A KIT juxtamembrane PY567 -directed pathway provides nonredundant signals for erythroid progenitor cell development and stress erythropoiesis

Abstract

Objective: KITL/KIT can elicit diverse sets of signals within lymphoid, myeloid, mast, and erythroid lineages, and exert distinct effects on growth, survival, migration, adhesion, and secretory responses. Presently, we have applied a PY-mutant allele knockin approach to specifically assess possible roles for KIT-PY567 and KIT-PY719 sites, and coupled pathways, during erythropoiesis.

Materials and methods: Mouse models used to investigate this problem include those harboring knocked-in KIT(Y567F/Y567F), KIT(Y569F/Y569F), KIT(Y719F,Y719F), and KIT(Y567F/Y567F:Y569F/Y569F) alleles. The erythron was stressed by myelosuppression using 5-fluorouracil, and by phenylhydrazine-induced hemolysis. In addition, optimized systems for ex vivo analyses of bone marrow and splenic erythropoiesis were employed to more directly analyze possible stage-specific effects on erythroid cell growth, survival, development and KIT signaling events.

Results: In Kit(Y567F/Y567F) mice, steady-state erythropoiesis was unperturbed while recovery from anemia due to 5-fluorouracil or phenylhydrazine was markedly impaired. Deficiencies in erythroid progenitor expansion occurred both in the bone marrow and the spleen. Responses to chronic erythropoietin dosing were also compromised. Ex vivo, Kit(Y567F/Y567F) (pro)erythroblast development was skewed from a Kit(pos)CD71(high) stage toward a subsequent Kit(neg)CD71(high) compartment. Proliferation and, to an extent, survival capacities were also compromised. Similar stage-specific defects existed for erythroid progenitors from Kit(Y567F/Y567F:Y569F/Y569F) but not KIT(Y719F/Y719F) mice. Kit(Y567F/Y567F) erythroblasts were used further to analyze KIT-PY567-dependent signals. MEK-1,2/ERK-1,2 signaling was unaffected while AKT, p70S6K, and especially JNK2/p54 pathways were selectively attenuated.

Conclusions: Nonredundant KIT-PY567-directed erythroblast-intrinsic signals are selectively critical for stress erythropoiesis. Investigations also add to an understanding of how KIT directs distinct outcomes among diverse progenitors and lineages.

Figure 1. In Kit^Y567F/Y567F mice, high-dose 5-fluorouracil (5-FU) administration leads to fatal anemia

(A) Following a single dose of 5-FU at 300 mg/kg, Kit^Y567F/Y567F mice exhibited sustained low-level hematocrits, and compromised survival (mean ± standard error; n = 5). (B) Failed splenic erythropoiesis in Kit^Y567F/Y567F mice following 5-FU dosing—As analyzed at day 15 post 5-FU, splenic hypertrophy faltered in Kit^Y567F/Y567F mice, and Ter119^pos erythroblasts were underrepresented. (C) Cytospin analyses of wild-type Kit^+/+ (wt) and Kit^Y567F/Y567F bone marrow preparations (day 15 post 5-FU injection, upper panels). As assessed by flow cytometry, Ter119^pos cells also were notably underrepresented in bone marrow among Kit^Y567F/Y567F mice. Data shown are representative of two independent analyses.

Figure 2. Lower level 5-fluorouracil (5-FU) dosing of Kit^Y567F/Y567F mice leads to sustained macrocytic anemia

Following a single injection of 5-FU at 150 mg/kg, red blood cell numbers (RBC), hematocrits, mean red cell volumes (MCV) and platelet levels were determined. From day 14 through 21, Kit^Y567F/Y567F mice exhibited decreased RBCs and lowered hematocrits. For Kit^Y567F/Y567F red cells, also note the increase in MCV at day 18. Graphed values are means ± standard error (n = 10 mice per group). The Student’s t-test assuming equal variances between the two samples was applied to determine statistical significance.

Figure 3. Defective erythropoiesis in Kit^Y567F/Y567F mice during phenylhydrazine-induced anemia

(A) Wild-type Kit^+/+ (wt) and Kit^Y567F/Y567F mice were treated with phenylhydrazine (PHZ) (60 mg/kg at 0 and 24 hours). At the indicated time points, hematocrits (left panel) and spleen size (right panel) were assessed. To determine statistical significance between samples the Student’s t-test assuming equal variances was used. (B) At day 5 post-PHZ treatment, frequencies of CD71^posTer119^pos erythroblasts in spleen were analyzed (by flow cytometry). For Kit^Y567F/Y567F erythroblasts, note the approximate fivefold underrepresentation. Data are representative of three independent analyses.

Figure 4. Ex vivo, Kit^Y567F/Y567F erythroid progenitors are underrepresented at a Kit^posCD71^high erythroblast stage, and become overrepresented within a Kit^negCD71^high compartment

(A) Kit^pos progenitor cells were isolated from bone marrow preparations from Kit^Y567F/Y567F mice and wild-type Kit^+/+ (wt) littermates, and expanded in SP34-ex medium. Among developing erythroblasts, CD117/KIT and CD71 marker expression was assayed over 6-days of culture. For Kit^Y567F/Y567F cells, note the limited representation of Kit^posCD71^high progenitors, and increased relative frequencies of Kit^negCD71^high erythroblasts (especially at days 4 and 5). Data shown are representative of three independent experiments. (B) Also graphed (as mean ± standard error, n = 3) are ratios of Kit^neg/Kit^pos erythroblasts for wild-type and Kit^Y567F/Y567F cells at 48, 72, and 96 hours of culture.

