In these studies, we show that the two major RARs expressed in hematopoietic cells, RARα and RARγ, have distinct roles in the regulation of immature progenitors and HSCs. We demonstrate that RARγ−/− mice have increased numbers of committed hematopoietic progenitors () and that this is accompanied by significantly reduced numbers of long-term repopulating HSCs ( and ). These abnormalities are not observed in RARα−/− mice ( and Fig. S3). The survival and cell cycle properties of enriched populations of RARγ−/− HSCs were not different to those of RARγ+/+ HSCs, suggesting that the impaired repopulating potential of the RARγ−/− HSCs were not the result of altered apoptosis or cycling of these HSCs (). Furthermore, we show that ATRA enhances HSC self-renewal () and that RARγ, but not RARα, is required for the potentiating effects of ATRA in extended ex vivo culture systems ().
These data collectively demonstrate that RARγ is a key physiological regulator of HSCs. In RARγ+/+ HSCs, there is a balance between HSC self-renewal and differentiation decisions. When treated with pharmacological levels of ATRA, the HSCs undergo more self-renewal divisions (). In contrast, our observations that RARγ−/− mice display increased numbers of committed progenitors (, and ) and reduced numbers of long-term repopulating HSCs ( and ) suggests that the HSCs from RARγ−/− mice display an imbalance between self-renewal and differentiation, favoring differentiation divisions. Furthermore, loss of RARγ signaling results in the inability of the HSCs to maintain repopulating ability in response to ATRA treatment in ex vivo culture (). In addition, the haplo-insufficiency shown by RARγ+/− HSCs, which had an intermediate phenotype between that of RARγ−/− and RARγ+/+ HSCs (), also underscores the importance of RARγ signaling in HSCs. These data demonstrate that RARγ is a key regulator of HSC homeostasis, influencing the balance between self-renewal and differentiation decisions of the HSC.
In a previous investigation, Labrecque et al. reported that RARγ null mice do not have altered numbers of progenitor cells (9
). The major difference between the previous study and ours is that they used fetal BM (18.5 dpc), whereas we used BM from adult (8-wk-old) mice. In addition, in contrast with the increased numbers of progenitors in 8-wk-old mice, we observed significantly reduced numbers of progenitors in 12-mo-old RARγ null mice. These contrasting data therefore demonstrate that the age of the mice studied can also be crucial to the results obtained as can the subsequent interpretation of that data, when investigating hematopoietic defects such as that observed in RARγ−/−
The effects of RARγ were specific to this RAR, as RARα null mice did not display any HSC defects (), and RARα null HSCs retained their ability to self-renew in ex vivo culture in response to ATRA treatment (). RARβ2 was not apparently important to the HSC defect observed in RARγ−/− mice as it was expressed at equal levels in both RARγ+/+ and RARγ−/− LKS+ cells (unpublished data). Collectively, these data highlight the importance and nonredundant role of RARγ in mediating the HSC self-renewing effects induced by ATRA treatment.
In hematopoiesis, ATRA is predominantly known as a differentiating agent, potently inducing the terminal granulocytic maturation of primary leukemia cells from patients with acute promyelocytic leukemia (31
). We have also shown that, in contrast with its potentiating effects on HSC self-renewal, ATRA also has potent effects in enhancing normal primary granulocyte differentiation in vitro (11
). RARα appears to be the most potent of the three receptors in inducing these effects, as shown by its genetic involvement in acute promyelocytic leukemia (34
), and it has the most dramatic effects on neutrophil differentiation from both maturing committed granulocyte progenitors and more immature progenitors (35
In the current study, we have confirmed the predominant role of RARα in granulopoiesis, as RARα-overexpressing BM cells rapidly differentiated down the granulocytic pathway compared with both the control and RARγ1-overexpressing cells (Fig. S1). However, despite these profound effects of RARα in enhancing primitive cell differentiation down the granulocyte lineage, RARα does not appear to have a role in regulating HSCs, as we did not observe any defects in the numbers of primitive progenitors or HSCs in RARα null mice.
It was of interest to determine whether RARγ signaling affected other genes known to induce HSC self-renewal (3
). Despite having previously been reported to have an retinoic acid response element in its promoter region (37
), expression of the cyclin-dependent kinase inhibitor p21Cip1/Waf1
was not altered by ATRA treatment ( B), nor was it deregulated in RARγ−/−
cells (). We have also previously shown that p27Kip1
, which has roles in HSC homeostasis (38
), is in part required for RARα-, but not RARγ-, mediated responses (36
); therefore p27Kip1
is also unlikely to be involved in RARγ-induced HSC self-renewal.
The expression of Hoxb4 was markedly up-regulated in ATRA-treated LKS+
cells (), consistent with previous studies showing that retinoids regulate Hox gene expression in other organs (40
). Despite this increase, however, there were no differences in Hoxb4 expression between the RARγ−/−
cells (); hence, Hoxb4 is not likely a target gene of RARγ.
In contrast with Hoxb4 and p21Cip1/Waf1, Notch1 expression was markedly increased in both ATRA-treated RARγ+/+ LKS+ cells () and RARγ1-overexpressing BM cells () and significantly decreased in RARγ−/− LKS+ cells compared with wild-type LKS+ cells (). This was accompanied by similarly altered levels of expression of Hes1, a measure of Notch-mediated signaling, in these populations (). In contrast, Notch1 and Hes1 were not significantly down-regulated in RARα−/− LKS+ cells ().
Of interest, it has recently been shown that Notch signaling is down-regulated as HSCs differentiate (41
). Given both our observations of the reduced self-renewal/enhanced differentiation of RARγ−/−
HSCs and the reduced expression of Notch1 in RARγ−/−
cells, it is possible that altered Notch1 signaling was contributing to the HSC defects observed in RARγ null mice. Likewise, it is possible that increased signaling of Notch1 was contributing to the enhanced HSC self-renewal observed in ATRA-treated cultures of wild-type HSCs (). However, unlike RARγ null mice, Notch1 knockout mice do not have an HSC defect (7
); hence, altered Notch1 is unlikely to be the main contributing factor to the HSC defects observed in RARγ null mice.
Of therapeutic relevance is that HSCs were induced to self-renew in simple ex vivo cultures using pharmacological levels of ATRA, which is approved for use. Interestingly, a recent report demonstrated the potential beneficial use of ATRA for the expansion of human HSCs (42
). Our studies demonstrating the different roles of the RARs and especially the critical role for RARγ in regulating HSCs now suggest that specifically targeting RARγ could further improve on the ATRA-mediated ex vivo expansion of HSCs. Additional studies are therefore warranted to further investigate the effects of RAR-specific ligands in enhancing the expansion of HSCs for therapeutic purposes.