单磷酸腺苷脱氨酶3通过CREB/ADCY10信号通路调控造血干细胞骨髓重建能力

doi:10.3969/j.issn.1674-8115.2026.06.001

上海交通大学学报（医学版） ›› 2026, Vol. 46 ›› Issue (6): 693-704.doi: 10.3969/j.issn.1674-8115.2026.06.001

• 创新团队成果专栏 • 下一篇

单磷酸腺苷脱氨酶3通过CREB/ADCY10信号通路调控造血干细胞骨髓重建能力

张悦¹, 夏一秋¹, 赫肖肖², 陈迟琪¹, 张亚萍¹, 王宇¹, 郑俊克¹(), 谢莉¹(), 于卓¹()

^1.上海交通大学基础医学院病理生理学系，细胞分化与凋亡教育部重点实验室，上海 201318
^2.上海交通大学医学院附属新华医院血液内科，上海 200092

收稿日期:2026-02-28 接受日期:2026-03-30 出版日期:2026-06-28 发布日期:2026-06-29
通讯作者: 于卓，研究员，博士；电子信箱：yuzhuo78@aliyun.com
谢莉，实验师，博士；电子信箱：xieli100@126.com
郑俊克，研究员，博士；电子信箱：zhengjunke@shsmu.edu.cn。
基金资助:
国家自然科学基金(82570151;82430007;82470121;82370180;32571298;32371160);国家重点研发计划(2024YFA1803500)

Adenosine monophosphate deaminase 3 regulates the bone marrow reconstitution ability of hematopoietic stem cells through the CREB/ADCY10 signaling pathway

Zhang Yue¹, Xia Yiqiu¹, He Xiaoxiao², Chen Chiqi¹, Zhang Yaping¹, Wang Yu¹, Zheng Junke¹(), Xie Li¹(), Yu Zhuo¹()

^1.Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine, Shanghai 201318, China
^2.Department of Hematology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China

Received:2026-02-28 Accepted:2026-03-30 Online:2026-06-28 Published:2026-06-29
Contact: Yu Zhuo, E-mail: yuzhuo78@aliyun.com
Xie Li, E-mail: xieli100@126.com
Zheng Junke, E-mail: zhengjunke@shsmu.edu.cn.
Supported by:
National Natural Science Foundation of China(82570151;82430007;82470121;82370180;32571298;32371160);National Key Research and Development Program of China(2024YFA1803500)

摘要/Abstract

摘要：

目的·探究单磷酸腺苷脱氨酶3（adenosine monophosphate deaminase 3，AMPD3）在造血干细胞（hematopoietic stem cell，HSC）中的功能和对急性髓系白血病（acute myeloid leukemia，AML）的影响。方法·通过Cre/LoxP系统繁育造血系统条件性敲除Ampd3的小鼠（Vav1-Cre；Ampd3^fl/fl）和对照组小鼠（Ampd3^fl/fl）。体内骨髓竞争性移植实验检测Ampd3对HSC骨髓重建和谱系分化能力的影响。转录组测序（RNA sequencing，RNA-seq）比较Vav1-Cre；Ampd3^fl/fl和Ampd3^fl/fl小鼠HSC差异基因和通路。实时反转录定量PCR（real-time reverse transcription quantitative PCR，RT-qPCR）验证差异基因并确定潜在靶点，构建潜在靶点的短发夹RNA（short hairpin RNA，shRNA），体内、外挽救实验验证相关靶点的功能，并通过Western blotting、免疫荧光染色和双荧光素酶报告基因实验等探究其上游调控因子。构建MLL-AF9诱导的AML模型并进行体内系列移植实验观察Ampd3对疾病进展的影响。结果·在骨髓竞争性移植实验中，Vav1-Cre；Ampd3^fl/fl小鼠骨髓重建能力显著高于Ampd3^fl/fl组，但其向髓系细胞、T淋巴细胞、B淋巴细胞谱系分化比例没有差异。RNA-seq分析发现Vav1-Cre；Ampd3^fl/fl小鼠HSC中环磷酸腺苷（cyclic adenosine monophosphate，cAMP）信号通路等显著富集，差异基因中腺苷酸环化酶10（adenylate cyclase 10，Adcy10）显著上调。在32D细胞中敲低Ampd3后细胞增殖速度显著增加，但同时敲低Adcy10后细胞增殖速度显著减慢；体内挽救实验显示，在敲除Ampd3的造血干祖细胞中进一步敲低Adcy10，可显著降低造血干祖细胞的骨髓重建能力，提示Adcy10是Ampd3的下游调控靶点。此外，Vav1-Cre；Ampd3^fl/fl小鼠HSC中磷酸化的环磷酸腺苷反应元件结合蛋白（phosphorylated cAMP response element-binding protein，p-CREB）表达水平相对于Ampd3^fl/fl小鼠显著增加，免疫荧光染色也观察到p-CREB在Vav1-Cre；Ampd3^fl/fl小鼠HSC细胞核中表达更强；同时双荧光素酶报告基因实验证实CREB能够激活Adcy10转录，且伴随着p-CREB和总CREB表达的增加；CREB抑制剂666-15能够显著下调Adcy10的mRNA和蛋白表达水平。但是在造血系统敲除Ampd3不影响AML进展。结论·在小鼠造血系统敲除Ampd3后，p-CREB的表达增高，激活Adcy10转录，从而促进HSC的骨髓重建能力；而Ampd3对AML的进展并无影响，提示Ampd3主要参与应激状态下HSC功能的调控。

