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G Krystal

Publications and source records attributed to G Krystal.

At least 55 records · Page 3Linked to original sources
PubMed1996

Interleukin-3 (IL-3) inhibits erythropoietin-induced differentiation in Ba/F3 cells via the IL-3 receptor alpha subunit.

Introduction of erythropoietin receptors (EpoRs) into the interleukin-3 (IL-3)-dependent murine hemopoietic cell line, Ba/F3, enables these cells to not only proliferate, after an initial lag in G1, but also to increase beta-globin mRNA levels in response to erythropoietin (Epo). With IL-3 and Epo costimulation, IL-3-induced signaling appears to be dominant since no increase in beta-globin mRNA occurs. Differentiation and proliferation signals may be uncoupled since EpoRs lacking all eight intracellular tyrosines were compromised in proliferative signaling but retained erythroid differentiation ability. Intriguingly, a chimeric receptor of the extracellular domain of the EpoR and the transmembrane and intracellular domains of IL-3RbetaIL-3 chain (EpoR/IL-3RbetaIL-3) was capable of Epo-induced proliferative and differentiating signaling, suggesting either the existence of a second EpoR subunit responsible for differentiation or that the alpha subunit of the IL-3 receptor (IL-3R) prevents it. Arguing against the former, a truncated EpoR lacking an intracellular domain was incapable of promoting proliferation or differentiation. An EpoR/IL-3Ralpha chimera, in contrast, was capable of transmitting a weak Epo-induced proliferative signal but failed to stimulate accumulation of beta-globin mRNA. Most significantly, coexpression of the EpoR/IL-3Ralpha chimera with either EpoR/IL-3Rbeta or wild-type EpoRs suppressed Epo-induced beta-globin mRNA accumulation. Taken together, these results suggest an active role for the IL-3Ralpha subunit in inhibiting EpoR-specific differentiating signals.

Animals↗
PubMed1996

Cloning and characterization of human SHIP, the 145-kD inositol 5-phosphatase that associates with SHC after cytokine stimulation.

We recently cloned and sequenced a cDNA encoding a 145-kD protein from the murine hematopoietic cell line B6SUtA, that becomes tyrosine phosphorylated and associated with Shc after cytokine stimulation. Based on its domains and enzymatic activity, we named this protein SHIP for SH2-containing inositol phosphatase (Damen et al, Proc Natl Acad Sci USA 93:1689, 1996). We describe here the cloning of the human homologue of murine SHIP (mSHIP) from a human megakaryocytic cell line (MO7e) lambda gt11 cDNA library using two nonoverlapping mSHIP cDNA fragments as probes. Northern blot analysis suggests that human SHIP (hSHIP) is expressed as a 5.3-kb mRNA in human bone marrow and a wide variety of other tissues. Sequence analysis of this cDNA predicts a protein of 1188 amino acids exhibiting 87.2% overall sequence identity with mSHIP. Contained within the defined open reading frame is an N-terminal, group l src homology 2 (SH2) domain; three NXXY motifs that, if phosphorylated, could be bound by phosphotyrosine binding (PTB) domains; a C-terminal proline-rich region; and two centrally located inositol polyphosphate 5-phosphatase motifs. Fluorescence in situ hybridization, using the full-length hSHIP cDNA as a probe, mapped hSHIP to the long arm of chromosome 2 at the border between 2q36 and 2q37.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Contribution of both STAT and SRF/TCF to c-fos promoter activation by granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that has been shown to support call proliferation in murine fibroblasts engineered to stably express both chains of the human GM-CSF receptor (NIH-GMR). Because the proto-oncogene c-fos is believed to provide a link between short-term signals elicited at the membrane and long-term cellular response, we chose to study the mechanism of GM-CSF-dependent cell regulation using c-fos promoter activity as a molecular marker in both NIH-GMR transfectants and in the CD34+ cell line TF-1. The importance of c-fos and related AP-1 activity in GM-CSF signalling was suggested by a tight correlation between GM-CSF-dependent activation of the c-fos promoter and cell proliferation and by the inhibitory effect of a trans-dominant c-fos mutant on cell growth. To evaluate the contribution of the serum response factor (SRF) associated with the ternary complex factor (TCF) and of STAT proteins to c-fos promoter activation in response to GM-CSF, the SRF binding site (SRE) and/or the STAT binding site (SIE) were inactivated. In serum-free medium, both SRE and SIE are essential to c-fos promoter activation by GM-CSF in NIH-GMR transfectants and in TF-1 cells. No response to GM-CSF was observed when both sites were mutated. The nature of the STAT family member was further investigated by Wester blots and DNA retardation assays using an SIE probe. Our data indicate that GM-CSF induced DNA binding of both STAT1 and STAT3 in NIH-GMR and mainly of STAT3 in TF-1 cells. STAT5 tyrosine phosphorylation was also observed in TF-1 cells. Finally, expression of a dominant negative MAPK mutant, ERK192A, resulted in a decrease of both SRE- and SIE-dependent activation of c-fos promoter by GM-CSF, suggesting that STAT1/3 are regulated not only by tyrosine kinases, but also partially by MAPK.

