In this study we analyzed the effect of targeted disruption of all PIM family members on murine development and hematopoietic differentiation. Surprisingly, the combined deficiency for all PIM family members resulted in a rather mild phenotype for the resultant compound Pim mutant mice. In light of the strong conservation of Pim genes between different species, this mild phenotype argues against a role for PIM family members in critical developmental processes. Apparently, life is compatible with the absence of PIM. On the other hand, the absence of an obvious phenotype in vivo under homeostatic conditions might in part be explained by the nature of Pim genes as early-response genes responding to growth factors and cytokines. In fact, critical functions of PIM family members might only be revealed under conditions of high stress, which severely disrupt the homeostatic situation.
We demonstrated that PIM proteins are important for body growth, since Pim1 Pim2 Pim3
compound KO mice display a profound reduction in body size at birth and throughout their entire live span. The decrease in body size was predominantly caused by a reduction in the number of cells rather than cell size, although a minor cell-size difference was consistently observed between PIM-proficient and -deficient animals. The reduction in body size (approximately 30%) observed at birth is, as such, reminiscent of a defect in insulin-like growth factor (IGF)-insulin signaling. Mice carrying defects in either of these pathways display a reduction in body size ranging from 20 to 60%, depending on the nature of the defect (for a review, see reference 1
). However, it remains to be determined whether IGF-insulin signaling is indeed affected in Pim
The majority of hematopoietic growth factors are efficient inducers of Pim1
, and Pim3
. In addition, a role for PIM1 in the proliferative response of hematopoietic cells to the presence of IL-3 and IL-7 was demonstrated in experiments with Pim1
KO bone marrow cells (9
). The generation of Pim1−/− Pim2−/− Pim3−/−
compound KO mice and the subsequent isolation of PIM-deficient bone marrow cells demonstrated that PIM1, PIM2, and PIM3 can act redundantly in stimulating the colony formation of bone marrow cells in response to the presence of IL-3, IL-5, SCF, and TPO. It should, however, be noted that it seems that PIM1 is the most and PIM2 is the least crucial PIM protein in these responses. Since PIM2 is more efficiently induced than PIM3 by a number of these growth factors, PIM2 might have a role distinct from the functions of PIM1 and PIM3 in these settings. In contrast to results seen with respect to the response to IL-3, IL-5, SCF, and TPO, we did not observe an effect of PIM deficiency on the GM-CSF response. This is very remarkable, in particular because the receptor for GM-CSF is highly related to the IL-3 and IL-5 receptors, as they share the same common-β-chain, which acts as the signaling module. Thus, although GM-CSF signaling leads to transcriptional activation of Pim
), the PIM proteins appear not to be necessary for efficient transduction of the GM-CSF signals. This is in contrast to the very similar IL-3 and IL-5 signaling, in which PIM kinases seem to play an important role.
In addition to the impaired colony formation of PIM-deficient bone marrow cells in response to a variety of growth factors, PIM-deficient early pre-B cells showed a reduced proliferative response to IL-7 in vitro. This was reflected in vivo by an attenuated progression of pro-B cells through the IL-7-driven proliferative stage that follows successful rearrangement of the heavy chain. Since differentiating B cells in the bone marrow of Pim-deficient animals show only marginally decreased IL-7Rα expression levels, it seems unlikely that the attenuated proliferative response to IL-7 can be attributed to lower IL-7R expression levels. In young animals, peripheral B-cell numbers were reduced, whereas in older animals, this was not the case. The observation that the number of B cells is unaltered in the periphery of older animals is likely explained by the continuous supplying of newly formed B cells from the bone marrow into the peripheral B-cell compartment and homeostatic proliferation of peripheral B cells (for a review, see reference 42
The differentiation of T cells in the thymus is also characterized by a series of alternating phases of genomic recombination (resting cells) and IL-7-driven proliferation. We did not observe any effect of the absence of PIM on T-cell differentiation in the thymus. Although Pim
mutant mice do not display abnormalities in thymic T-cell differentiation, the ability of peripheral T cells to proliferate in response to synergistic IL-2 and TCR activation was markedly impaired. The proliferative response of Pim
-deficient and Pim
-proficient T cells to a dilution series of CD3-cross-linking antibodies was identical. Likewise, serial dilutions of IL-2 on peripheral T cells elicited the same responses in terms of proliferation and STAT5 phosphorylation in both Pim
-deficient and -proficient T cells. Yet when the two stimuli are combined, Pim
