Background
The steady-state production of B lineage lymphocytes in bone marrow is contingent on the developmental-stage-specific expression and combinatorial activities of multiple regulatory proteins including transcription factors, signaling molecules, microRNAs, and epigenetic modifiers. A critical early event in B cell development is lymphoid lineage specification within the multipotential progenitor (MPP) pool which we and others have shown is dependent, in part, on the homeodomain transcription factor Hoxa9 and signaling via the receptor tyrosine kinase Flt3 [
1‐
4]. Germline knockout of Hoxa9 or Flt3-ligand largely phenocopy each other with respect to reductions in numbers of MPPs, the lymphoid progenitor subsets ALP and BLP, and B cell precursors in BM. Importantly, Hoxa9 and Flt3 signaling function synergistically to regulate a critical early checkpoint in lymphoid development, as combined loss of Hoxa9 and Flt3 signaling ablates the generation of lymphoid primed multipotential progenitors [
4].
Pim-1 is a member of the Pim family of proto-oncogenes that encode a distinct class of serine/threonine kinases. Pim-1 was originally identified as a target for proviral activation in Moloney murine leukemia virus induced T cell lymphomas [
5]. PIM proteins are widely expressed in most tissues and co-expression of family members has been found in various tissues [
6]. The transcription of Pim family members is induced by a variety of cytokines and mitogens that transduce their primary signal through the JAK/STAT pathway [
7]. A role for Pim-1 in B cell development was previously demonstrated through analysis of
pim1-/- and
Eμ-Pim1 transgenic mice [
8,
9].
Pim1-/- Pre-B cells exhibited impaired in vitro proliferation in response to IL-7 and stem cell factor (SCF) that was rescued by expression of a functional Pim-1 transgene [
10]. In contrast, overexpression of Pim-1increased numbers of IL-7 + SCF responsive B cell colonies. These combined data provided the first evidence that Pim-1 was an important regulator of B lymphopoiesis in mice, and linked Pim-1 to the IL-7R signaling pathway.
Cytokine signaling plays an essential role in early lymphoid/B cell development. Threshold levels of Flt3 signaling are required for the proliferation, survival, and maintenance of MPPs competent to generate B cell precursors [
1,
11]. Flt3 signaling is mediated by the Ras and STAT5 pathways [
12]. A dominant negative form of Ras was shown to phenocopy the B lineage developmental block in
flt3-/- mice, impairing the proliferation of common lymphoid progenitors and Pre-Pro-B cells. The same study showed that Ras promoted STAT5-dependent Pro-B differentiation by enhancing expression of IL-7Rα [12]. Pim-1 is induced downstream of Jak2/STAT5 signaling and has also been implicated in playing a role in the proliferation and/or differentiation of myeloid progenitors [
13‐
15]. Importantly, a role for Pim-1 in regulation of the early lymphoid/B cell progenitor pool, prior to expression of CD45R/B220, has not been reported.
Functional studies have confirmed a role for Pim-1 in regulating hematopoietic stem cell (HSC) proliferation and survival. HSCs from
pim1-/- mice exhibited impaired repopulating capacity in competitive transplantation experiments [
16]. In vitro assays revealed decreased cytokine mediated cell growth and differentiation of hematopoietic progenitors [
7]. In contrast, overexpression of human Pim-1 driven by
vav hematopoietic regulatory elements and SV40 showed enhanced hematopoietic progenitor function in vitro and in vivo [
16]. The hematopoietic defects exhibited by
pim1-/- mice are strikingly similar to those in
hoxa9-/- mice as loss of
hoxa9 also impaired the proliferation and repopulating ability of HSCs [
17]. Consistent with this observation,
pim1 is a direct target of Hoxa9 [
18]. Somatic ablation of
pim1,
hoxa9, or
flt3 causes select reductions in hematopoietic progenitor subsets and B cell precursors. However, an obligate role for Pim-1 in regulation of lymphoid and/or early B cell development has not been investigated. In this study we evaluated the role of Pim-1 in murine lymphoid lineage specification and B cell development through comparative flow cytometric analysis of
hoxa9-/- x Eμ-Pim1Tg,
flt3l-/- x Eμ-Pim1Tg,
Eμ-Pim1 transgenic,
pim1-/-,
hoxa9-/-, and
flt3l-/- mice. Our experimental findings revealed that Pim-1 dysregulation has developmental-stage-specific effects on B lymphopoiesis and early myeloid, but not erythroid progenitors. Furthermore, we show that
Pim1-deficiency is not the basis of the lymphoid or early B cell developmental defects in
hoxa9-/- or flt3l-/- mice.
