Agosti Valter, Karur Vinit, Sathyanarayana Pradeep, Besmer Peter, Wojchowski Don M
Developmental Biology Program, Sloan-Kettering Institute, New York, NY, USA.
Exp Hematol. 2009 Feb;37(2):159-71. doi: 10.1016/j.exphem.2008.10.009. Epub 2008 Dec 18.
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.
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.
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.
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.
KITL/KIT可在淋巴、髓系、肥大细胞和红系谱系中引发多种信号，并对生长、存活、迁移、黏附和分泌反应产生不同影响。目前，我们应用了PY突变等位基因敲入方法，以特异性评估KIT-PY567和KIT-PY719位点以及相关信号通路在红细胞生成过程中的可能作用。
用于研究此问题的小鼠模型包括携带敲入KIT(Y567F/Y567F)、KIT(Y569F/Y569F)、KIT(Y719F/Y719F)和KIT(Y567F/Y567F:Y569F/Y569F)等位基因的模型。通过使用5-氟尿嘧啶进行骨髓抑制以及苯肼诱导溶血来应激红细胞生成。此外，采用优化的体外分析系统对骨髓和脾脏红细胞生成进行分析，以更直接地分析对红系细胞生长、存活、发育和KIT信号事件的可能阶段特异性影响。
在Kit(Y567F/Y567F)小鼠中，稳态红细胞生成未受干扰，但因5-氟尿嘧啶或苯肼导致的贫血恢复明显受损。骨髓和脾脏中的红系祖细胞扩增均出现缺陷。对慢性促红细胞生成素给药的反应也受到损害。在体外，Kit(Y567F/Y567F)（原）红细胞的发育从Kit(pos)CD71(high)阶段偏向随后的Kit(neg)CD71(high)区室。增殖以及一定程度上的存活能力也受到损害。Kit(Y567F/Y567F:Y569F/Y569F)小鼠的红系祖细胞存在类似的阶段特异性缺陷，但KIT(Y719F/Y719F)小鼠的则没有。进一步使用Kit(Y567F/Y567F)红细胞分析KIT-PY567依赖性信号。MEK-1,2/ERK-1,2信号未受影响，而AKT、p70S6K，尤其是JNK2/p54通路被选择性减弱。
非冗余的KIT-PY567导向的红细胞内在信号对应激红细胞生成具有选择性关键作用。这些研究也有助于理解KIT如何在不同祖细胞和谱系中指导不同的结果。
