p38α plays differential roles in hematopoietic stem cell activity dependent on aging contexts. p38α plays differential roles in hematopoietic stem cell activity dependent on aging contexts

Hematopoietic stem cells (HSCs) and their progeny sustain lifetime hematopoiesis. Aging alters HSC function, number, and composition and increases risk of hematological malignancies, but how these changes occur in HSCs remains unclear. Signaling via p38MAPK has been proposed as a candidate mechanism underlying induction of HSC aging. Here, using genetic models of both chronological and premature aging, we describe a multimodal role for p38α, the major p38MAPK isozyme in hematopoiesis, in HSC aging. We report that p38α regulates differentiation bias and sustains transplantation capacity of HSCs in the early phase of chronological aging (from young to 1-year-old). However, p38α decreased HSC transplantation capacity in the late progression phase of chronological aging (from 1- to 2-years-old). Furthermore, co-deletion of p38α in mice deficient in Ataxia-telangiectasia mutated (Atm), a model of premature aging, exacerbated aging-related HSC phenotypes seen in Atm single mutant mice. Mechanistically, p38α makes a positive contribution to inflammation during the late phase aging, resulting in defects in 2-year-old HSCs. Overall, we propose multiple functions of p38MAPK, which both promotes and suppresses HSC aging context-dependently. Overall design: we injected CAG-CreERT2: p38αflox/flox (p38αfl/fl) mice aged approximately 1 year (50–60 weeks) with TAM to delete p38α, and then analyzed those mice and corresponding controls 1 year later (i.e., at 2 years-of-age) (p38αcKO). lineage-Sca-1+c-Kit+CD34-Flt3- cells (LT-HSC) in the bone marrow (femur and tibia) from 2-year-old wild type (WT) or p38αcKO C57BL/6J mice (Ly5.2) were sorted into SF-O3 medium. Then, sorted cells were centrifuged at 340 x g for 5 min at 4°C and lysed with 75µL RLT buffer + 0.75µL 2-ME. RNA extraction, cDNA synthesis, microarray analysis, and data normalization were outsourced to DNA Chip Research Inc.

造血干细胞（Hematopoietic stem cells, HSCs）及其子代细胞可终生维持造血功能。衰老会改变HSC的功能、数量与组成，并增加血液系统恶性肿瘤的发病风险，但HSC内此类变化的发生机制仍有待阐明。p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase, p38MAPK）信号通路曾被提出是介导HSC衰老的潜在候选机制。本研究借助生理性衰老与早衰性衰老的遗传模型，揭示了造血系统中主要p38MAPK同工型p38α在HSC衰老过程中的多维度调控作用。研究发现，在生理性衰老的早期阶段（从年轻小鼠至12月龄小鼠），p38α可调控HSC的分化偏倚并维持其移植能力；而在生理性衰老的晚期进展阶段（从12月龄至24月龄小鼠），p38α反而会降低HSC的移植能力。此外，在共济失调毛细血管扩张症突变（Ataxia-telangiectasia mutated, Atm）缺陷的早衰模型小鼠中联合敲除p38α，会加重Atm单突变小鼠中已观察到的衰老相关HSC表型。机制层面，p38α在衰老晚期可正向调控炎症反应，进而导致24月龄HSC的功能缺陷。综上，我们提出p38MAPK具有多重功能，其对HSC衰老的调控作用具有情境依赖性，既可促进也可抑制HSC衰老。

总体实验设计：我们对约12月龄（50~60周龄）的CAG-CreERT2:p38αflox/flox（p38αfl/fl）小鼠注射他莫昔芬（Tamoxifen, TAM）以敲除p38α，并在1年后（即小鼠达到24月龄时）对该条件性敲除小鼠（p38αcKO）及其对应对照小鼠进行分析。从24月龄野生型（WT）C57BL/6J（Ly5.2）小鼠或p38α条件性敲除（p38αcKO）小鼠的骨髓（股骨与胫骨）中分选谱系阴性-Sca-1+c-Kit+CD34-Flt3-细胞（长期造血干细胞，long-term hematopoietic stem cells, LT-HSC）至SF-O3培养基中。随后将分选得到的细胞于4℃下以340×g离心5分钟，并用75μL RLT缓冲液+0.75μL 2-巯基乙醇（2-mercaptoethanol, 2-ME）进行裂解。RNA提取、cDNA合成、芯片分析及数据标准化工作均委托至DNA Chip Research Inc.完成。