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Blood First Edition Paper, prepublished online October 1, 2014; DOI 10.1182/blood-2014-05-575308
Growth hormone receptor signaling is dispensable for HSC function and aging.
Morag H. Stewart1,2, Paula Gutierrez-Martinez1,2, Isabel Beerman1,2, Brian Garrison1,2,
Emily. J. Gallagher3, Derek LeRoith3 and Derrick J. Rossi1,2*
1
Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, USA. 2
Department of Stem Cell and Regenerative Medicine, Harvard University, Cambridge,
USA. 3Division of Medicine, Endocrinology, Diabetes, and Bone Diseases, Department
of Medicine, Mount Sinai School of Medicine, New York, USA. *corresponding author.
*corresponding author:
Derrick J. Rossi
Email: [email protected]
phone: 617-713-8900
fax: 617-713-8910
Running Title: Ghr is dispensable for HSC function and aging.
Abstract word count: 134
Text word count: 1300 (+123 acknowledgments and contributions)
Figures: 2
Tables: 0
References: 27
1
Copyright © 2014 American Society of Hematology
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Key Points
1. Ghr is specifically expressed on HSCs within the hematopoietic system and is
dynamically regulated upon HSC aging and activation
2. Ghr is dispensable for HSC function.
Abstract
Growth hormone receptor (Ghr) signaling is important in a wide variety of cellular
processes including aging, however the role of Ghr signaling in hematopoietic stem cell
(HSC) biology remains unexplored. Within the hematopoietic system, Ghr is expressed
in a highly HSC-specific manner and is significantly upregulated during aging. Exposure
of young and old HSCs to recombinant growth hormone (rGh) ex vivo led to diminished
short-term reconstitution, and restored B-cell output from old HSCs. Hematopoieticspecific genetic deletion of Ghr neither impacted steady-state hematopoiesis nor serial
transplantation potential. Repeat challenge with 5-fluorouracil showed that Ghr was
dispensable for HSC activation and homeostatic recovery in vivo and, following
challenge, Ghr-deficient HSCs functioned normally through serial transplantation. These
results indicate that while exogenous Gh induces age-dependent HSC effects, Ghr
signaling appears largely dispensable for HSC function and aging.
2
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Introduction
Upon aging the hematopoietic system displays diminished regenerative potential,
reduced immune competence, and a predisposition towards myelogenous disease1. The
importance of cell autonomous regulation of hematopoietic stem cell (HSC) potential
throughout aging is well established, though emerging evidence suggests that HSC
potential may also be regulated by environmental cues that are subject to age-related
variation2.
Growth hormone (Gh) signaling has been implicated in a variety of age-related
hematopoietic phenotypes3-5 and exogenous Gh can enhance or restore young or aged
hematopoietic cellularity and function, respectively5-11. Studies have proposed that Gh
mediates its hematopoietic effects indirectly through non-hematopoietic cells within the
BM7,12, however as the Gh responsive cell was not identified in these studies, it remains
unclear whether Gh directly targets hematopoietic stem/progenitor cells in a cell
autonomous manner. Here, we have addressed the cell intrinsic impact of Gh signaling
on HSCs during aging using gain-of-function and loss-of-function approaches. We found
that Ghr is specifically expressed on HSCs within the hematopoietic system and ex vivo
exposure of HSCs to Gh compromised the short-term reconstitution potential of young
but not old HSCs and led to restored B-lymphocyte potential in old HSCs. Hematopoietic
deletion of Ghr surprisingly did not impact hematopoietic steady-state homeostasis or
HSC activity upon 5-fluorouracil (5-FU) challenge or serial transplantation. These results
show that while exogenous Gh exposure elicits age-dependent effects in HSCs, Ghr
signaling is non-essential to HSC biology and aging.
Methods
Mice. Ghrfl/fl mice were bred with Vav1Cre/+ mice to generate Ghrfl/fl;Vav1Cre/+ experimental
and Ghr+/+;Vav1Cre/+ control mice. All mice were maintained according to Boston
Children’s Hospital Animal Facility protocols. Procedures were performed with
consent from local ethics committees.
