Growth hormone receptor signaling is dispensable for HSC function and aging

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 ex vivo led to diminished short-term reconstitution and restored B-cell output from old HSCs. Hematopoietic-specific 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, after challenge, Ghr-deficient HSCs functioned normally through serial transplantation. Although exogenous Gh induces age-dependent HSC effects, these results indicate that Ghr signaling appears largely dispensable for HSC function and aging.

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) Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of Ghr expression in HSCs (LSKCD34⁻Flk2⁻), multipotent progenitor-1 (MPP1; LSKCD34⁺Flk2⁻) and myeloid progenitors (MP) (Lin-Sca1⁻cKit⁺) young (4-month-old) mice. (C) Expression of Ghr in young, middle age, and old HSC (LSKFlk2⁻CD34⁻) and MPP1 (LSKCD34⁺Flk2^–) populations. (D) The 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. (F) 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 after transplantation of rGh-treated or control-treated young and old HSCs showing (G) donor engraftment at 4 and 17 weeks posttransplant. (H) Fold-change in PB engraftment between by young and old untreated and rGh-treated HSCs at week 17 posttransplant. (I) Lineage reconstitution at 17 weeks posttransplant. Unpaired Student t test: *P < .05; **P < .01; ***P < .001. ns, not significant; WBM, whole BM.

Figure 2

Ghr is dispensable for HSC function. (A-B) BM analysis of steady-state hematopoiesis in Ghr^fl/fl;Vav1^Cre/+ experimental and Ghr^+/+;Vav1^Cre/+ control mice showing BM frequency of (A) lineage marker negative, Sca1⁺, and c-Kit⁺ (LSK), MP, and common lymphoid progenitor (CLP) populations, and (B) LSK compartment including HSCs. (C) Experimental design for analysis of recipient mice competitively transplanted with Ghr^fl/fl;Vav1^Cre/+ and Ghr^+/+;Vav1^Cre/+ BM. (D-E) Primary transplant analysis showing (D) donor PB reconstitution and (E) donor BM reconstitution. (F-G) Secondary transplant analysis showing (D) donor PB reconstitution and (E) donor BM reconstitution. (H) Experimental overview of serial 5-FU exposure followed by competitive BM transplantation of Ghr^fl/fl;Vav1^Cre/+ experimental and Ghr^+/+;Vav1^Cre/+ control mice. (I) Analysis of white blood cell (WBC) counts after post-5-FU injection over the indicated time-course in Ghr^fl/fl;Vav1^Cre/+ (gray line) and Ghr^+/+;Vav1^Cre/+ (black line) mice. Arrows indicate time points of 5-FU injection. (J) The 1° transplant analysis showing donor reconstitution in PB at 4 and 16 weeks posttransplant. (K) Secondary transplant analysis showing donor reconstitution in PB at 4 and 16 weeks posttransplant. CBC, complete blood counts; i.p., intraperitoneal.