3T3 Cells↗
PubMed1996

Negative signaling in B lymphocytes induces tyrosine phosphorylation of the 145-kDa inositol polyphosphate 5-phosphatase, SHIP.

Stimulation of the B cell Ag receptor (BCR) has been reported to induce tyrosine phosphorylation of a 145-kDa protein and its association with the adapter protein Shc. We have identified this protein as the novel inositol polyphosphate 5-phosphatase (SHIP). Further analysis revealed that both maximal phosphorylation of SHIP and its association with Shc require co-clustering with the Fc receptor for IgG (Fc gamma RII) rather than stimulation of the BCR alone. Since co-clustering of the BCR and Fc gamma RII also down-regulates proliferation induced by Ag receptor stimulation, we hypothesize that tyrosine phosphorylation of SHIP and its association with Shc contribute to negative signaling through effects on inositol and phosphatidylinositol metabolism.

Animals↗
PubMed1996

Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis.

Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation.

Animals↗
PubMed1996

The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase.

A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain, two consensus sequences that are targets for phosphotyrosine binding domains, a proline-rich region, and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties, we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Cardiac hypertrophy in the Dahl rat is associated with increased tyrosine phosphorylation of several cytosolic proteins, including a 120 kDa protein.

Because of the well established role that tyrosine phosphorylation (tyr phos) plays in growth factor signalling and regulating cell growth, we hypothesized that cardiac hypertrophy might be associated with altered tyr phos of certain cellular proteins in the heart. Furthermore, we hypothesized that angiotensin II (ang II), a putative growth factor for cardiac cells, might be useful as a probe to highlight any differences in intracellular signalling between normal and hypertrophied hearts. The heart and, for comparison, skeletal muscle, from Dahl S rats, which are predisposed to cardiac hypertrophy, and Dahl R rats, which are not, were examined. Antiphosphotyrosine immunoprecipitation and immunoblotting of heart cell extracts revealed the presence of a constitutively tyr phos 120 kDa cytosolic protein. Hearts from Dahl R rats on a high salt diet displayed a smaller amount of constitutive tyr phos of this protein. In the hearts of both Dahl R and S rats maintained on low salt diets there was little evidence of constitutive tyr phos of this protein. Ang II induced tyr phos of this protein in Dahl S rats on a low salt diet and Dahl R rats on a high salt diet, both of which show mild cardiac hypertrophy. In contrast, the markedly hypertrophied ventricle showed a minimal response to Ang II. Thus the severity of cardiac hypertrophy correlated directly with the tyr phos level of this protein. In an attempt to identify this protein, immunoblotting was carried out with antibodies to the signal transducing proteins rasGAP, JAK2 iNOS, p125FAK, and the Src substrate, pp120, but all proved negative. Ang II also stimulated an increase in tyr phos of proteins with apparent molecular masses of 42, 55, and 69 to 85 kDa in hearts from Dahl S rats on high salt diet. By comparison, there was no 120 kDa tyr phos protein in skeletal muscle even in response to Ang II. Silver stained sodium dodecyl sulfate gels demonstrated that this 120 kDa tyr phos protein is present in substantial amounts in the ventricles of rats fed high salt diets. Thus cardiac hypertrophy is characterized by an abundant 120 kDa cytosolic tyr phos protein, which is apparent with Ang II stimulation in milder degrees of cardiac hypertrophy, and is most likely an as yet uncharacterized protein.

