CoChat
Sign Up
Cited by 45Hematopoietic Stem Cell TransplantationMesenchymal stem cell researchCytokine Signaling Pathways and Interactions

PKCδ-Induced PU.1 Phosphorylation Promotes Hematopoietic Stem Cell Differentiation to Dendritic Cells

Open access
Hamdorf, Matthias, Berger, André, Schüle, Silke, Reinhardt, Jens, Flory, Egbert·2011·Stem Cells
DOI 10.1002/stem.564
Open this paper in CoChat — free accountOpen PDF

Abstract

Human CD34+ hematopoietic stem cells (HSCs) exhibit the potential to differentiate into a variety of specialized blood cells. The distinct intracellular mechanisms that control cell fate and lineage commitment of these multipotent cells are not well defined. In this study, we investigate and modulate the signaling processes during HSC differentiation toward myeloid dendritic cells (mDCs). DC differentiation induced by the cytokines Granulocyte macrophage colony-stimulating factor (GM-CSF) and Interleukin-4 (IL-4) led to activation of the Extracellular-signal-regulated kinase (ERK), protein kinase C (PKC), and Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) but not the SAPK/c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase signaling pathways. From the activated signaling pathways the PKC isoform δ was found to phosphorylate the transcription factor PU.1, which is described as one of the key factors for myeloid HSC differentiation. On molecular level, PKCδ regulated PU.1 activity by affecting its transactivation activity, whereas its DNA binding activity remained unaffected. This was accompanied by PKCδ-induced phosphorylation of the PU.1 transactivation domain. Furthermore, treatment with PKC- and ERK1/2-specific signaling inhibitors impaired both HSC differentiation toward mDCs as well as phosphorylation-mediated transactivation activity of PU.1. Taken together, these results provide new insights into the molecular mechanisms promoting the differentiation process of HSCs toward mDCs and introduce the PKC isoform δ as critical mediator.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports that GM-CSF/IL-4-driven differentiation of human CD34+ hematopoietic stem cells into myeloid dendritic cells depends on ERK and PKCδ signaling, with PKCδ directly phosphorylating PU.1’s transactivation domain to enhance its transcriptional activity. It positions PKCδ as a critical mediator of this lineage choice.

Key findings

  • In cord blood CD34+ HSC cultures, GM-CSF/IL-4 rapidly activated ERK (peak around 5 minutes), JAK2/STAT5, and PKCδ (phosphorylation still detectable at 75 minutes), while JNK and p38 were not activated; PI3K was also not activated.
  • TPA also generated DC-like cells and activated ERK and PKCδ but not STAT5, suggesting JAK/STAT signaling is not required for DC differentiation itself; JAK inhibition reduced colony formation.
  • In vitro kinase assays showed PKCδ, but not ERK2, phosphorylated PU.1, and the PKC inhibitor Gö6983 blocked this. PU.1 DNA binding was constitutive and unchanged by TPA or GM-CSF/IL-4, whereas TPA increased PU.1 transactivation about sixfold; MEK/ERK inhibition reduced it by about 50% and PKC inhibition by up to 66%. In reporter assays, MEK/ERK inhibitors reduced PU.1 activity fourfold, PKC inhibition fivefold, ERK knockdown impaired it twofold, and PKCδ knockdown reduced it 10-fold relative to ERK knockdown.
  • ERK or PKC inhibitors blocked TPA-induced HSC differentiation toward DCs; after inhibitor removal and restimulation, cells regained DC morphology by day 10.

Methods

  • Human CD34+ cells from umbilical cord blood were expanded and then differentiated with GM-CSF/IL-4 or TPA; DC phenotype and function were assessed by flow cytometry, RT-PCR, cytokine array, and colony-forming assays.
  • Signaling was analyzed by phospho-specific immunoblot, immunoprecipitation/in vitro kinase assays with recombinant ERK2 or PKCδ, EMSA for PU.1 DNA binding, and luciferase reporters for PU.1 transactivation; siRNA knockdown in HeLa cells and pharmacological inhibitors (U0126, ERK inhibitor, Gö6983, JAK inhibitor I) were used.
  • Cell lines included HeLa, NIH 3T3, 293T, A3.01, U937, KG-1a, K-562, HOS, Alpha 1, and others.

