Mutation of STAT1/3 binding sites in gp130(FXXQ) knock-in mice does not alter hematopoietic stem cell repopulation or self-renewal potential

Abstract

Interleukin (IL)-6 family cytokine signaling through gp130 and signal transducer and activator of transcription (STAT) activation is believed important for early hematopoiesis. To determine whether gp130/STAT1/3 physical interaction is required, we compared hematopoietic repopulating activities of embryonic day (E)14.5 fetal liver cells from gp130(FXXQ/FXXQ) knock-in mice, which have four mutated STAT1/3 binding sites. In hematopoietic cells, failure to tyrosine phosphorylate STAT3 by gp130 did not cause any significant effects on myeloid progenitor colony forming units (CFU) in vitro and or on competitive multilineage hematopoietic reconstitution. Serial transplantation of fetal liver (FL) cells was unaffected throughout primary, secondary, and tertiary transplants indicating normal self-renewal capacity. Even gp130(FXXQ/FXXQ) on the background of STAT5 deficiency, with known hematopoietic stem cell (HSC) repopulating dysfunction, did not further impair HSCs beyond that of STAT5 alone. Overall, the defective gp130-mediated STAT1/3 signaling is surprisingly dispensable for HSC function. However, since these mice lack both STAT1/3 binding sites there are several possible explanations for this result and these are discussed.

Figure 1

Effect of gp130^FXXQ/FXXQ mutations on STAT3 activation, HSC multilineage repopulation, and self-renewal. (A) Western blot analysis for phospho-STAT3 and STAT3. FL was harvested from E14.5 embryos of the indicated genotypes, starved for four hours in PBS with 2% FBS, and stimulated with or without 50 ng/ml IL-6 for 30 min. 20 μg of total protein was separated by SDS-PAGE and transferred to PDVF membrane. Phospho-STAT3 and STAT3 were detected by antibodies from Cell Signaling Technology. (B) A representative secondary competitive repopulation assay between gp130^FXXQ/FXXQ and gp130^+/+ FL grafts. FL cells were obtained from E14.5 fetuses from mating of gp130^+/FXXQ (CD45.2) or wild type Boy J mice (CD45.1), genotyped by PCR, and then either gp130^FXXQ/FXXQ or gp130^+/+ cells were mixed with wild type Boy J FL cells at a 1:1 ratio. The cells were transplanted into lethally-irradiated adult Boy J recipient mice and analyzed 12-16 weeks later for the relative contribution of donor engraftment. Secondary competitive repopulation experiments were then set up using BM cells from primary recipients and transplanted into lethally-irradiated Boy J mice at one donor to five recipient mice, and analyzed 12-16 weeks later for CD45.2 cells co-staining for GR-1, B220, Ter119 or CD4. (C) Primary, secondary and tertiary FL repopulation assay between gp130^FXXQ/FXXQ and gp130^+/+ grafts. FL cells with either gp130^FXXQ/FXXQ or gp130^+/+ genotypes were transplanted into lethally-irradiated adult Boy J recipient mice and analyzed 12-16 weeks later for relative contribution of donor engraftment in each lineage. Secondary or tertiary transplantation were carried out using BM cells from primary or secondary recipient mice and then transplanted into lethally-irradiated Boy J mice respectively.

Figure 2

Cross of gp130^FXXQ/FXXQ mutations onto a STAT5-deficient background and analysis of CFU and HSC activity from FL cells. (A). Colony-forming ability of STAT5ab (abbreviated as S for the figure) S^+/+gp130^+/+, S^+/+gp130^FXXQ/FXXQ, S^ΔN/ΔNgp130^+/+, and S^ΔN/ΔNgp130^FXXQ/FXXQ FL cells stimulated with hematopoietic cytokines in vitro. The frequency of CFU-Cs assayed in methylcellulose was multiplied by the total FL cellularity to derive the absolute number of FL CFU-Cs per FL. (B). A representative secondary transplantation for the repopulation assay among S^+/+gp130^+/+, S^+/+gp130^FXXQ/FXXQ, S^ΔN/ΔNgp130^+/+, and S^ΔN/ΔNgp130^FXXQ/FXXQ FL cells. FL cells with the above four genotypes were transplanted into lethally-irradiated adult Boy J recipient mice at the ratio of one donor to five recipients and analyzed 12-16 weeks later for relative contribution of donor engraftment in each lineage. Secondary transplantation was carried out using BM cells from primary recipient mice, transplanted into lethally-irradiated Boy J mice and analyzed for the donor contribution in each lineage. Results are presented as the mean ± SD (n=5-10 mice per transplant).

Figure 3

Gp130 mediated IL-6 family of cytokines signaling pathway. (Left) In the wild type gp130 mouse, IL-6 family of cytokines can activate STAT family proteins especially STAT3 and STAT1 depending on the YXXQ phosphorylation motif on four distinct tyrosines of gp130 or they can activate the SHP-2/ERK MAPK pathway depending on the YXXV phosphorylation motif on Y759 . (Right) In gp130FXXQ knockin mouse, tyrosines at the 767, 814, 905, and 915 YXXQ motifs was blocked by phenylalanine replacement thus preventing STAT family binding to gp130. Therefore, gp130FXXQ mice have lost gp130-mediated STAT1/3 activation. There are three possible explanations: (1) STAT3 is still activated in HSC via non-receptor tyrosine kinase (2) Function of STAT3 is perfectly counterbalanced by STAT1. (3) Function of STAT3 is exactly counterbalanced by unknown effectors that also dock with gp130FXXQ.