G protein-coupled receptor kinase 3 (GRK3) regulation of receptor signaling in stem cell function and transplantation

 描述(申请人提供)：造血细胞移植(HCT)用于治疗恶性和非恶性疾病，但HCT是一种高成本的程序，具有不同的免疫重建结果，包括发病率和死亡风险。为了改善治疗结果，有必要更好地了解骨髓来源的造血干细胞(HSCs)和影响免疫重建的环境(称为生态位)。造血干细胞(HSCs)是一种多能细胞，可以分化为所有的造血系。在骨髓间充质干细胞与基质间充质干细胞(MSCs)密切接触，MSCs分泌CXCL12刺激HSCs上的G蛋白偶联受体CXCR4。CXCL12/CXCR4连接通过细胞内G蛋白激活信号，在维持静止的HSCs和干细胞的保留、归巢和动员中起着至关重要的作用。G蛋白偶联受体激酶3(GRK3)通过触发脱敏、糖蛋白募集和GPCR内化来调节CXCR4；然而，GRK3在干细胞中的调节功能以及其他对造血至关重要的GPCRs尚不清楚。我们假设GRK3对GPCR信号相互作用的调节控制着MSCs和HSCs的细胞功能。我们发表的工作表明，GRK3缺陷(-/-)小鼠减少了CXCR4在造血细胞上的内化，从而增强了CXCL12/CXCR4信号转导和细胞迁移。我们还观察到GRK3--小鼠骨髓细胞增多，HSCs增多。我的体外初步数据显示，GRK3-/-HSC对CXCL12的反应增加了增殖，在CXCR4拮抗剂存在的情况下，GRK3-/-HSCs的增殖减少到野生型(WT)水平。我对GRK3-/-供者骨髓移植到GRK3-/-受者体内的初步短期移植研究显示，与对照WT供者骨髓相比，GRK3-/-受者的造血细胞增殖显著增加。重要的是，GRK3-/-供者骨髓进入WT受者和WT供者骨髓进入GRK3-/-受者与对照组相比有中等程度的增殖增加表型，提示GRK3缺乏对HSCs和基质BM生态位成分的影响。GRK3-/-MSCs具有促进增殖和向CXCL12丰富的网状(CAR)样细胞和成骨前细胞发展的倾向，两者都能促进造血。虽然我们有数据支持GRK3对白细胞和HSC上CXCL12/CXCR4信号相互作用的调节作用，但GRK3调节MSCs以诱导前面讨论的增强表型的GPCR(S)尚不清楚。为了验证我们的假设，我将确定GRK3调控CXCL12/CXCR4信号影响HSC功能的机制(S)(AIM 1)，并测定GRK3调节MSCs的GPCR3(AIM2)(S)。最后，我将通过进行长期的HCT研究来调查GRK3缺乏是否改善了HCT结局(AIM 3)。如果我们揭示GRK3调节两个在造血中起关键作用的干细胞群体--造血干细胞和骨髓间充质干细胞--那么我们就已经确定了潜在的增强HCT的候选治疗靶点。

 DESCRIPTION (provided by applicant): Hematopoietic cell transplantation (HCT) is utilized to treat malignant and non-malignant diseases, but HCT is a high cost procedure with variable immune reconstitution outcomes that include morbidity and mortality risks. A better understanding of the bone marrow (BM)-derived hematopoietic stem cells (HSCs) and environment, termed niche, that affects immune reconstitution is warranted for improvement in therapeutic outcomes. Hematopoietic stem cells (HSCs) are multipotent cells that can differentiate into all hematopoietic lineages. In the BM niche, HSCs are in intimate contact with stromal mesenchymal stem cells (MSCs), which secrete CXCL12 to stimulate G protein-coupled receptor (GPCR) CXCR4 on HSCs. CXCL12/CXCR4 ligation activates signaling via intracellular G-proteins and plays a crucial role in the maintenance of quiescent HSCs and stem cell retention, homing, and mobilization. G protein-coupled receptor kinase 3 (GRK3) regulates CXCR4 by triggering desensitization, recruitment of ß-arrestin, and GPCR internalization; however, the regulatory function of GRK3 in stem cells, as well as other GPCRs critical to hematopoiesis, is unknown. We hypothesize regulation of GPCR signaling interactions by GRK3 controls cellular functions of both MSCs and HSCs. Our published work shows GRK3 deficient (-/-) mice have reduced CXCR4 internalization on hematopoietic cells, which consequently enhances CXCL12/CXCR4 signaling and cellular migration. We also observed GRK3-\- mice have a hypercellular BM with increased HSCs. My ex vivo preliminary data show GRK3-/- HSCs have increased proliferation in response to CXCL12, which is reduced to wildtype (WT)-level in the presence of CXCR4 antagonist. My preliminary short-term transplant investigations of GRK3-/- donor marrow transplanted into GRK3-/- recipients showed significantly increased hematopoietic cell proliferation compared to control WT donor marrow into WT recipients. Importantly, GRK3-/- donor marrow into WT recipients and WT donor marrow into GRK3-/- recipients had intermediate phenotypes of increased proliferation compared to control, suggesting an effect of GRK3 deficiency on HSCs and stromal BM niche components. GRK3-/- MSCs have enhanced proliferation and a propensity to develop into CXCL12-abundant reticular (CAR)-like cells and pre-osteoblasts, both known to enhance hematopoiesis. Although we have data to support a regulatory role of GRK3 on CXCL12/CXCR4 signaling interactions on leukocytes and HSCs, the GPCR(s) that GRK3 regulates on MSCs to induce the enhanced phenotypes previously discussed is unknown. To test our hypothesis, I will define the mechanism by which GRK3 regulation of CXCL12/CXCR4 signaling affects HSC function(s) (AIM 1) and determine the GPCR(s) on MSCs regulated by GRK3 (AIM 2). Lastly, I will investigate whether GRK3 deficiency improves HCT outcomes by performing long-term HCT studies (AIM 3). If we reveal GRK3 regulates two stem cell populations with crucial roles in hematopoiesis-- HSCs and MSCs-- then we will have identified a candidate therapeutic target for the potential enhancement of HCT.