关键词: 造血干细胞, 单磷酸腺苷脱氨酶3, 骨髓重建, 腺苷酸环化酶10, 环磷酸腺苷反应元件结合蛋白, 急性髓系白血病

Abstract:

Objective ·To investigate the role of adenosine monophosphate deaminase 3 (AMPD3) in hematopoietic stem cells (HSCs) and acute myeloid leukemia (AML). Methods ·Hematopoietic system-specific Ampd3 knockout mice (Vav1-Cre;Ampd3^fl/fl) and control mice (Ampd3^fl/fl) were generated using the Cre/LoxP system. An in vivo competitive bone marrow transplantation assay was performed to examine the effects of Ampd3 on the bone marrow reconstitution and lineage differentiation capacities of HSCs. RNA sequencing (RNA-seq) was performed to compare differentially expressed genes and enriched pathways in HSCs from Vav1-Cre;Ampd3^fl/fl and Ampd3^fl/fl mice. Real-time reverse transcription quantitative PCR (RT-qPCR) was used to validate differentially expressed genes and identify potential targets. Short hairpin RNAs (shRNAs) targeting the identified geneswere constructed, and in vivo and in vitro rescue experiments were conducted to validate their functions. Western blotting, immunofluorescence staining, and dual-luciferase reporter assays were then employed to explore the upstream regulatory factors. An MLL-AF9-induced AML model was established, and serial in vivo transplantation experiments were performed to observe the impact of Ampd3 on disease progression. Results ·In the competitive bone marrow transplantation assay, Vav1-Cre;Ampd3^fl/fl mice exhibited significantly higher bone marrow reconstitution ability than the Ampd3^fl/fl mice, but no differences were observed in myeloid, T lymphocyte, or B lymphocyte lineage differentiation. RNA-seq analysis revealed significant enrichment of the cyclic adenosine monophosphate (cAMP) signaling pathway and other pathways in HSCs from Vav1-Cre;Ampd3^fl/fl mice, and among the differentially expressed genes, adenylate cyclase 10 (Adcy10) was significantly upregulated. Knockdown of Ampd3 in 32D cells significantly accelerated cell proliferation, whereas simultaneous knockdown of Adcy10 markedly slowed the proliferation. In vivo rescue experiments showed that further knockdown of Adcy10 in Ampd3-deficient hematopoietic stem and progenitor cells significantly reduced their bone marrow reconstitution capacity, suggesting that Adcy10 was a downstream target of Ampd3. Furthermore, the expression level of phosphorylated cAMP response element-binding protein (p-CREB) in HSCs from Vav1-Cre;Ampd3^fl/fl mice was significantly increased compared with that in Ampd3^fl/fl mice. Immunofluorescence staining also revealed stronger nuclear localization of p-CREB in HSCs from Vav1-Cre;Ampd3^fl/fl mice. Dual-luciferase reporter assays confirmed that CREB was able to activate the transcription of Adcy10, accompanied by increased expression of both p-CREB and total CREB. The CREB inhibitor 666-15 significantly downregulated the mRNA and protein expression levels of Adcy10. However, hematopoietic-specific knockout of Ampd3 did not affect AML progression. Conclusion ·Hematopoietic-specific deletion of Ampd3 in mice leads to elevated p-CREB expression, which activates Adcy10 transcription and thereby enhances the bone marrow reconstitution capacity of HSCs. Ampd3 does not affect AML progression, suggesting that Ampd3 is mainly involved in regulating HSC function under stress conditions.