Limitations

  • Stated: no PKCδ-specific inhibitor was available, so Gö6983 was used despite also inhibiting other PKC isoforms; PKCδ knockdown in HeLa cells supported specificity. siRNA knockdown in primary HSCs was unsuccessful due to low transfection efficiency, so inhibitor studies were used instead. The putative ERK-dependent PU.1-activating cofactor was not identified.
  • Evident: many mechanistic conclusions rely on non-hematopoietic or leukemic cell lines (HeLa, NIH 3T3, A3.01, U937) rather than primary HSCs, and no in vivo model is presented.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1002/stem.564

Cites

55 of 55 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

10 of 40 cited sources checked · 4 supported · 1 flagged · accuracy not yet measured

  • Findingown data

    JAK signaling is required for broad HSC differentiation capacity by influencing cell proliferation.

    “We found JAK signaling to be indispensable for broad HSC differentiation capacity per se by influencing proliferative properties of the cells, which is in line with earlier findings that JAK/ STAT signaling promotes proliferation and self-renewal of HSCs [42].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [42] Schuringa, 2004, Constitutive activation of STAT5A promotes human hematopoietic stem cell self-renewal and erythroid differentiation, J Exp Med source
  • Findingown data

    PKCδ phosphorylates transcription factor PU.1.

    “From the activated signaling pathways the PKC isoform d was found to phosphorylate the transcription factor PU.1”
    Quote in the paperSubstantial claim
  • Findingown data

    As many as 95% of expanded cells expressed CD34 and CD117.

    “Up to 95% of the expanded cells were positive for the stem cell markers CD34 and CD117 (Fig. 1A).”
    Quote in the paperNot a substantial claim
  • Findingown data

    After GM-CSF/IL-4 stimulation, cells developed DC-characteristic veils by day 7.

    “Until day 7, the cells developed DC-characteristic veils (Fig. 1A).”
    Quote in the paperNot a substantial claim
  • Findingown data

    PKCδ induces phosphorylation of the PU.1 transactivation domain.

    “This was accompanied by PKCd-induced phosphorylation of the PU.1 transactivation domain.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 28
  • May combine two claims. 31

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In vitro, GM-CSF plus IL-4 drive immature DC differentiation, while TNF-α and TGF-β promote maturation.

    “It has been described that Granulocyte macrophage colony-stimulating factor (GM-CSF) and Interleukin-4 (IL-4) induce the differentiation to immature DCs, whereas tumor necrosis factor alpha (TNF-a) [2] and Transforming growth factor beta TGF-b [3] stimulation supports maturation in vitro.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [2] Sallusto, 1994, Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha, J Exp Med source
    • cites [3] Geissmann, 1998, Transforming growth factor beta1, in the presence of granulocyte/macrophage colony-stimulating factor and interleukin 4, induces differentiation of human peripheral blood monocytes into dendritic Langerhans cells, J Exp Med source

      Citation [3] is attached to the claim that TGF-β supports maturation in vitro. In Results and Discussion (page unavailable), the paper instead reports that TGF-β1, with GM-CSF and IL-4, drives differentiation into Langerhans cells that remain immature; it reports activation-associated changes after TNF-α and IL-1 are added. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    p38 and JNK pathways are indispensable for erythroid development.

    “Hierarchically located in parallel, the stress-induced p38 and the JNK pathway are indicated to be indispensable for erythroid development [8,9].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [8] Nagata, 1999, Requirement of activation of JNK and p38 for environmental stress-induced erythroid differentiation and apoptosis and of inhibition of ERK for apoptosis, Blood source
    • cites [9] Uddin, 2004, Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells, Proc Natl Acad Sci USA source

      The Results report that inhibiting p38α reduced a marker of differentiation in cultured primary erythroid progenitors (36% versus 65%), and the Abstract says p38α and p38δ promote late-stage differentiation. This supports a role for p38, but not the stronger claim that it is indispensable for erythroid development. The cited paper does not establish that claim for JNK. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Myeloid progenitor differentiation to monocytes and macrophages after cytokine stimulation requires PKCα, PKCδ, PKCε, and PKCζ, while lymphoid progenitor differentiation involves PKCγ.