Ex vivo rGh administration
For transplantation experiments, FACS isolated HSCs were cultured in S-Clone 0.75%
BSA, 50ng/mL each SCF, TPO and IL12 +/– 100ng/mL recombinant mouse Gh (rGh) for
6 days. Media was changed at day 2 and day 4. At day 6, each well was transplanted
into 3 recipient mice with 3x105 competitive BM.
5FU treatment. Four doses of 5-fluorouracil (5-FU) at 150 mg/kg were administered
every 3 weeks by intraperitoneal (i.p.)
injection to Ghrfl/fl;Vav1Cre/+ (n=4) and Ghr+/+;Vav1Cre/+ (n=4) littermates. Genotyping
primers, Supplemental Table 1.
See supplemental file for full Methods.
3
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Results and Discussion
Ghr is HSC-specific and age-regulated
We identified Ghr as a gene whose expression was highly restricted to HSCs in
comparison to their downstream progeny through analysis of a number of
comprehensive expression profiling studies generated by ourselves and others13-15 (Fig
1A-B). Interestingly, Ghr was substantially upregulated (3.7-fold) in HSCs isolated from
old mice whereas no change in downstream MPP1s was observed during aging (Figure
1C-D). Protein analysis confirmed that Ghr was expressed on HSCs (Supplemental
Figure 1A), however in contrast to the differential age regulation at the mRNA level, no
difference in protein expression was observed during aging (Supplemental Figure 1B).
As the activity of Ghr signaling has been shown to be aging dependent16-18, we assessed
expression of Ghr signaling targets Igf119 and suppressor of cytokine signaling 2
(Socs2)20 in purified HSCs from young and old mice following exposure to recombinantGh (rGh). rGh exposure marginally upregulated Igf1 expression in young HSCs, while
Socs2 was significantly upregulated in old HSCs (Supplemental Figure 1C-D).
To determine whether Ghr was dynamically regulated upon HSC activation, we
examined its expression in young and old HSCs over 24 hours post-cytokine stimulation
in vitro (Figure 1E)21. Interestingly, Ghr exhibited a dynamic age-dependent response in
which old but not young HSCs rapidly upregulated Ghr upon ex vivo stimulation peaking
at 6 hours (Figure 1E); both young and old HSCs downregulated Ghr starting at 12
hours post-stimulation. Together, these results show that Ghr expression is largely HSCspecific within the hematopoietic system and that Ghr signaling cascades are
dynamically regulated with age upon HSC activation.
Ex vivo rGh exposure impacts HSC function in an aging-dependent manner
To investigate the possibility that the differential Ghr signaling we observed may
contribute to the functional changes observed during HSCs aging, we cultured purified
young and old HSCs in the presence of rGh and then assessed their potential by
competitive transplantation (Figure 1F). This allowed us to assay Gh signaling in HSCs
cell autonomously without confounding effects arising from systemic treatment with Gh.
Interestingly, whereas old HSCs exhibited similar reconstitution kinetics independent of
rGh exposure, young HSCs exposed to rGh exhibited significantly diminished short-term
reconstitution that largely recovered at later time points post-transplant (Figure 1G).
Comparison of young and old cohorts showed that total reconstitution was significantly
diminished with aging independent of rGh treatment (Figure 1H). Interestingly however,
exposure to rGh partially mitigated the loss in B-cell potential (Figure 1I), that is
characteristic of old HSCs 22. We also observed a significant increase in T-cell potential
arising from rGh treated young HSCs, which was not observed with the old (Figure 1I).
BM analysis performed at week 20 post-transplant revealed that donor-derived
chimerism and frequency of progenitors and HSCs within the BM were not impacted by
exposure to rGh (Supplemental Figure 2A-D). We did not observe changes in myeloid
potential reported in earlier studies that employed systemic rGh treatment5,7 suggesting
that these previous observations reflect non-cell autonomous changes in hematopoietic
lineage potential. Together our results show that exogenous rGh treatment differentially
affects lineage potential of young and old HSCs, and induces a more balanced lineage
output from aged HSCs.