Key words: hematopoietic stem cell (HSC), adenosine monophosphate deaminase 3 (AMPD3), bone marrow reconstitution, adenylate cyclase 10 (ADCY10), cAMP response element-binding protein (CREB), acute myeloid leukemia (AML)

中图分类号:

R363.2

张悦, 夏一秋, 赫肖肖, 陈迟琪, 张亚萍, 王宇, 郑俊克, 谢莉, 于卓. 单磷酸腺苷脱氨酶3通过CREB/ADCY10信号通路调控造血干细胞骨髓重建能力[J]. 上海交通大学学报（医学版）, 2026, 46(6): 693-704.

Zhang Yue, Xia Yiqiu, He Xiaoxiao, Chen Chiqi, Zhang Yaping, Wang Yu, Zheng Junke, Xie Li, Yu Zhuo. Adenosine monophosphate deaminase 3 regulates the bone marrow reconstitution ability of hematopoietic stem cells through the CREB/ADCY10 signaling pathway[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026, 46(6): 693-704.

图/表 5

图1 血液系统 Ampd3 条件性敲除小鼠的构建Note： A/B. Expression profiles of AMPD3/Ampd3 in human (A) and mouse (B) hematopoietic cells. Data were obtained from the Bloodspot database. C. RT-qPCR analysis of Ampd3 expression in various mouse hematopoietic cell populations. D. Schematic diagram for the strategy for the construction of the Ampd3 conditional knockout mouse line. E. Representative electrophoretic images of PCR genotyping for the WT allele, Ampd3fl/wt allele, Ampd3fl/fl allele, and Vav1-Cre transgene in mice. F. Ampd3 knockout efficiency in hematopoietic cells from Vav1-Cre;Ampd3fl/fl mice by RT-qPCR analysis. G. AMPD3 protein levels in hematopoietic cells from Vav1-Cre;Ampd3fl/fl and Ampd3fl/fl measured by Western blotting. BM—bone marrow; WT—wild type; KO—knockout.

Fig 1 Generation of hematopoietic-specific Ampd3 conditional knockout mice

图2 血液系统特异性敲除 Ampd3 后HSC骨髓重建能力增强Note： A. The frequencies of LT-HSCs, ST-HSCs, and MPPs in the bone marrow from Vav1-Cre;Ampd3fl/fl and Ampd3fl/flmice detected by flow cytometry. B. The proportions of myeloid cells (Mac1+Gr1+), B cells (B220+), and T cells (CD3e+) in peripheral blood (PB) from Vav1-Cre;Ampd3fl/fl and Ampd3fl/fl mice quantitated by flow cytometric analysis. C. Representative flow cytometric analyses of competitive reconstitution assay with donor cells isolated from CD45.1 recipient mice receiving Vav1-Cre;Ampd3fl/fl and Ampd3fl/fl (CD45.2+) mice bone marrow cells at 16 weeks after transplantation. D. Quantification data of the flow cytometric analyses. The repopulation of CD45.2+ donor cells was measured at 4 to 16 weeks after transplantation (n=5, ①P=0.036, ②P=0.007). E. The frequencies of HSCs from donor cells of Vav1-Cre;Ampd3fl/fl and Ampd3fl/fl mice in the bone marrow of CD45.1 recipients as measured by flow cytometric analysis at 16 weeks post-transplantation (n=3). F. The multilineage contribution of donor cells to myeloid cells, B cells, and T cells in PB quantitated by flow cytometric analysis at 16 weeks post-transplantation (n=3).

