NGF monoclonal antibody (MoAb) that neutralized by 84% +/- 4.1% (n = 5) UT-7 clone formation. However, NGF did not act alone, and several arguments demonstrated the synergistic action of MS-5-derived SCF: (1) an anti-c-kit partially inhibited UT-7 cells clone formation in coculture assays, (2) SCF and NGF synergized in an H3-TdR incorporation assay, and (3) the stimulatory effect of 10x-concentrated MS-5 supernatant was completely inhibited by an anti-c-kit but not by an anti-NGF, and levels of soluble NGF (1.2 ng/mL) detected by enzyme-linked immunosorbent assay in 10x supernatant of MS-5 cells cultures were below the biologically active concentrations. In contrast, although MS-5 cells also promoted the differentiation of very primitive CD34+/CD38- human stem cells both in colony assays and long-term cultures, we could not incriminate MS-5-derived NGF in the observed effect: an anti-NGF MoAb did not inhibit the synergistic effect of MS-5 cells in colony assays or long-term cultures nor did soluble muNGF duplicate MS-5 effect and survival of CD34+/CD38- clonogenic progenitor cells promoted by MS-5 was unaffected by an anti-NGF and was not induced by soluble NGF alone or combined with SCF. In contrast, NGF in synergy with SCF supported the short-term maintenance of high numbers of CD34+/CD38+ mature erythroid progenitors probably through an indirect mechanism implying macrophages. These results suggest that NGF, in which the primary target cells are outside the hematopoietic system, is present in the marrow environment and might act at some steps of hematopoietic stem cell development. These results also underline that the response of cell lines and normal stem cells to stromal cells is mediated by different pathways.

Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of CD34++/CD38- long-term culture-initiating cells.

Very primitive human hematopoietic progenitor cells are identified indirectly by their ability to give rise to clonogenic progenitors in the presence of either human or murine stromal cells. These long-term culture-initiating cell (LTC-IC) assays are usually performed in the presence of hydrocortisone based on the initial observation that hydrocortisone was required for prolonged hematopoiesis in standard long-term bone marrow cultures. In this report, we investigated the role of hydrocortisone in LTC-IC assays initiated with CD34++/CD38- cells seeded onto either human bone marrow LTC-derived adherent cells or a murine marrow-derived stromal cell line, MS-5. It was found that weekly addition of hydrocortisone to the cultures reduced the frequency of LTC-IC (from 1/5 to 1/20) calculated from limiting dilution experiments and also reduced fivefold to 10-fold the number of their progeny clonogenic cells detected after 4 to 5 weeks. In contrast, the frequency and differentiative potential of CD34++/CD38- grown in the presence of human marrow feeders was unaltered by the addition of glucocorticoids. Data are consistent with the hypothesis that hydrocortisone inhibited LTC-IC differentiation by downregulating the expression of a synergistic factor produced by MS-5 cells. (1) In the absence of hydrocortisone, the number of clonogenic progenitors generated by LTC-IC was much higher in cultures seeded on MS-5 than in cultures seeded on human marrow adherent cells, which was also true when cytokines were added to the cocultures. However, based on the phenotype of the colonies, progenitors produced in MS-5 cocultures were more mature than those generated on human marrow adherent cells. (2) Hydrocortisone counteracted the stimulatory effect of recombinant human cytokines (interleukin-3, interleukin-6, and steel factor) in assays performed on MS-5 but not on human marrow feeders. (3) Hydrocortisone led to a 50% decrease in the numbers of colony-forming units-granulocyte-macrophage found in methycellulose colony assays of CD34++/CD38- cells performed in the presence of MS-5 cells. Taken together, our results indicate that hydrocortisone acts differently on a murine stromal cell line and on marrow-derived human stromal cells and may suppress the expression by MS-5 cells of an activity selectively promoting amplification of clonogenic cells derived from primitive LTC-IC.

Age-related alterations in erythroid and granulopoietic progenitors in Diamond-Blackfan anaemia.

Mechanisms involved in the erythroid failure characterizing Diamond-Blackfan anaemia (DBA) remain unidentified. The general consensus is that the defect is intrinsic to the marrow erythroid progenitor, but the target progenitor cell has not been precisely identified, and in vitro studies have revealed considerable heterogeneity between patients. In order to understand better the meaning of such a biological heterogeneity, we examined the in vitro response of erythroid progenitors CFU-E (colony-forming unit-erythroid) and BFU-E (burst-forming unit-erythroid) to erythropoietin (Epo), interleukin-3 (IL-3) and stem cell factor (SCF) in a large series of 24 patients from 1 month to over 20 years of age. Results of colony assays revealed a striking correlation between the age of the patient and the extent of the abnormalities detected in vitro. Therefore, despite profound anaemia, 80% (7/10) of the patients studied within 1 year of diagnosis had normal numbers of both CFU-E and BFU-E which exhibited a normal response to cytokines. In contrast, 12/14 patients followed up for more than 3 years had decreased numbers of erythroid progenitors, in seven cases associated with decreased colony-forming unit granulocyte-macrophage (CFU-GM). The number of CFU-E and BFU-E was not normalized even by the addition of high concentrations of combined Epo, IL-3 and SCF.(ABSTRACT TRUNCATED AT 250 WORDS)

A murine stromal cell line promotes the proliferation of the human factor-dependent leukemic cell line UT-7.

In long-term human bone marrow cultures, stromal cells of human origin are usually used on the assumption that human primitive progenitor cells do not respond to cytokines produced by stromal cells from other species. There is accumulating evidence, however, that murine stromal cells also promote maintenance and differentiation of very primitive human stem cells, which suggests the existence of novel stromal activities that cross species barriers. In this study, we show that a murine bone marrow-derived stromal cell line, MS-5, allows the proliferation of the human leukemic cell line UT-7. The long-term growth of UT-7 is usually supported only by human interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo). None of these three cytokines was involved in the observed effect, since murine GM-CSF and IL-3 do not act on human cells and MS-5 cells do not produce Epo. Soluble stem cell factor (SCF) induced UT-7 cell proliferation. However, S1/S1 mutant fibroblasts also supported UT-7 cell growth and anti-c-kit antibodies only partially abolished UT-7 cell proliferative response to MS-5 cells. These observations excluded a major role of SCF in this system. MS-5-derived growth-promoting activity was diffusible, but attempts to grow UT-7 cells in high levels of known soluble murine stromal-derived cytokines active on human cells showed no or minimal response, suggesting that MS-5's proliferative effect was not mediated by known cytokines. Finally, involvement of an autocrine loop of activation induced by MS-5 was excluded: RT-PCR analysis did not detect increased transcripts for GM-CSF, IL-3, IL-6, SCF, or Epo in UT-7 cells cocultured for 2 to 6 days with MS-5. In addition, UT-7 cell proliferation on MS-5 was not inhibited by neutralizing antibodies against the human GM-CSF receptor or the human IL-6 receptor alpha chain. Whether UT-7 cell proliferation triggered by MS-5 reflects the existence of novel stromal cytokines or results from synergistic interactions on the MS-5 cell surface between extracellular matrix proteins and cytokines will require further investigation.