Angiotensin II↗
PubMed1996

Erythropoietin therapy in neonates at risk of having bronchopulmonary dysplasia and requiring multiple transfusions.

OBJECTIVES: To determine whether treatment with recombinant human erythropoietin (r-HuEPO) reduces transfusion requirements in premature neonates at risk of having bronchopulmonary dysplasia and requiring multiple transfusions. STUDY DESIGN: A double-blind, randomized, controlled trial. SUBJECTS: Fifty-five infants appropriate in weight for gestational age (less than 1250 gm birth weight) who, at 10 days of age, were predicted to have a greater than 75% probability of having bronchopulmonary dysplasia. This criterion had previously been shown to identify infants requiring multiple transfusions. Twenty-seven infants were randomly assigned to receive r-HuEPO therapy and 28 to a control group. r-HuEPO was administered in a dosage of 20 U/kg body weight, subcutaneously, three times a week for 6 weeks. Control infants received sham treatment. RESULTS: Infants treated with r-HuEPO required significantly fewer transfusions than control infants during their entire hospital stay (mean 3.48 +/- 1.58 vs 5.68 +/- 2.30; p = 0.0001) and had a higher mean reticulocyte count (p < or = 0.0005) and a higher mean hemoglobin concentration (p < or = 0.005) during the treatment period. At follow-up, 4 months after term, there were no significant differences between the groups in mean reticulocyte count (p = 0.86) or mean hemoglobin concentration (p = 0.56). However, two infants in each group had low serum ferritin values indicative of depleted iron stores. CONCLUSIONS: Treatment with r-HuEPO effectively stimulated erythropoiesis in premature infants at high risk of having bronchopulmonary dysplasia and requiring multiple transfusions; the result was a reduction in transfusion requirements. This treatment, together with other strategies to reduce the need for transfusions, is appropriate in this population. Unrelated to r-HuEPO treatment, these infants may be at risk of having iron deficiency later in infancy.

Bronchopulmonary Dysplasia↗
PubMed1996

The expression of stem cell factor and its receptor, c-kit in human endometrium and placental tissues during pregnancy.

Stem cell factor (SCF) and its receptor Kit regulate the proliferation and survival of early hematopoietic cell types as well as germ cells and melanocytes. As SCF augments the effects of several hematopoietic growth factors that are produced in reproductive tissues during pregnancy and also plays an important role in cell migration, proliferation, and survival, we studied the expression and localization of this receptor/ligand in human endometrial and placental tissues. Kit was detected by Western blot analysis in early decidual and placental tissues (8-19 weeks gestation) and in term placenta. Immunohistochemistry localized Kit mainly in trophoblast and to a lesser extent in scattered cells in the placental villous core and decidual stroma. Ribonuclease protection assay showed that SCF messenger ribonucleic acid (mRNA) expression increased 3-fold in decidua from early pregnancy compared to proliferative and secretory endometrium (P < 0.05). Placental tissues expressed 4- to 8-fold higher levels of SCF mRNA compared to decidus (P < 0.05). Isolated placental villous core expressed 7-fold higher levels of SCF mRNA than did trophoblast (P < 0.05). Thus, SCF and its receptor Kit are expressed in human endometrium and placental tissues during pregnancy, and the pattern of receptor/ligand expression suggests that endometrial and placental villous core SCF may have a paracrine effect on trophoblast through the receptor Kit.

Base Sequence↗
PubMed1996

Early events in erythropoietin-induced signaling.