    “Cytokine-induced differentiation of myeloid progenitor cells toward monocytes and macrophages was described to involve activation of PKCa, PKCd, PKCe, and PKCf [11], whereas lymphoid progenitor differentiation was associated with PKCy activity [12].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [11] Redig, 2007, The protein kinase C (PKC) family of proteins in cytokine signaling in hematopoiesis, J Interferon Cytokine Res source
    • cites [12] Barouch-Bentov, 2005, Protein kinase C-theta is an early survival factor required for differentiation of effector CD8+ T cells, J Immunol source

      The citation [12] is attached to the lymphoid-progenitor and PKCγ part of the sentence; the myeloid-progenitor claim is attached to [11]. Our copy instead discusses PKCθ in antigen-stimulated CD8+ T-cell differentiation (Discussion, pp. 8–9). Searches for lymphoid progenitors and PKCγ found no passage addressing the attributed association. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Active ERK is needed for myeloid and megakaryocyte differentiation, while erythroid differentiation requires ERK to be inactive.

    “Several groups described that mitogen-activated protein kinase kinase 1 (MEK-ERK) activation is critical for myeloid and megakaryocyte differentiation [5,6], whereas inactive ERK is a prerequisite for erythroid differentiation [7].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [5] Miranda, 2005, Cytokine-induced myeloid differentiation is dependent on activation of the MEK/ERK pathway, Leuk Res source
    • cites [6] Racke, 1997, Sustained activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway is required for megakaryocytic differentiation of K562 cells, J Biol Chem source

      The Results (p. 3) show that inhibiting MEK–ERK activation blocked megakaryocytic differentiation of K562 cells. The paper does not establish the same requirement for myeloid differentiation. The erythroid statement is attached to a different citation, [7], and is not assessed here. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [7] Matsuzaki, 2000, Induction of erythroid differentiation by inhibition of Ras/ERK pathway in a friend murine leukemia cell line, Oncogene source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    ERK may regulate PU.1 activity through interacting proteins such as GATA-1, CBP, TLE-4, and TBP.

    “As PU.1 is not phosphorylated by ERK, we suggest that PU.1 activity might be mediated by ERK-regulated proteins that are known to interact with PU.1 in a modulating manner such as GATA binding protein 1 (GATA-1), CREB binding protein (CBP), transducin-like enhancer of split 4 (TLE-4), and TATA box binding protein (TBP) [44][45][46][47].”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [44] Biggs, 1998, Activation of the mitogen-activated protein kinase pathway in U937 leukemic cells induces phosphorylation of the amino terminus of the TATA-binding protein, Cell Growth Differ
    • cites [45] Carter, 2000, A constitutive active MEK --> ERK pathway negatively regulates NF-kappa B-dependent gene expression by modulating TATA-binding protein phosphorylation, J Biol Chem source

      The TBP phosphorylation results (page unavailable) show that activating the MEK–ERK pathway inhibits TBP phosphorylation and suggest that this modulates NF-κB-dependent transcription. The paper does not establish that TBP interacts with PU.1 or that ERK regulates PU.1 activity through TBP; it addresses only the ERK–TBP part of the cited suggestion. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [46] Linderson, 2004, Corecruitment of the Grg4 repressor by PU. 1 is critical for Pax5-mediated repression of B-cell-specific genes, EMBO Rep source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PU.1 is bifunctional: CBP binding activates PU.1 transcription, while TLE-4 supports PU.1-dependent repression.