Genetic ablation reveals that Ghr is dispensable for HSC function.
4
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To address how loss of Ghr signaling would impact HSC potential, we generated
Ghrfl/fl;Vav1Cre/+ experimental and Ghr+/+;Vav1Cre/+ control mice23. Genomic PCR of FACS
isolated BM cells confirmed efficient hematopoietic deletion of Ghr in the experimental
mice (Supplemental Figure 3A). To assess how loss of Ghr in HSCs impacted steadystate hematopoiesis, we analyzed complete blood cell counts (CBCs) and peripheral
blood (PB) composition of age-matched experimental and control mice. A significant
decrease in the number of platelets was observed in the absence of Ghr but no other
lineages were altered (Supplemental Figure 3B-F). Analysis of progenitor compartments
in the BM of Ghrfl/fl;Vav1Cre/+ and Ghr+/+;Vav1Cre/+ mice revealed that all were maintained
at comparable frequencies regardless of Ghr status (Figure 2A-B).
To address the possibility that deletion of Ghr from HSCs might preserve or enhance
HSC potential, we competitively transplanted whole BM cells from Ghrfl/fl;Vav1Cre/+ and
Ghr+/+;Vav1Cre/+ mice (Figure 2C). PB analysis over 16 weeks did not reveal any
differences in the reconstitution or lineage potential (Figure 2D, Supplemental Figure
4A). BM analysis at 17 weeks post-transplant similarly revealed no difference in donor
chimerism or in stem and progenitor compartments (Figure 2E, Supplemental 4B-C). To
examine the impact of Ghr deletion on HSC self-renewal, we performed secondary
transplants. PB analysis showed that loss of Ghr did not alter reconstitution or lineage
potential (Figure 2F, Supplemental Figure 4D). BM analysis at 20 weeks post-secondary
transplant revealed diminished total chimerism, though this did not reach significance
(Figure 2G). Similarly, progenitors and HSCs were not impacted by loss of Ghr
(Supplemental Figure 4E-F).
Due to the dynamic regulation of Ghr expression in HSCs during ex vivo activation
(Figure 1E), we examined the functional response of HSCs subjected to repeated cycles
of activation induced by 5-FU exposure (Figure 2H). 5-FU exposure activates HSCs24-26
and we have shown repeat exposure compromises HSC functional potential27. CBC
analysis showed that Ghr deletion did not compromise the ability of HSCs and
progenitors to mount an effective recovery of WBCs, RBCs or platelets following each 5FU exposure (Figure 2I, Supplemental Figure 5 & 6A-B).
In order to assess how serial 5-FU exposure impacted HSC potential in Ghrfl/fl;Vav1Cre/+
and Ghr+/+;Vav1Cre/+ mice, competitive BM transplantation was performed (Figure 2H).
Deletion of Ghr did not impact PB engraftment or lineage potential (Figure 2J,
Supplemental Figure 6C), and BM analysis at 26 weeks post-transplantation showed no
significant difference in the level of engraftment or the frequency of BM progenitors and
HSCs (Supplemental Figure 6D-F). To assess HSC self-renewal, competitive secondary
transplants were performed. No difference in PB engraftment or lineage reconstitution
potential was observed over 16 weeks post-secondary transplant (Figure 2K,
Supplemental Figure 6G) and no change in BM reconstitution or in the frequency of BM
progenitors or HSCs was found at 20 weeks (Supplemental Figure 6H-J). The lack of
differential recovery of the hematopoietic system in steady-state or following
transplantation as a consequence of Ghr deletion demonstrated, surprisingly, that Ghr is
dispensable for HSC activation and recovery, and further, serial activation of HSCs did
not affect the engraftment or lineage potential of Ghr null HSCs.
In conclusion, our results suggest that while exogenous Gh can impact HSC potential in
an age-dependent manner, Ghr signaling appears largely dispensable to HSC function
and aging.