The cloning of the erythropoietin receptor (EpoR) in 1989 has allowed very rapid progress in our understanding of the early intracellular events that may be triggered by erythropoietin (Epo) in erythroid progenitor cells. From studies carried out primarily with cell lines expressing exogenous wild-type and mutant EpoRs, it appears that the activated EpoR is capable of triggering many of the same cascades that are utilized by receptors possessing endogenous tyrosine kinase domains. The major challenge over the next decade lies in seeing if these same signaling pathways are also utilized by normal Epo-responsive erythroid progenitors, discriminating between proliferation and differentiation inducing events in these cells, and determining whether various hematologic disorders can be attributed to aberrations in these signaling pathways.

Animals↗
PubMed1995

Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation.

While previous studies with truncated erythropoietin receptors (EpRs) have suggested that the tyrosine phosphorylation of the EpR does not play a role in Ep-induced proliferation, we have found, using a more subtle, full length EpR mutant, designated Null, in which all eight of the intracellular tyrosines have been substituted with phenylalanine residues, that Null cells require substantially more Ep than wild-type cells in order to proliferate as efficiently. A comparison of Ep-induced proliferation with Ep-induced tyrosine phosphorylation patterns, using wild-type and Null EpR-expressing cells, revealed that Stat5 tyrosine phosphorylation and activation correlated directly with proliferation. Moreover, studies with a Y343F EpR point mutant and various EpR deletion mutants revealed that both Ep-induced proliferation and Stat5 activation were mediated primarily through Y343, but that other tyrosines within the EpR could activate Stat5 in its absence.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Phosphorylation of tyrosine 503 in the erythropoietin receptor (EpR) is essential for binding the P85 subunit of phosphatidylinositol (PI) 3-kinase and for EpR-associated PI 3-kinase activity.

We recently reported that phosphatidylinositol (PI) 3-kinase becomes associated with the activated erythropoietin receptor (EpR), most likely through the Src homology 2 (SH2) domains within the p85 subunit of PI-3 kinase and one or more phosphorylated tyrosines within the EpR. We have now investigated this interaction in more detail and have found, based on both blotting studies with glutathione S-transferase-p85-SH2 fusion proteins and binding of these fusion proteins to SDS-denatured EpRs, that this binding is direct. Moreover, both in vitro competition studies, involving phosphorylated peptides corresponding to the amino acid sequences flanking the eight tyrosines within the intracellular domain of the EpR, and in vivo studies with mutant EpRs bearing tyrosine to phenylalanine substitutions, indicate that phosphorylation of Tyr503 within the EpR is essential for the binding of PI 3-kinase. The presence of PI 3-kinase activity in EpR immunoprecipitates from DA-3 cells infected with wild-type but not Y503F EpRs confirms this finding. Our results demonstrate that the SH2 domains of p85 can bind, in addition to their well established Tyr-Met/Val-X-Met consensus binding sequence, a Tyr-Val-Ala-Cys motif that is present in the EpR. A comparison of erythropoietin-induced tyrosine phosphorylations and proliferation of wild-type and Y503F EpR-infected DA-3 cells revealed no differences. However, the PI-3 kinase inhibitor, wortmannin, markedly inhibited the erythropoietin-induced proliferation of both cell types, suggesting that PI 3-kinase is activated in Y503F EpR expressing cells. This was confirmed by carrying out PI 3-kinase assays with anti-phosphotyrosine immunoprecipitates from erythropoietin-stimulated Y503F EpR-infected DA-3 cells and suggested that PI 3-kinase has a role in regulating erythropoietin-induced proliferation, but at a site distinct from the EpR.

Amino Acid Sequence↗
PubMed1995

Identification and characterization of an interleukin-3 receptor-associated 110-kDa serine/threonine kinase.

