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

项目摘要 味觉是由多细胞味蕾调节的，每个味蕾容纳50-100个快速更新的味觉。 受体细胞(TrCs)。TRCs主要分为三种类型：I型神经胶质样细胞、II型细胞 甜味、苦味或鲜味，以及能察觉酸味的III型细胞。当TRC续订时，这三种类型将在 比例相对稳定，使味觉在一段时间内保持稳定。然而，快速的周转 味觉细胞使味觉系统容易受到某些药物和疾病的干扰。患有癌症的患者 转移性肾细胞癌(MRCC)在接受酪氨酸激酶抑制剂(TKIs)治疗时经常遇到 味觉障碍，或称味觉障碍。TKI用于治疗肾细胞癌的主要靶点是受体酪氨酸 在味觉组织中不表达的酪氨酸激酶(RTK)、血管内皮生长因子受体(VEGFR)和血管内皮生长因子受体β。然而，这些将军澳也 抑制许多非靶向RTK，如Met、Ret、PDGFRα和c-Kit。根据我们的单细胞RNA测序 (ScRNAseq)数据，这些RTK在味觉组织中的祖细胞和分化的味觉亚群中表达 细胞。这表明，抑制这些非靶标RTK可能是导致动作障碍的原因，部分或全部 这些RTK对于正确的TRC更新和味道动态平衡是必要的。测试脱离目标的RTK的作用 在味觉稳态方面，我用TKIS Axitinib，Cabozantinib和Sunitinib治疗舌质有机化合物，这三种药物 抑制不同的脱靶RTK组合，并经常导致患者的运动障碍。我发现了这些 药物不影响祖细胞增殖，反而降低某些分化基因的表达 味觉细胞标记。具体地说，这三个TKI都降低了甜味细胞标记Tas1r2的表达，并且 Tas1r2是唯一受这三种药物影响的标志物。重要的是，唯一被所有人禁止的目标外RTK 有三种药物是c-Kit，我们在scRNAseq数据中发现，这种药物在甜味感受型II型中表达最高 TRC。这些数据强烈表明c-Kit参与了甜蜜细胞的动态平衡。进一步分析我们的scRNAseq数据 揭示了c-Kit的配体干细胞因子(SCF)是由I型和III型TRCs表达的，提高了 C-Kit介导味蕾内串扰的可能性。总而言之，这些数据让我得出了c-Kit的假设 在与其他TRC类型的串扰的刺激下，信号对于分化和/或 甜味感受型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 通过TKI治疗导致体内甜味缺陷通过阿西替尼治疗小鼠并进行 行为、电生理和细胞分析。我的第二个目标是确定c-Kit对于 甜味细胞分化或存活。我将在不同的CRE驱动程序下使用两种遗传c-Kit基因敲除模型 敲除TRC分化过程中不同时间点c-Kit的表达。最后，我将调查c-Kit介导的 通过从味蕾中从基因上敲除SCF来进行相声。这一项目的完成将照亮 先前未研究过的RTK c-Kit在味觉动态平衡中的作用。

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.