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S. Fañanas-Baquero et al.
We have described a positive effect of E2 or E4 treatment on the engraftment of human cells, with the impact of E4 being more significant. It is important to note that E4 is synthesized exclusively by the human liver during preg- nancy. It is detected at 9 weeks of pregnancy and reaches high levels in the second trimester, with concentrations ris- ing steadily towards the end of pregnancy.16 The fetal liver is a hematopoietic organ during the last half of gestation. During the hematopoietic stage of the fetal liver, different signaling pathways are coordinated to promote both mas- sive expansion of HSC through the activation of the HSC cycle and massive production of erythroid cells. After birth, the HSC migrate from the liver to the adult BM, where the most primitive HSC are largely quiescent.1,31 The concurrence of E4 synthesis in the fetal liver, when it is a hematopoietic organ, may suggest an indirect link between this estrogen and the expansion of human HSPC during pregnancy. The association between estrogens and hematopoietic development has previously been described during zebrafish development,32 in mice19,27 and in the hematopoietic differentiation of human induced pluripo- tent stem cells.28 So estrogens have a clear impact on HSC emergence. Oguro et al. demonstrated coordination between E2 and 27-hydroxycholesterol in the regulation of hematopoiesis during pregnancy.27 Consequently, we hypothesize that E4 likely plays a role in modulating early human hematopoiesis during embryonic development.
As previously reported, the observations could be attrib- uted to an intricate regulation mediated by the estrogens.17 The complexity of estrogen signaling pathways starts with the existence of several estrogen receptors. Three of these receptors (ESR1, ESR2 and GPER33) are expressed in hematopoietic cells, but only ESR1 has been described to play a role in the regulation of HSC.18,19,24 A second level of complexity is that the expression of these estrogen recep- tors tends to differ among hematopoietic subpopulations24 (Figure 2). Moreover, different estrogens vary in their binding affinity for different estrogen receptors; for exam- ple, E2 has a 7-fold higher affinity for ESR1 (inhibition constant, Ki=0.21 nM) than for ESR2 (Ki=0.015 nM), and E4 has a 400-fold higher affinity for ESR1 (Ki=4.9 nM) than for ESR2 (Ki=19 nM).34 Once the estrogen and recep- tor are bound, specific cell responses are triggered by two different mechanisms: (i) gene expression programs, which can be initiated through estrogen nuclear signaling, and (ii) the estrogens acting through membrane-initiated steroid signaling (MISS), which is a rapid extra-nuclear cel- lular response to the estrogen signal.15 These two types of estrogen signaling may also explain the differences we observed between the effects of E4 and E2, from their tox- icity and expansion in vitro (Figure 3; Online Supplementary Figure S3) to their in vivo effects (Figure 6). E4 uncouples nuclear activation and MISS, in contrast to E2 which does not.15 For example, E4 acts as an estrogen antagonist on breast cancer cells.16,35 Moreover, the lower affinity of E4 for estrogen receptors, in comparison with the affinity of E2, might suggest a very limited effect of E4 on HSPC; however, the E4 doses, whose effects on HSPC were observed (Figures 3 and 4), were the same doses used by Abbot et al. for which ERE transcriptional activity could be detected.15 Furthermore, given that E4 lacks MISS activity, it is possible that the impact of E2 and E4 on human HSPC is due to their nuclear signaling, with similar transcription- al output, but this point will have to be analyzed in depth. Additionally, the presence of E2 or E4 increased the levels
of both ESR1 and ESR2 and modified their cellular local- ization. The increment of cells in S/G2/M-phase mediated by estrogens could be partially blocked by ESR1 and GPER antagonists in the case of E2, and by an ESR2 antagonist in the case of E4 (Online Supplementary Figure S3J). The impli- cation of this is that different estrogen receptors in human HSPC might be involved in the signaling triggered by E2 or E4; however, this point will require more in-depth study. More interestingly, E2 might activate estrogen receptor-mediated MISS, since a clear polarized location of the estrogen receptors in the cytoplasm membrane was found (Online Supplementary Figure S3O and P). On the other hand, E4 might activate nuclear estrogen signaling, since E4 is unable to induce MISS15 and a clear increment of ESR2 in the cytoplasm was detected (Online Supplementary Figure S3N). The consequences of ESR1 and ESR2 upregulation and localization should be explored in future experiments. Additionally, Oguro et al.27 described two different ESR1 ligands, E2 and 27-hydroxycholesterol, which regulated HSPC differently during pregnancy. Both ESR1 ligands collaborated to induce HSC proliferation, mobilization and extramedullary hematopoiesis. In a sim- ilar way, E2 and E4 might collaborate together with a dif- ferential impact on human HSPC.
We identified that the underlying mechanism mediated by estrogens is activation of the cell cycle in vitro, as previ- ously described, which promotes the expansion of hematopoietic progenitors.19,24 However, estrogens might also have other effects, including the activation of telom- erase activity to facilitate the expansion of HSPC,20-22 or an increase of the unfolded protein response to promote hematopoietic regeneration after a proteotoxic stress, such as irradiation.23,36 In our in vivo model, E2 or E4 might acti- vate the gene signaling involved in the cell cycle,19,24 telom- erase activity20-22 or unfolded protein response,23 but these estrogens could also activate apoptosis24,27 when high doses are used (Figure 3; Online Supplementary Figure S3G and H). Surprisingly, the estrogen-mediated expansion observed in vitro was not enough to explain the improve- ment in human hematopoietic engraftment. Indeed, the in vitro proliferation of human HSPC induced by the estro- gens was counterproductive to the enhancement of hematopoietic engraftment (Figure 6A). This might be due to the reduction of long-term engraftment ability of cycling HSPC, and the decoupling of HSPC expansion and stem cell properties in vitro.37 As HSC quiescence, self- renewal and differentiation are controlled through intrin- sic HSC signaling and extrinsic niche signaling and we observed that the co-culture of HSPC with human BM- MSC was able to expand hematopoietic cells (Figure 4) and maintain engraftment potential (Figure 6B), in vitro expansion of HSPC might be compensated by niche sig- naling. In accordance with this, estrogens could also mod- ulate hematopoiesis by affecting the capacity of MSC to promote osteogenesis.30,38 Furthermore, osteogenic differ- entiation might favor the proliferation of HSPC.25 The beneficial effect of E2 on the expansion of both HSPC and MSC was noted previously by Kitajima et al.39 As shown in Figure 6, an increase in MSC was also detected in our in vivo model after E4 treatment. Besides, the presence of estrogen might favor the recovery of MSC after irradiation (Figure 6E; Online Supplementary Figure S6E-G), as previ- ously described for HSPC.23,36 Consequently, the impact of estrogens on promoting human hematopoietic engraft- ment in immunodeficient mice might be mediated
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haematologica | 2021; 106(6)