We recently reported that interleukin-3 (IL-3) stimulation of the murine IL-3-responsive cell line, B6SUtA1, results in the rapid phosphorylation of the beta subunit of the IL-3 receptor (IL-3R), not only on tyrosine residues but on serine/threonine (Ser/Thr) residues as well. Since this occurred even at 4 degrees C, it suggested that a Ser/Thr-specific kinase might be closely associated with the IL-3R. To test this possibility, IL-3R complexes were isolated with anti-IL-3R (alpha IL-3R) antibodies, and in vitro phosphorylation studies were undertaken. These revealed the presence of a 110-kDa protein that was heavily phosphorylated in vitro on serine and threonine residues and that bound selectively to gamma-ATP-Sepharose beads. Moreover, this protein, which was not the 110-kDa subunit of phosphatidylinositol 3-kinase, was tyrosine phosphorylated in response to IL-3 and was specifically labeled in vitro with azido-[32P]ATP. These data, together with in vitro kinase inhibitor studies, suggest that an as yet uncharacterized H7- and staurosporine-sensitive 110-kDa Ser/Thr kinase may be constitutively associated with the IL-3R and activated following IL-3 stimulation. A comparison of IL-3R and erythropoietin receptor complexes suggests that this 110-kDa protein may be preferentially associated with the IL-3R.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Upstream organization of and multiple transcripts from the human folylpoly-gamma-glutamate synthetase gene.

Folylpoly-gamma-glutamate synthetase (FPGS) is essential for the survival of proliferating mammalian cells and central to the action of all "classical" folate antimetabolites. We report the isolation of cDNAs corresponding to the 5' ends of FPGS mRNA from both human and hamster cells which include a start codon upstream of and in-frame with the AUG in the previously reported FPGS open reading frame. The predicted hamster and human amino-terminal extension peptides have features consistent with a mitochondrial targeting sequence. Ribonuclease protection and 5'-rapid amplification of cDNA ends assays indicated multiple transcriptional start sites consistent with the sequence of the promoter region of this gene, which was highly GC-rich and did not contain TATA or CCAAT elements. These start sites would generate two classes of transcripts, one including the upstream AUG and one in which only the downstream AUG would be available for translation initiation. Transfection of the full length human cDNA into cells lacking FPGS restored their ability to grow in the absence of glycine, a product of mitochondrial folate metabolism, as well as of thymidine and purines. Therefore, we propose that the mitochondrial and cytosolic forms of FPGS are derived from the same gene, arising from the use of the two different translation initiation codons, and that the translation products differ by the presence of a 42-residue amino-terminal mitochondrial leader peptide.

Amino Acid Sequence↗
PubMed1995

Erythropoietin and interleukin-3 induce distinct events in erythropoietin receptor-expressing BA/F3 cells.

To compare the signal transduction pathways used by erythropoietin (Ep) and interleukin-3 (IL-3), the cDNA for the murine erythropoietin receptor (EpR) was introduced into the IL-3-responsive cell lines Ba/F3 and DA-3 using retrovirally mediated gene transfer. After selection in G418 and IL-3, clones expressing comparable levels of cell surface EpR were identified using biotinylated Ep and flow cytometry. A comparison of the effects of Ep and IL-3 on these cells showed that most EpR+ Ba/F3 clones, when first exposed to Ep, dramatically increased their levels of beta-globin mRNA. The kinetics of appearance of this message after exposure to Ep varied considerably from clone to clone, with some clones showing a marked increase in beta-globin mRNA within 1 hour, while others required several days before an increase was observed. Interestingly, not only was this increase not seen with IL-3, but IL-3 prevented the Ep-induced appearance of beta-globin message. On the other hand, none of the EpR+ DA-3 cell clones tested increased their levels of beta-globin mRNA in response to Ep. While the EpR+ DA-3 clones showed identical proliferative responses to IL-3 and Ep, most EpR+ Ba/F3 clones displayed a marked, albeit transient, proliferative lag when first exposed to Ep. This was manifested as both an increased doubling time in liquid culture and a decreased colony size in methylcellulose. Plating efficiencies of EpR+ Ba/F3 cells in methylcellulose, however, were identical in response to IL-3 and Ep, suggesting that the Ep-induced lag in proliferation reflected a growth delay of the entire population of cells to Ep rather than a selection of an Ep-responsive subpopulation. Flow cytometric analysis established that this growth delay was due to a lengthening of the first G1 period after exposure to Ep. Interestingly, this Ep-induced delay in entry into the S phase was not detected in cells stimulated with both Ep and IL-3 nor in EpR+ Ba/F3 cell clones that did not show an increase in beta-globin mRNA in response to Ep. Thymidine-induced growth arrest, however, showed that delaying entry into S phase alone was not sufficient to stimulate beta-globin mRNA in the absence of Ep. Further studies established that the Ep-induced increase in beta-globin mRNA could be inhibited by the tyrosine kinase inhibitor genistein and the protein kinase C inhibitor Compound 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differentiation↗
PubMed1995