    “PU.1 is a bifunctional protein as for instance CBP binding leads to PU.1 transcriptional activation [47], whereas TLE-4 was found to support PU.1-dependent repres-sion [46].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [47] Yamamoto, 1999, Physical and functional interactions between the transcription factor PU. 1 and the coactivator CBP, Oncogene source
    • cites [46] Linderson, 2004, Corecruitment of the Grg4 repressor by PU. 1 is critical for Pax5-mediated repression of B-cell-specific genes, EMBO Rep source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PU.1 forms complexes with IRF-4 and IRF-8 to drive cofactor-dependent expression, such as Toll-like receptor 4.

    “Notably, besides transcription of genes that is directly dependent on PU.1mediated transactivation, PU.1 is also able to form complexes with cofactors as IRF-4 and IRF-8 to induce cofactordependent expression, for example, of Toll-like receptor 4 [50][51][52].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [50] Kanno, 2005, Immune cell-specific amplification of interferon signaling by the IRF-4/8-PU. 1 complex, J Interferon Cytokine Res source
    • cites [51] Escalante, 2002, Crystal structure of PU. 1/IRF-4/DNA ternary complex, Mol Cell source
    • cites [52] Perkel, 1998, A two-step mechanism for recruitment of Pip by PU. 1, J Immunol source

      The paper's Abstract (page not provided) identifies Pip as IRF4 and describes its recruitment by PU.1 to immunoglobulin enhancer DNA-binding sites. The passages support PU.1–IRF4 cooperation, but do not address PU.1 complexes with IRF8 or cofactor-dependent expression of Toll-like receptor 4. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    PKC- and ERK1/2-specific signaling inhibitors reduce both mDC-directed HSC differentiation and phosphorylation-dependent PU.1 transactivation.

    “Furthermore, treatment with PKC-and ERK1/2-specific signaling inhibitors impaired both HSC differentiation toward mDCs as well as phosphorylation-mediated transactivation activity of PU.1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PKCδ regulates PU.1 activity by affecting its transactivation activity, while PU.1 DNA binding activity remains unaffected.

    “On molecular level, PKCd regulated PU.1 activity by affecting its transactivation activity, whereas its DNA binding activity remained unaffected.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    After 5 days of expansion, HSCs kept a round, blast-like stem cell morphology.

    “HSCs were expanded for 5 days and thereby maintained a typical round and blast stem cell morphology.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    TPA treatment specifically activated ERK and PKCδ, whereas STAT5 phosphorylation was not induced.

    “Interestingly, a specific activation of ERK and PKCd was detected, whereas STAT5 phosphorylation was not induced by TPA (Fig. 3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    On LPS exposure, generated DCs secreted IL-6, MCP-1 (CCL2), and RANTES (CCL5) within 6 or 24 hours, and TNF-α within 6 hours.

    “The functional analysis of DCs in terms of cytokine secretion on Lipopolysaccharides (LPS) exposure revealed secretion of IL-6, monocyte chemoattractant protein-1 (MCP-1; CCL2)MCP-1) (CCL2), and RANTES (CCL5) within 6 or 24 hours, respectively (Fig. 1C) as well as TNF-a within 6 hours (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PU.1 DNA binding in HSCs is constitutive and remains unchanged after TPA or GM-CSF/IL-4 stimulation.

    “We observed a constitutive DNA binding of PU.1, which was not altered in the presence of TPA or GM-CSF/IL-4 stimulation (Fig. 6A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PU.1 is phosphorylated by PKCd but not by ERK2.

    “On molecular level, we show that the transcription factor PU.1, whose activation was reported to induce DC differentiation [43], is phosphorylated by PKCd but not by ERK2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    TPA-induced DC formation is faster but yields significantly fewer effector cells.

    “resulting in a more rapid DC formation but with a significantly lower number of effector cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    PU.1 was detected in HSCs, KG-1a, and A.301, but not in Molt 4/8 T, HOS, 293T, HeLa, or Alpha 1.