5
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Acknowledgements
The authors would like to sincerely thank A. Zguro for animal husbandry and technical
assistance as well as all members of the D.J.R. lab. This work was supported by grants
from National Institutes of Health (to D.J.R., R00AG029760 to D.J.R., UO1DK072473-01
to D.J.R.), the Leona M. and Harry B. Helmsley Charitable Trust (to D.J.R.), the New
York Stem Cell Foundation (to D.J.R.), and the Harvard Stem Cell Institute (to D.J.R.).
D.J.R. is a New York Stem Cell Foundation Robertson Investigator.
Authorship Contributions and Disclosure of Conflicts of Interest
M.H.S designed and performed experiments and wrote manuscript. I.B, P.G-M and B.G
helped with experiments. E.J.G and D.L. provided Ghrfl/fl animals. D.J.R designed
experiments and wrote manuscript. The authors declare no conflict of interest.
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Figure Legends
Figure 1: Dynamic regulation of Ghr on HSCs induces age-dependent effects upon
ex vivo rGH stimulation. A) Expression of Ghr in hematopoiesis in the indicated
populations as revealed by microarray analysis. B) qRT-PCR of Ghr expression in HSCs
(LSKCD34–Flk2–), MPP1(LSKCD34+Flk2–) and myeloid progenitors (MP, Lin-Sca1–
cKit+) young (4 months) mice. C) Expression of Ghr in young, mid and old HSC
(LSKFlk2-CD34-) and MPP1 (LSKCD34+Flk2–) populations. D) qRT-PCR of Ghr
expression in young and old HSCs. E) Ghr expression in young and old HSCs over 24
hours of ex vivo culture. E) Experimental design of ex vivo rGh treatment of isolated
young and old HSCs (LSKCD34–Flk2–CD150+) followed by in vivo functional analysis.
G-I) PB analysis following transplantation of rGh treated or control treated young and old
HSCs showing G) Donor engraftment at 4 and 17 weeks post-transplant; H) Foldchange in PB engraftment between by young and old untreated and rGh-treated HSCs
at week 17 post-transplant, and; I) Lineage reconstitution at 17 weeks post-transplant.
Unpaired t-test: *p<0.05, **p<0.01, ***p<0.001.
Figure 2: Ghr is dispensable for HSC function. A-B) BM analysis of steady-state
hematopoiesis in Ghrfl/fl;Vav1Cre/+ experimental and Ghr+/+;Vav1Cre/+ control mice showing
BM frequency of A) LSK, MP and CLP populations and B) LSK compartment including
HSCs. C) Experimental design for analysis of recipient mice competitively transplanted
with Ghrfl/fl;Vav1Cre/+ and Ghr+/+;Vav1Cre/+ BM. D-E) Primary transplant analysis showing;
D) Donor PB reconstitution; E) Donor BM reconstitution. F-G) Secondary transplant
analysis showing; D) Donor PB reconstitution; E) Donor BM reconstitution. H)
Experimental overview of serial 5-FU exposure followed by competitive BM
transplantation of Ghrfl/fl;Vav1Cre/+ experimental and Ghr+/+;Vav1Cre/+ control mice. I)
Analysis of WBC counts following post-5-FU injection over the indicated time-course in
Ghrfl/fl;Vav1Cre/+ (grey line) and Ghr+/+;Vav1Cre/+ (black line) mice. Arrows indicate time
points of 5-FU injection. J) 1o transplant analysis showing donor reconstitution in PB at 4
and 16 weeks post-transplant and K) Secondary transplant analysis showing donor
reconstitution in PB at 4 and 16 weeks post-transplant .
8
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Prepublished online October 1, 2014;
doi:10.1182/blood-2014-05-575308
Growth hormone receptor signaling is dispensable for HSC function and
aging
Morag H. Stewart, Paula Gutierrez-Martinez, Isabel Beerman, Brian Garrison, Emily J. Gallagher, Derek
LeRoith and Derrick J. Rossi
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