G1 cell cycle arrest and apoptosis are induced in NIH 3T3 cells by KN-93, an inhibitor of CaMK-II (the multifunctional Ca2+/CaM kinase).

CaMK-II (the type II multifunctional Ca2+/calmodulin kinase) is a ubiquitous serine/threonine protein kinase that is activated by Ca2+ and calmodulin (CaM) and has been implicated in cell cycle control. NIH 3T3 fibroblast cytosolic extracts contain CaMK-II enzymatic activity and two major Ca2+/CaM-dependent phosphoproteins of M(r) 55,000 and 115,000. Reverse transcription-PCR indicates that the gamma B and gamma C isozymes of CaMK-II are predominately expressed. KN-93, a novel membrane-permeant synthetic inhibitor of purified neuronal CaMK-II, inhibits serum-induced fibroblast cell growth in a comparable dose-dependent fashion to its inhibition of CaMK-II activity. After 2 days of KN-93 treatment, 95% of cells are arrested in G1. G1 arrest is reversible; 1 day after KN-93 release, a peak of cells had progressed into S and G2-M. KN-92, a similar but inactive compound, had no effect on CaMK-II activity or cell growth. KN-93 also blocked cell growth stimulated by basic fibroblast growth factor, platelet-derived growth factor-BB, epidermal growth factor, and insulin-like growth factor-1. After 3 days of KN-93-induced G1 arrest, cell size and viability decreased and DNA fragmented, indicating apoptosis. These data suggest that CaMK-II is necessary for cell cycle progression through G1 and operates at a site common to the transduction of signals from growth and/or survival factors.

3T3 Cells↗
PubMed1994

Transforming growth factor beta 1 is an inducer of erythroid differentiation.

Normal human bone marrow cells, highly enriched for burst-forming units-erythroid (BFU-E), were cultured in serum-free medium, in the presence and absence of various factors, to investigate the mechanisms involved in regulating erythroid differentiation. In cultures containing interleukin 3 (IL-3), Steel factor (SF), and erythropoietin (Ep), benzidine-positive erythroblasts first became detectable on day 6. Their numbers then rapidly increased until, by day 16, > 99% of the cells, which were 20,000-fold amplified over input numbers, were benzidine-positive. It is interesting to note that omission of either SF or Ep from this assay markedly enhanced the rate of differentiation and reduced total cell numbers, whereas omission of IL-3 had no effect on the rate of differentiation and only slightly reduced cell numbers. Of various agents tested, the most potent erythroid differentiation inducer (and inhibitor of cell proliferation) was found to be transforming growth factor beta 1 (TGF-beta 1). This cytokine stimulated both the rapid appearance of hemoglobin-positive cells and an early cessation of cell proliferation. Using fluorescently tagged antibodies to glycophorin A and fluorescence-activated cell sorter (FACS) analysis, this phenomenon was shown to be due to an early induction of erythroid differentiation rather than an aberrant production of hemoglobin. Methylcellulose assays indicated that the well-documented reduction of BFU-E colony numbers observed with TGF-beta 1 may actually be due to a TGF-beta 1-induced "conversion" of BFU-E into colony-forming units-erythroid (CFU-E). Thus, in vivo, TGF-beta 1 might serve, in part, to decrease the number of mature erythrocytes by stimulating BFU-E to skip a number of cell divisions and differentiate early.

Animals↗