    “Initially, we confirmed PU.1 expression in HSCs, the CD34 þ myeloid leukemia cell line KG-1a, the lymphatic cell line A.301 but not in Molt 4/8 T, HOS, 293T, HeLa, and Alpha 1 cells (Fig. 4A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    TPA drives HSC differentiation and activates PU.1.

    “As TPA induces HSC differentiation and activation of PU.1, we analyzed its putative stimulation-dependent mode of regulation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    JAK/STAT signaling is not involved in TPA-mediated differentiation, unlike GM-CSF/IL-4-triggered differentiation.

    “Interestingly, JAK/STAT signaling was not involved in TPA-mediated differentiation compared with GM-CSF/IL-4 triggered differentiation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After GM-CSF/IL-4 treatment, Jak2 activation persisted to 5-7 minutes and then dephosphorylated by 15-30 minutes.

    “Jak2 was found activated until 5 and 7 minutes after treatment and dephosphorylation was observed between 15 and 30 minutes poststimulation (Fig. 2B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Recombinant PKCd phosphorylates both GST-PU.1-TAD and full-length HA-PU.1 in vitro, and Go6983 prevents this.

    “In vitro kinase assays with GST-PU.1-TAD and fulllength HA-PU.1 showed that both were phosphorylated by recombinant PKCd, but not in presence of the PKC inhibitor Go ¨6983 (Fig. 6C)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PKCd directly phosphorylates PU.1 in vitro, and the PKC inhibitor Go6983 blocks this phosphorylation.

    “Interestingly, we found a significant PKCd-dependent phosphorylation of PU.1, which was suppressed by incubation with the PKC-specific inhibitor Go ¨6983 (Fig. 4C).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PU.1 phosphorylation correlates with PKCδ and PKCζ activation in B cells.

    “Moreover, PU.1 phosphorylation was found to correlate with PKCd and f activation in B cells [21]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Ikizawa, 2001, PKCdelta and zeta mediate IL-4/IL-13-induced germline epsilon transcription in human B cells: A putative regulation via PU. 1 phosphorylation, Biochem Biophys Res Commun source

  • May combine two claims.

    Study design and conductown data

    ERK2 activity was confirmed by phosphorylation of Elk-1, which was significantly reduced by ERK inhibitor.

    “The activity of ERK2 was confirmed by phosphorylation of the ERK substrate Elk-1, which was significantly circumvented in the presence of the ERKinhibitor (Fig. 4B).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Among myeloid cells, PKCδ is the main isoform.

    “PKCd was reported to be the predominant isoform present in myeloid cells [13]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [13] Mischak, 1991, Expression of protein kinase C genes in hemopoietic cells is cell-type- and B cell-differentiation stage specific, J Immunol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Novel PKC isoforms are predominantly expressed in hematopoietic cells.

    “Previous findings revealed that, in comparison with other members of the family, novel isoforms are predominantly expressed in hematopoietic cells [10]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Bassini, 1999, Lineage-restricted expression of protein kinase C isoforms in hematopoiesis, Blood source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The specificity of kinase activity was verified using ERK inhibitor or PKC inhibitor Go6983.

    “The specificity of the kinase activity was verified using 30 lM ERK inhibitor or 1 lM PKC inhibitor Go ¨6983 (Calbiochem, Darmstadt).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    PKCδ raises PU.1 activity through transactivation-domain phosphorylation, thereby driving HSC differentiation toward mDCs.

    “our results indicate that PKCd induces PU.1 activity by phosphorylation of its transactivation domain (TAD) to promote mDC-directed HSC differentiation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    PU.1 is needed for myeloid-derived DC development but not for lymphoid-related DCs.

    “PU.1 acts on stimuli for granulocyte-macrophage lineage commitment and is critical for the development of myeloid-derived but not lymphoid-related DCs [20].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Guerriero, 2000, PU. 1 is required for myeloid-derived but not lymphoid-derived dendritic cells, Blood source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PU.1 is required for hematopoietic commitment to myeloid or lymphoid lineages and for erythroid or thrombocytic development from progenitors.

