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

 描述（由申请人提供）：造血细胞移植（HCT）用于治疗恶性和非恶性疾病，但HCT是一种高成本手术，具有可变的免疫重建结果，包括发病率和死亡率风险。更好地了解骨髓（BM）来源的造血干细胞（HSC）和环境，称为小生境，影响免疫重建是必要的，以改善治疗结果。造血干细胞（HSCs）是一种多能性细胞，可分化为各种造血细胞系。在BM生态位中，HSC与基质间充质干细胞（MSC）密切接触，后者分泌CXCL 12以刺激HSC上的G蛋白偶联受体（GPCR）CXCR 4。CXCL 12/CXCR 4连接通过细胞内G蛋白激活信号传导，并在维持静止HSC和干细胞保留、归巢和动员中起关键作用。G蛋白偶联受体激酶3（GRK 3）通过触发脱敏、募集β-抑制蛋白和GPCR内化来调节CXCR 4;然而，GRK 3在干细胞中的调节功能以及对造血至关重要的其他GPCR尚不清楚。我们假设GRK 3对GPCR信号相互作用的调节控制MSC和HSC的细胞功能。我们发表的工作表明GRK 3缺陷（-/-）小鼠减少了造血细胞上的CXCR 4内化，从而增强了CXCL 12/CXCR 4信号传导和细胞迁移。我们还观察到GRK 3 - 1-小鼠具有细胞过多的BM和增加的HSC。我的离体初步数据显示GRK 3-/-HSC响应于CXCL 12具有增加的增殖，其在CXCR 4拮抗剂存在下降低至野生型（WT）水平。我对移植到GRK 3-/-受体的GRK 3-/-供体骨髓的初步短期移植研究显示，与对照WT供体骨髓移植到WT受体相比，造血细胞增殖显著增加。重要的是，与对照相比，GRK 3-/-供体骨髓进入WT受体和WT供体骨髓进入GRK 3-/-受体具有增殖增加的中间表型，表明GRK 3缺乏对HSC和基质BM生态位组分的影响。GRK 3-/-MSC具有增强的增殖和发育成CXCL 12丰富的网状（CAR）样细胞和前成骨细胞的倾向，两者都已知增强造血。虽然我们有数据支持GRK 3对白细胞和HSC上的CXCL 12/CXCR 4信号相互作用的调节作用，但GRK 3对MSC调节以诱导先前讨论的增强的表型的GPCR是未知的。为了验证我们的假设，我将定义GRK 3调节CXCL 12/CXCR 4信号传导影响HSC功能的机制（AIM 1），并确定GRK 3调节的MSC上的GPCR（AIM 2）。最后，我将通过进行长期HCT研究（AIM 3）来研究GRK 3缺陷是否改善HCT结果。如果我们揭示GRK 3调节两个在造血中起关键作用的干细胞群体-HSC和MSC-那么我们将确定一个潜在增强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.