-deficient T cells display a decreased response to the combined activities when either stimulus is present at suboptimal levels. PIM mutant peripheral T cells express lower levels of common-γ-chain but normal levels of the IL-2Rα and IL-2Rβ chains. This might indicate that common-γ-chain-transduced signals contribute to the decreased proliferation of PIM-deficient T cells. However, we did not find differences in STAT5 phosphorylation between PIM-proficient and PIM-deficient T cells. Moreover, it has been shown that the common-γ-chain is not required for antigen-driven proliferation of naïve T cells (21
), indicating that additional mechanisms are likely to contribute to the decreased proliferation of peripheral T cells in response to the IL-2 and TCR activation. Further analysis of this proliferation defect demonstrates that PIM appears to be important for cell cycle entry of resting peripheral T cells at suboptimal levels of TCR signaling. PIM2 appeared to be the most critical PIM protein for this response, although PIM1 and PIM3 also contribute. These results indicate that the PIM proteins can augment the response to growth factor signals either directly or indirectly. The indirect pathway could involve signaling via the IGF receptors, as both IGF-I and -II can potentiate proliferation in B and T cells (14
). Such a role for PIM would also be consistent with previous results showing that overexpression of PIM1 can rescue the reduced thymic cellularity in common-γ-chain-deficient mice but only in the context of a functional CD3 complex (18
We found no evidence that PIM deficiency compromises the survival of peripheral T cells. The results of other studies indicated that in vitro overexpression of PIM proteins protects cells against apoptosis (13
). This apparent discrepancy emphasizes the notion that gene inactivation and overexpression studies do not necessarily reveal the same physiologically relevant functions of a protein. On the one hand, ectopic overexpression of proteins might evoke effects that are not representative of its function under physiological conditions. On the other hand, inactivation might elicit compensation by other proteins, obscuring or mitigating the normal role of the protein of interest. With respect to PIM proteins, which are usually expressed during a short time period due to the short half-lives of both mRNA and protein, overexpression studies using cells with enhanced PIM activity for prolonged periods are likely to yield data different from data obtained with studies using cells from which PIM activity had been deleted. The former situation might be more reflective of the role of PIM proteins during cellular transformation as observed in M-MuLV-induced tumors, whereas the latter could give more insight into the physiological role of PIM proteins in nontransformed cells. Therefore, the observation that an absence of PIM proteins does not give rise to cells which are more susceptible to apoptosis is not in contradiction with the notion that overexpression of PIM proteins might provide protection from apoptosis. The results from our study appear to be in agreement with Pim1
- and Pim2
-deficient mouse results from retroviral insertional mutagenesis screenings designed to identify genes that can substitute for PIM function in lymphomagenesis (25
). The genes identified in this screen (e.g., Kit
) all regulate the G1
to S transition. Therefore, although we cannot exclude the possibility that rescue from apoptosis is indeed a function of PIM proteins in lymphomagenesis, it seems that regulation of cell cycle is at least as important.
expression is directly regulated by the JAK/STAT pathway (36
), the Pim
mutant phenotypes described here might provide clues about the biological significance of the STAT proteins for transactivation of the Pim
genes. In fact, Stat5a Stat5b
compound KO bone marrow cells display a reduced ability to form colonies in response to the presence of IL-3, IL-5, IL-7, SCF, GM-CSF, and TPO (37
). Thus, the inability of STAT-deficient cells to induce Pim
expression might be responsible for some of the observed signaling defects. In particular, STAT-deficient T cells cannot proliferate in response to TCR/IL-2 signaling (27
). Although this phenotype is much stronger than the defect observed in Pim
mutant T cells, our data show that the induction of PIM proteins has a significant effect on TCR/IL-2-mediated proliferation.
In summary, the phenotypes of the Pim1 Pim2 Pim3 compound-KO mice illustrate a role for the PIM proteins in single-growth-factor signaling and in synergistic signaling pathways. The results with respect to synergistic growth factor signaling would suggest a view that PIM proteins might act as sensitizers of these pathways. This view would fit the induction of early transcription of the Pim genes by growth factors and the instability of the PIM mRNAs and proteins. Although such interplay between growth factor signals has been postulated before, the mediators have remained obscure. We show here that PIM proteins might fulfill such a role.