    “including PU.1, a prominent factor essential for differentiation processes such as the hematopoietic commitment toward the myeloid or lymphoid lineage and the erythroid or thrombocytic system from its progenitors [16][17][18][19].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16][17][18][19]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Overexpression of PKCδ in COS7 cells and murine myeloid 32D cells allows differentiation toward macrophages when treated with phorbol esters.

    “its overexpression within COS7 cells and the murine myeloid cell line 32D allowed differentiation toward macrophages when treated with phorbol esters [14,15].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [14] Wang, 1997, The catalytic domain of protein kinase C-delta in reciprocal delta and epsilon chimeras mediates phorbol ester-induced macrophage differentiation of mouse promyelocytes, J Biol Chem source
    • cites [15] Kovanen, 1999, Inhibitors keep cytokines in check, Curr Biol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The structural integrity of HA-PU.1 was confirmed by casein kinase II-induced phosphorylation.

    “The structural integrity of HA-PU.1 was confirmed by incubation with casein kinase II, which expectedly led to phosphorylation of HA-PU.1 (see Supporting Information Fig. 5).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    The cell-type pattern of PU.1 expression aligns with its known importance in hematopoietic and differentiation-competent cells.

    “These cell-specific expression patterns coincide with the observed significance of PU.1 in differentiation processes of hematopoietic cells and differentiation-competent cell lines [38].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Fisher, 1998, Role of PU. 1 in hematopoiesis, Stem Cells source

  • May combine two claims.

    Interpretationreasoning

    Blocking PKC signaling causes a differentiation block that ERK1/2 signaling cannot compensate, and the reverse is also true.

    “suggesting that the inhibition of the PKC pathway results in a differentiation block, which cannot be compensated by the ERK1/2 signaling pathway and vice versa.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    For HSC-derived DC formation, MAPK/ERK and PKCd activation is essential and sufficient, whereas JAK/STAT activation is not.

    “However, in terms of HSC-derived DC formation, our data indicate that the activation of the MAPK/ERK and PKCd signaling pathway, but not JAK/STAT signaling pathway activation, is essential and sufficient.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Expanding HSCs and treating them with GM-CSF/IL-4 produced functional myeloid DCs.

    “Taken together, these data demonstrate that HSC expansion and GM-CSF/IL-4 stimulation led to development of functional myeloid DCs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductcitation

    The study expanded undifferentiated HSCs from umbilical cord blood in a human ex vivo culture system and verified the phenotypical and physiological properties of HSC-derived myeloid DCs.

    “We used a human ex vivo cell culture system to expand undifferentiated HSCs [25,26] derived from umbilical cord blood and verified the phenotypical and physiological properties of HSC-derived myeloid DCs [1,27].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [25] Heike, 2002, Ex vivo expansion of hematopoietic stem cells by cytokines, Biochim Biophys Acta source
    • cites [26] Zandstra, 1998, Ontogeny-associated changes in the cytokine responses of primitive human haemopoietic cells, Br J Haematol source
    • cites [1] Curti, 2001, Dendritic cell differentiation from hematopoietic CD34+ progenitor cells, J Biol Regul Homeost Agents
    • cites [27] Luft, 1998, A serum-free culture model for studying the differentiation of human dendritic cells from adult CD34+ progenitor cells, Exp Hematol

  • Classified as probably not a substantial claim.

    Premisecitation

    Activation of PU.1 induces dendritic cell differentiation.

    “whose activation was reported to induce DC differentiation [43]”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [43] Bakri, 2005, Balance of MafB and PU. 1 specifies alternative macrophage or dendritic cell fate, Blood source

  • May combine two claims.

    Interpretationown data

    TPA-induced PU.1 activity depends strictly on PKCd and only to a minor degree on ERK1/2.

    “Therefore, these results support that TPAinduced PU.1 activity strictly depends on the presence of the PKCd isoform and to a minor degree on the presence of ERK1/2.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Super-shift experiments with a PU.1-specific antibody controlled the PU.1 DNA binding data.

