Not so sweet: Investigating the role of c-Kit in sweet cell homeostasis

不太甜：研究 c-Kit 在甜细胞稳态中的作用

• 批准号: 10620211
• 负责人: Christina M Piarowski
• 金额: $ 3.59万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10620211
• 关键词: Adult; Affect; Behavioral; Binding; Cancer Patient; Categories; Cell Death; Cell Differentiation process; Cell Survival; Cells; Cellular Assay; Cellular biology; Cre driver; Data; Differentiation Antigens; Disease; Dysgeusia; Electrophysiology (science); Environment; Functional disorder; Genetic; Homeostasis; Human; KDR gene; Knock-out; Lead; Ligands; Mediating; Metastatic Renal Cell Cancer; Modeling; Monitor; Mus; Nerve; Organoids; Patients; Pharmaceutical Preparations; Platelet-Derived Growth Factor alpha Receptor; Play; Predisposition; Proliferating; Protein Tyrosine Kinase; Proto-Oncogene Protein c-kit; Quality of life; Receptor Cell; Receptor Protein-Tyrosine Kinases; Role; Signal Pathway; Signal Transduction; Specific qualifier value; Stem Cell Factor; Supporting Cell; Symptoms; Taste Buds; Taste Perception; Testing; Therapeutic; Time; Tissues; Tongue; Treatment outcome; Tumor Angiogenesis; Type II Epithelial Receptor Cell; Type III Epithelial Receptor Cell; Tyrosine Kinase Inhibitor; Tyrosine Kinase Receptor Inhibition; cell type; experience; experimental study; in vivo; novel; pharmacologic; precursor cell; preference; prevent; progenitor; response; single-cell RNA sequencing; small molecule; stem cell proliferation; stem cells; sweet taste perception; taste system

Project Summary The sense of taste is mediated by multicellular taste buds that each house 50-100 rapidly renewing taste receptor cells (TRCs). TRCs are categorized into three main types: type I glial-like cells, type II cells that detect sweet, bitter or umami, and type III cells that detect sour. As TRCs renew, the three types are maintained in relatively stable proportions, allowing the sense of taste to remain stable over time. However, rapid turnover of taste cells makes the taste system prone to disruption by certain drugs and diseases. Cancer patients with metastatic renal cell carcinoma (mRCC) being treated with tyrosine kinase inhibitors (TKIs) often experience taste dysfunction, or dysgeusia. The primary targets of TKIs used to treat mRCC are the receptor tyrosine kinases (RTKs) VEGFR and PDGFRβ, which are not expressed in taste tissue. However, these TKIs also inhibit many off-target RTKs like Met, Ret, PDGFRα, and c-Kit. According to our single-cell RNA sequencing (scRNAseq) data, these RTKs are expressed in taste tissue in subsets of progenitors and differentiated taste cells. This suggests inhibition of these off-target RTKs may be the cause of dysgeusia and that some or all of these RTKs are necessary for proper TRC renewal and taste homeostasis. To test the role of off-target RTKs in taste homeostasis, I treated lingual organoids with the TKIs Axitinib, Cabozantinib and Sunitinib, which inhibit different combinations of off-target RTKs and frequently cause dysgeusia in patients. I found these drugs did not affect progenitor cell proliferation but instead decreased the expression of certain differentiated taste cell markers. Specifically, all three TKIs decreased expression of the sweet cell marker Tas1r2, and Tas1r2 was the only marker affected by all three drugs. Importantly, the only off-target RTK inhibited by all three drugs is c-Kit, which we find in our scRNAseq data to be most highly expressed in sweet-sensing type II TRCs. These data strongly implicate c-Kit in sweet cell homeostasis. Further analysis of our scRNAseq data reveals that c-Kit's ligand - stem cell factor (SCF), is expressed by type I and type III TRCs, raising the possibility of c-Kit mediated crosstalk within taste buds. These data in sum lead me to my hypothesis that c-Kit signaling, stimulated by crosstalk with other TRC types, is necessary for the differentiation and/or survival of sweet-sensing type II TRCs. To test this hypothesis, my first aim is to determine if c-Kit inhibition by TKI treatment leads to deficits in sweet taste in vivo by treating mice with Axitinib and performing behavioral, electrophysiological and cellular assays. My second aim is to determine if c-Kit is necessary for sweet cell differentiation or survival. I will use two genetic c-Kit knockout models under different Cre drivers to knock out c-Kit expression at different points in TRC differentiation. Lastly, I will investigate c-Kit mediated crosstalk by genetically knocking out SCF from within taste buds. Completion of this project will illuminate the role of the previously unstudied RTK c-Kit in taste homeostasis.

项目摘要 味觉是由多细胞味蕾介导的，每个味蕾有50-100个快速更新的味道。 受体细胞（TRCs）。TRC分为三种主要类型：I型胶质样细胞，II型细胞，检测 甜味、苦味或鲜味，以及检测酸味的III型细胞。在储税券续期时， 相对稳定的比例，允许味觉随时间保持稳定。然而， 味觉细胞使味觉系统易于被某些药物和疾病破坏。癌患者 用酪氨酸激酶抑制剂（TKI）治疗的转移性肾细胞癌（mRCC）通常会经历 味觉功能障碍或味觉障碍。用于治疗mRCC的TKI的主要靶点是受体酪氨酸 激酶（RTK）VEGFR和PDGFRβ，它们在味觉组织中不表达。然而，这些TKI也 抑制多种脱靶RTK，如Met、Ret、PDGFRα和c-Kit。根据我们的单细胞RNA测序 根据scRNAseq（scRNAseq）数据，这些RTK在味觉组织中的祖细胞和分化的味觉细胞亚群中表达。 细胞这表明这些脱靶RTK的抑制可能是味觉障碍的原因，并且部分或全部的 这些RTK对于正确的TRC更新和味觉稳态是必需的。测试非目标RTK的作用 在味觉稳态方面，我用TKI阿西替尼、卡博替尼和舒尼替尼治疗舌类器官， 抑制脱靶RTK的不同组合，并经常导致患者味觉障碍。我发现这些 药物并不影响祖细胞增殖，而是降低了某些分化的 味觉细胞标志物具体来说，所有三种TKI都降低了甜细胞标志物Tas 1 r2的表达， Tas 1 r2是唯一受这三种药物影响的标记物。重要的是，唯一的脱靶RTK抑制所有 三种药物是c-Kit，我们在scRNAseq数据中发现它在甜味敏感II型中表达最高， 储税券。这些数据强烈暗示c-Kit在甜细胞内稳态中。我们的scRNAseq数据的进一步分析 揭示了c-Kit的配体-干细胞因子（SCF），由I型和III型TRC表达，提高了TRC的表达水平。 c-Kit介导的味蕾内串扰的可能性。总之，这些数据使我得出一个假设， 由与其他TRC类型的串扰刺激的信号传导对于区分和/或 甜味敏感II型TRCs的存活。为了验证这一假设，我的第一个目标是确定c-Kit抑制是否 通过用阿西替尼治疗小鼠并进行实验， 行为、电生理和细胞分析。我的第二个目标是确定是否需要c-Kit 甜细胞分化或存活。我将在不同的Cre驱动程序下使用两个基因c-Kit敲除模型， 在TRC分化的不同点敲除c-Kit表达。最后，我将研究c-Kit介导的 通过基因敲除味蕾内的SCF来实现串话。该项目的完成将阐明 以前未研究的RTK c-Kit在味觉稳态中的作用。