Fig 2 Enhanced bone marrow reconstitution capacity of HSCs following hematopoietic-specific deletion of Ampd3

图3 Adcy10 是 Ampd3 介导信号的潜在下游靶点Note： A. KEGG pathway enrichment analysis showing the top 20 significantly enriched pathways identified by RNA-seq in HSCs from Vav1-Cre;Ampd3fl/flvsAmpd3fl/fl. B. Volcano plot showing DEGs in HSCs from Vav1-Cre;Ampd3fl/flvsAmpd3fl/fl. C. RT-qPCR analysis of the potential candidate genes in HSCs from Vav1-Cre;Ampd3fl/flvsAmpd3fl/fl(n=3, ①P=0.001, ②P=0.002, ③P=0.013, ④P=0.006). D. ADCY10 activity in HSCs from Vav1-Cre;Ampd3fl/flvsAmpd3fl/fl determined by ELISA (n=5, ①P=0.012). E. cAMP levels in HSCs from Vav1-Cre;Ampd3fl/flvsAmpd3fl/fldetermined by ELISA (n=3, ①P=0.004). F/G. Knockdown efficiencies of shRNAs targeting murine Ampd3 (F) and Adcy10 (G) determined by RT-qPCR (n=3). H. Proliferation of 32D cells following knockdown of Ampd3 or/and Adcy10 (n=3, ①P<0.001). I. Quantification data of the frequencies of donor cells in peripheral blood at 4 and 8 weeks after transplantation in the in vivo rescue assay. Donor bone marrow Lin- cells isolated from Vav1-Cre;Ampd3fl/fl and Ampd3fl/fl mice were infected with either a non-targeting control shRNA or an Adcy10-targeting shRNA and subsequently transplanted into lethally irradiated CD45.1 recipient mice (n=5, ①P=0.010, ②P<0.001, ③P=0.006). FC—fold change.

Fig 3 Adcy10 is a potential downstream target of Ampd3-mediated signaling

图4 Ampd3 通过上调p-CREB转录激活 Adcy10Note： A. Protein levels of p-CREB and total CREB in HSCs from Vav1-Cre;Ampd3fl/fl or Ampd3fl/flmice determined by Western blotting. B. Immunofluorescence staining of p-CREB in LT-HSC from Vav1-Cre;Ampd3fl/fl or Ampd3fl/fl mice (×400). C. Relative luciferase activities in HEK-293T cells co-transfected with a Creb plasmid and a luciferase reporter construct containing the Adcy10 promoter (n=3, ①P=0.022, ②P=0.007, ③P=0.003, ④P<0.001). D. Protein levels of p-CREB and total CREB in HEK-293T cells co-transfected with a Creb plasmid and a luciferase reporter construct containing the Adcy10 promoter, as determined by Western blotting. E. ADCY10 protein levels in 32D cells treated with DMSO or the CREB inhibitor 666-15, as determined by Western blotting. F. Relative Adcy10 mRNA levels in 32D cells treated with DMSO or 666-15, as determined by RT-qPCR (n=3, ①P=0.009, ②P=0.017).

Fig 4 Ampd3 regulates Adcy10 expression through upregulation of p-CREB

图5 造血系统敲除 Ampd3 不影响AML进展Note： A/B. Representative flow cytometric analyses of the proportion of myeloid cells among YFP+ leukemia cells (A) and quantification of the data shown in panel A (B, n=5). C/D. Representative flow cytometric analyses of YFP+ leukemia cells in peripheral blood following primary transplantation (C) and quantification of the data shown in panel C (D, n=5). E. Overall survival of recipient mice transplanted with MLL-AF9+ bone marrow cells derived from Vav1-Cre;Ampd3fl/fl or Ampd3fl/fl in the primary transplantation assay (n=5). F–H. Representative image of spleens and livers from recipient mice following primary transplantation (F), and quantification of relative spleen (G) and liver (H) weights. I. Overall survival of recipient mice transplanted with MLL-AF9+ bone marrow cells derived from Vav1-Cre;Ampd3fl/fl or Ampd3fl/fl in the secondary transplantation assay (n=5).

Fig 5 Hematopoietic-specific deletion of Ampd3 does not affect AML progression

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单磷酸腺苷脱氨酶3通过CREB/ADCY10信号通路调控造血干细胞骨髓重建能力

Adenosine monophosphate deaminase 3 regulates the bone marrow reconstitution ability of hematopoietic stem cells through the CREB/ADCY10 signaling pathway

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