    “These data were controlled by super-shift experiments using a PU.1-specific antibody (Fig. 6A).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Prior work used TPA to generate DCs from KG-1 CD34+ cells and primary CD34+ progenitors.

    “The PKC activator Phorbol-12-myristate-13-acetate (TPA) was used previously for DC generation derived from the CD34 þ cell line KG-1 [35] and primary CD34 þ progenitor cells [36].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [35] Shoji, 1988, Effects of thioether phospholipid BM 41.440 on protein kinase C and phorbol ester-induced differentiation of human leukemia HL60 and KG-1 cells, Cancer Res
    • cites [36] Davis, 1998, Phorbol esters induce differentiation of human CD34+ hemopoietic progenitors to dendritic cells: Evidence for protein kinase C-mediated signaling, J Immunol source

  • May combine two claims.

    Interpretationown data

    GM-CSF/IL-4-driven HSC differentiation toward DCs involves ERK, JAK2/STAT5, and PKCδ activation.

    “Overall, these observations indicate that DC-directed HSC differentiation by GM-CSF and IL-4 stimulation is associated with activation of ERK, JAK2/STAT5, and PKCd.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The results in Figure 4 represent two independent experiments.

    “The results shown represent two independent experiments.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    PU.1 transactivation capacity is positively regulated by both PKCd and ERK.

    “Instead, our results demonstrate that the PU.1 transactivation capacity is positively regulated in a PKCd-and ERK-dependent manner.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The results in Figure 1 are representative of three independent experiments.

    “The results shown are representative of three independent experiments.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Both pharmacological inhibitors were previously verified for specificity and cell toxicity in a dose-dependent manner.

    “Both pharmacological inhibitors were previously verified according to specificity and cell toxicity in a dose-dependent fashion (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    PKCd plays a prominent role in DC generation, reinforcing the importance of PKC isoforms in hematopoietic signaling.

    “Besides ERK, our findings underline the prominent role of PKCd in DC generation and strengthen the importance of PKC isoforms in hematopoietic signaling as reported by other groups [10].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Bassini, 1999, Lineage-restricted expression of protein kinase C isoforms in hematopoiesis, Blood source

  • May combine two claims.

    Interpretationown data

    PKCd phosphorylation of the PU.1 transactivation domain is associated with increased transactivation activity, promoting DC generation.

    “According to PU.1-dependent transcription in HSCs, we show that phosphorylation of the PU.1 TAD by PKCd is associated with its enhanced transactivation activity thereby promoting DC generation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The results provide new insights into HSC differentiation toward myeloid DCs and highlight the role of distinct PKC isoforms in hematopoietic processes.

    “Overall, these results provide new insights in the molecular mechanisms of HSC differentiation toward mDCs and underline the outstanding role of distinct PKC isoforms in hematopoietic processes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The results in Figure 5 represent one of three independent experiments.

    “The results shown here represent one of three independent experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The results in Figure 2 are representative of three independent experiments.

    “The results shown are representative of three independent experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The results in Figure 3 are representative of five independent experiments.

    “The results shown are representative of five independent experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The experiments in Figure 6 were performed at least twice with similar results.

    “Each of the shown experiments was performed at least twice showing similar results.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Successful knockdown was confirmed by immunoblot analysis.

    “Successful knockdown was confirmed by immunoblot analysis (Fig. 5C).”
    Quote in the paperSubstantial claim
53 of 53

  • The paper relies on this without support.

    How intracellular signaling governs HSC fate and lineage choice remains poorly defined.

    “The distinct intracellular mechanisms that control cell fate and lineage commitment of these multipotent cells are not well defined.”

  • The paper relies on this without support.

    It is unresolved whether the intracellular response to GM-CSF/IL-4 and its signaling pathways are required for HSC differentiation into mDCs.

    “However, whether the distinct intracellular translation of GM-CSF and IL-4-induced stimulation and the associated signaling pathways are essential for differentiation of HSCs toward myeloid dendritic cells (mDCs) is still elusive.”

  • The paper relies on this without support.