Figure 5. Unlike Kit^Y567F/Y567F erythroblasts, Lyn^−/− erythroid progenitors accumulate ex vivo within a Kit^posCD71^high compartment

Within SP34-ex cultures of Kit^Y567F/Y567F, Lyn^−/−, and wild-type Kit^+/+ (wt) erythroid progenitor cells, the development of Kit^posCD71^high and Kit-^negCD71^high erythroblasts was assayed. For Lyn^−/− cells, note the increased frequencies of Kit^posCD71^high erythroblasts (lower subpanel).

Figure 6. Kit^Y567F/Y567F proerythroblasts exhibit decreased proliferation and survival capacities

(A) Ex vivo growth profiles are illustrated for bone marrow–derived (pro)erythroblasts from Kit^+/+, Kit^Y567F/Y567F, and Lyn^−/− mice. Kit^pos progenitors (as isolated from bone marrow of Kit^Y567F/Y567F and congenic control mice) were cultured in SP34-ex media in the presence of stem cell factor (SCF; 100 ng/mL) and erythropoietin (Epo; 2.5 U/mL). Viable cells counts were performed daily. Note the marked defect in expansion potential of Kit^Y567F/Y567F erythroblasts. (B) Left panel: bone marrow–derived progenitor cells were prepared from wild-type Kit^+/+ (wt-KIT) and Kit^Y567F/Y567F mice, and expanded in SP34-ex medium. At day 5, frequencies of Annexin-V positive vs negative cells among Kit^posCD71^high and Kit^negCD71^high erythroblasts were analyzed. (B) Right panel: For splenic erythroblasts produced in vivo in response to phenylhydrazine-induced anemia, substantial increases in frequencies of Annexin-V–positive apoptotic erythroblasts also were observed. (C) Left panel: At day 3.5 post-phenylhydrazine treatment of Kit^Y567F/Y567F and Kit^+/+ mice, BrdU was injected (tail vein). At 1.5 hours spleens were isolated. Post Ter119^pos cell depletion, CD71^high erythroid progenitors were retrieved via magnetic-activated cell sorting, and frequencies of S-phase (proliferating) CD71^high erythroid progenitors were estimated via flow cytometry. (C) Right panel: At day 3.5 post-phenylhydrazine treatment of Kit^Y567F/Y567F and Kit^+/+ mice, erythroid progenitors from spleen were prepared and cultured in the presence of KIT-L at 50 ng/mL (and Epo at 2.5 U/mL). At 16 hours of culture, cells were stained with YoPro1 to determine cell survival. (D) To test whether defects in Kit^Y567F/Y567F erythroblast expansion are exhibited during Epo-induced erythropoiesis, mice were injected with Epo (300 U/kg for 5 days) and increases in hematocrits were assayed over an 11-day period. Mean ± standard error are graphed (n = 4). Note the deficit response among Kit^Y567F/Y567F mice. (E) Kit^Y567F/Y567F and Kit^+/+ mice were dosed with both Epo (1800 U/kg) and stem cell factor (SCF) (100 µg/kg). At day 3 post dosing, bone marrow Kit^posCD71^high erythroid progenitors were isolated and cultured in SP34-ex media. Hematopoietic cytokines were withdrawn for 4 hours, and cells were then exposed to KIT-L at 50 ng/mL. At 8 hours, cell cycle phase distributions were determined via staining with DRAQ5.

Figure 7. KIT activation of AKT, p70S6K, and JNK2/p54 is attenuated in Kit^Y567F/Y567F erythroblasts

(A) Attenuated AKT and p70S6K activation in Kit^Y567F/Y567F erythroblasts—Bone marrow–derived progenitor cells from wild-type Kit^+/+ (wt-Kit) and Kit^Y567F/Y567F mice were expanded in SP34-ex medium. Kit^posCD71^high erythroblasts were then isolated (by magnetic-activated cell sorting) and incubated for 5.5 hours in the absence of hematopoietic cytokines. Following exposure to KITL (150 ng/mL) for the indicated intervals, levels of phospho- (and total) AKT (A1) and p70-S6K (A2) were assayed by Western blotting. For Kit^Y567F/Y567F erythroblasts, note the attenuated phosphorylation/activation of AKT (especially 5 minutes). Phospho-p70S6K levels also were diminished in Kit^{Y567F/ Y567F} erythroblasts (especially at 15 and 30 minutes). Signal quantitation was by densitometry (A3, A4). (B) Attenuated JNK2/p54 activation in Kit^Y567F/Y567F erythroblasts—Primary erythroblasts were expanded and prepared as above (A). KITL-induced levels of phospho- JNK2/p54 and JNK1/p46 were assayed by immunoblotting (B1). In Kit^Y567F/Y567F erythroblasts, note the selectively attenuated activation of JNK2/p54. In parallel, levels of phospho-ERK1,2 were determined (B2), including signal quantitation (B3, B4).