Discussion
Previous studies have implicated a role for Pim-1 in regulation of B cell development in BM and
pim-1 is a Hoxa9 target gene [
10,
18]. Furthermore,
hoxa9- and
pim1-deficient mice share similar hematopoietic phenotypes and forced expression of Pim-1 rescued the in vitro proliferation defect in
hoxa9-/- BM progenitors [
17,
18]. To determine if forced expression of Pim-1 was sufficient to restore the lymphoid progenitor/B cell precursor defect in
hoxa9-/- or
flt3l-/- mice, we generated
hoxa9-/- Eμ-Pim1Tg mice and
flt3l-/- Eμ-Pim1Tg mice. Unexpectedly,
Eμ driven expression of Pim-1 exacerbated the deficiencies in ALP and BLP in
flt3l-/-, but not
hoxa9-/- mice. The detrimental effect in
flt3l-/- Eμ-Pim1Tg mice was specific to the ALP and BLP stages of B cell differentiation, as the
Eμ-Pim1Tg did not further exacerbate the B cell deficiencies in
flt3l-/- mice from the Pre-Pro-B through the IgM
+ stages. The negative effect of
Eμ-Pim1Tg expression was not restricted to the ALP and BLP compartments. We previously showed that
Eμ is active from a very early stage in hematopoiesis and here we also show that
Eμ-Pim1Tg expression caused significant reductions in numbers of HSCs, MPPs, CMPs, and GMPs suggesting that Pim-1 expression levels must be carefully controlled during early hematopoietic differentiation [
11,
24]. In contrast, loss of Pim-1, which we hypothesized might have deleterious consequences on the early lymphoid/B cell progenitor pools, was dispensable for hematopoiesis with the exception of IgM
− BCPs [
10]. Consistent, with our findings, realtime PCR of
pim-1 transcription across the hematopoietic spectrum, showed significant levels limited to BCPs at the Pre-B cell stage. Finally, through direct comparative hematopoietic progenitor subset analysis of
hoxa9-/-,
flt3l-/-, and
pim1-/- mice, we suggest that loss of Pim-1 is unlikely to contribute significantly to the hematopoietic progenitor defects in either
hoxa9-/- or
flt3l-/- mice. Taken together, these comprehensive flow cytometric analyses demonstrate very select lineage and developmental-stage-specific consequences of dysregulated expression of Pim-1 expression on BM hematopoietic progenitor and B lineage subsets.
The serine threonine phosphokinase Pim-1 is highly expressed in hematopoietic cells in mouse and man [
20,
25]. The
pim-1 gene was originally discovered as a common insertion site in MoMuLV-induced T cell lymphomas and later found in B-cell lymphomas and erythroleukemias. Overexpression of Pim-1 driven by the
Eμ enhancer was shown to lead to a low incidence of T cell lymphomas and increased sensitivity to chemically induced T cell transformation. In the B lineage, overexpression of
Eμ − Pim-1 facilitated establishment of B cell progenitor cell lines blocked at the Pre-B cell stage of differentiation [
10]. The same study reported that marrows with the highest Pim-1 expression had a reduction in M-CSF responsive cells, and suggested the result could be due to impaired macrophage differentiation or on a macrophage progenitor. Our immunophenotypic analysis of
Eμ-Pim1 transgenic mice provides insight into the reductions in M-CSF responsive cells as we identified significant reductions in frequencies and absolute numbers of CMP and GMP.
PIM kinases are short-lived proteins induced at the level of transcription upon mitogenic stimulation [
26]. They do not have regulatory domains and are constitutively active when expressed. Under physiologic conditions, their short half-life limits their activity [
27]. Many cytokines that induce
pim gene transcription act through the JAK/STAT pathway. The
pim1 promoter has binding sites for STAT3 and STAT5 [
28,
29]. Pim-1 protein then, in turn, functions to inhibit the JAK/STAT pathway by binding and activating SOCS proteins [
30]. SOCS proteins support cytokine signaling through the Ras/MAPK pathway by binding the Ras inhibitor RasGAP [
30]. Interestingly, dysregulated Ras/SOCS function mediated by overexpression of Pim-1 provides a plausible cellular mechanism for the impaired lymphoid/early B lineage development exhibited by
Eμ − Pim1Tg mice. Ras proteins are involved in regulating proliferation and differentiation of various cell types in a tissue specific manner. Pertinent to this study, mice expressing a dominant negative form of human H-Ras have a severe block in B cell development at the Pre-Pro-B to Pro-B transition [
31]. Li, et al., previously showed that a Flt3/Ras dependent signal governs lymphoid progenitor (ALP + BLP) and Pre-Pro-B proliferation [
12]. In addition, they showed that Flt3/Ras suppresses expression of
socs2 and
socs3. They concluded that Flt3 activation of Ras primes B cell development by inducing a state of STAT5 responsiveness, a key event downstream of IL-7R signaling in lymphoid progenitors leading to induction of the B cell fate specification and commitment factor EBF1. Sustained expression of Pim-1 would be predicted to stabilize SOCS expression, impairing STAT5-mediated induction of
ebf1, suppressing EBF1-directed B cell differentiation. Indeed, this hypothetical molecular explanation is consistent with our findings that the Flt3 dependent ALP and BLP stages are exquisitely sensitive to dysregulated Pim-1 in
flt3l-/- mice. It also suggests that it is unlikely that elevated levels of Pim-1 are inhibitory for lymphoid/B cell development by accelerating B cell differentiation, given the critical role for STAT5/EBF1 in this process.