    Which PKC isoforms and mechanisms promote HSC differentiation toward myeloid DCs is not known.

    “However, the distinct PKC isoforms and key mechanisms that promote differentiation of HSCs toward myeloid DCs are still unknown.”

  • The paper relies on this without support.

    How much ERK- or PKC-mediated PU.1 activation contributes to HSC myeloid differentiation remains undescribed.

    “However, to which extend ERK-or PKC-mediated PU.1 activation contributes to myeloid cell differentiation of HSCs has not yet been described.”

  • The paper relies on this without support.

    How PU.1 is regulated molecularly during DC differentiation is unclear.

    “However, the molecular mode of PU.1 regulation in this process is still unclear.”

  • The paper relies on this without support.

    No PKCd-specific inhibitors are available.

    “Unfortunately, there are no PKCd-specific inhibitors available.”

  • The paper relies on this without support.

    Go6983 inhibits PKC isoforms a, b, c, and f in addition to PKCd.

    “Using Go ¨6983 in our experimental setups instead that, besides PKCd, also inhibits PKC isoforms a, b, c, and f might raise the concern that other PKC isoforms also contribute to TPA-induced PU.1 activation.”

  • The paper relies on this without support.

    U0126 inhibits MEK, the upstream kinase of ERK.

    “the inhibitor U0126 targeting the ERK upstream signaling kinase MEK”

  • The paper relies on this without support.

    TPA activates both ERK and PKC signaling pathways.

    “To activate the ERK and PKC signaling pathways, the transfected cells were stimulated with TPA.”
9 of 9

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • PU.1 transactivation experiment names different cell lines

    Major

    The results attribute the Figure 6B transactivation response to A.301 T cells, whereas the Figure 6B caption says HeLa cells were transfected for that experiment.

  • HSC expansion duration differs between methods and results

    Major

    The methods specify seven days of expansion before differentiation, whereas the results say the HSCs were expanded for five days.

  • PKCδ quantity in the kinase assay differs

    Major

    The kinase-assay methods specify 2.5 ng of recombinant PKCδ, while the captions for the reported PKCδ phosphorylation assays specify 20 ng.

  • MEK inhibitor identifier differs for the transactivation experiment

    Minor

    The results name the inhibitor U0216, while the methods and the referenced Figure 5B procedure name U0126.

4 of 4

Cites

Flagged first, then by citations

2 of the 57 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Hamdorf, M., Berger, A., Schüle, S., Reinhardt, J., & Flory, E.
Affiliations
Paul Ehrlich Institut (DE)
Corresponding author
Flory, Egbert
ORCID
Hamdorf, Matthias
Venue
Stem Cells
Publisher
Oxford University Press (OUP)
Published
2011-02-01
Volume
29
Issue
2
Pages
297-306
ISSN
  • 1066-5099 (ISSN-L)
  • 1549-4918
DOI
10.1002/stem.564
OpenAlex
W2102849896
Cited by
45
References
57
Citations by year
2026: 1 · 2024: 1 · 2023: 1 · 2022: 1 · 2021: 2 · 2020: 3 · 2019: 2 · 2018: 3 · 2017: 2 · 2016: 4 · 2015: 3 · 2014: 5 · 2013: 4 · 2012: 11
FWCI
3.06
Type
journal article
Language
en
Open access
Licence
academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
Locations
  • Stem Cells (publishedVersion)
  • PubMed (publishedVersion)
Funders
German BMBF grant (0312113)
Topics
  • Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
  • Health Sciences › Medicine › Genetics › Mesenchymal stem cell research
  • Health Sciences › Medicine › Oncology › Cytokine Signaling Pathways and Interactions
Keywords
PKCδ, PU.1, hematopoietic stem cells, dendritic cell differentiation, myeloid differentiation, transcriptional activation, ERK signaling, JAK/STAT signaling
MeSH terms
Proto-Oncogene Protein Spi-1, Animals, Cell Differentiation, Cell Line, Dendritic Cells, Hematopoietic Stem Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review