Pim kinases act as oncogenes by promoting cell cycle progression by phosphorylating and down-regulating the cyclin dependent kinase inhibitor p27Kip1. Pim kinases also have the ability to suppress
p27kip1 transcription through phosphorylation and inactivation of forkhead transcription factors, including FoxO1 and FoxO3 [
32]. Pim-1 inactivation of FoxO1 would have deleterious effects on B cell fate determination as FoxO1 is an integral member of a global network of transcription factors, including E2A and EBF1 [
33]. Importantly,
FoxO1-deficient mice also have an arrest in B cell development at the CLP stage, specifically at the BLP stage, and phenocopy
EBF1-deficiency [
34]. Importantly,
FoxO1-deficient BLPs have increased transcripts for
socs2, reminiscent of Pim-1 overexpression [
30]. Based on our immunophenotyping results, together with previous phenotypic, molecular, and cellular data in the literature database, we hypothesize that dyregulated expression on Pim-1, driven by
Eμ, impairs lymphoid/early B cell differentiation through upregulation of SOCS proteins and inactivation of FoxO1. Upregulation of SOCS proteins inhibits STAT5 mediated B cell differentiation leading to upregulation of EBF1. Inactivation of FoxO1 impairs the global transcriptional network orchestrated by E2A, EBF1, and FoxO1 that directs the early program of B lineage gene expression, requisite for B cell differentiation. In contrast, loss of Pim-1, would be predicted to suppress SOCS protein expression and stabilize FoxO1, favoring B cell developmental potential, providing an explanation for the slight, but not statistically significant, increase in BLP and Pre-Pro-B cells we observed in
pim1-/- mice. We note that additional studies are required to determine if this molecular explanation is valid.
The goal of this project was to investigate the role of Pim-1 in early lymphoid/B cell development. However, we also found reductions in numbers of HSC, MPP, CMP, and GMP in
Eμ − Pim1Tg mice. An, et al., reported an increase in frequencies and numbers of LSK
+ cells in
vav-Pim1Tg mice, whereas we report decreased frequencies and numbers of LSK
+ cells in
Eμ − Pim1Tg mice [
16]. The overall decrease in LSK
+ cells was the basis of the decreased numbers of HSCs and MPPs, as frequencies of CD150
+ CD34
− immunophenotypic HSC within the LSK
+ compartment were actually increased in
Eμ − Pim1Tg mice. We believe it unlikely that
Eμ − Pim1Tg expression has any functional consequence on HSC biology for two reasons. First, we previously showed that
Eμ driven reporter expression is confined to CD27
+ MPPs within the LSK
+ compartment and HSCs are CD27
− [
11,
24,
35]. Second, the frequencies of CD150
+ CD34
− HSCs were substantially increased in
Eμ − Pim1Tg mice.
The cellular and/or molecular basis of the inhibitory consequence of
Eμ − Pim1Tg expression on numbers of Flt3
+ MPPs in noteworthy, as Flt3
+ MPPs are the precursors of CMP and CLP and these hematopoietic subsets were the most sensitive to
Eμ − Pim1Tg expression.
Eμ − Pim1Tg expression did not impact Flt3
+ CMP or CLP by altering the expression levels of Flt3. However, Flt3
+ CMP and CLP were particularly sensitive to
Eμ − Pim1Tg expression. These findings suggest that overexpression of Pim-1 might negatively impact signaling networks downstream of Flt3 stimulation in Flt3
+ progenitor subsets, impairing either their proliferation or survival. Regardless of the mechanism, differentiating CMP and CLP overcome the inhibitory effects and their downstream progeny are refractory to sustained expression of Pim-1. Overexpression of Pim-1 in myeloid progenitors is likely limited by inactivation of
Eμ due to lack of expression of critical activators necessary to maintain enhancer activity. It is interesting to note that Pim-1 driven by
vav regulatory elements was low in mature myeloid cells [
16].
Finally, although our flow cytometry analysis revealed that Pim1-deficiency does not provide a molecular explanation for the lymphoid/B cell deficiency in hoxa9-/- mice, pim1-/- and hoxa9-/- mice do share similar phenotypic and functional HSC defects. These observations and reported findings underscore developmental stage specific requirements for Hoxa9 and Pim-1 in hematopoietic development.