Targeting Grainyhead-Like 2 Suppresses Entry Factors of SARS-CoV-2 in Epithelial Cells of Oral Mucosa.

靶向 Grainyhead-Like 2 可抑制口腔粘膜上皮细胞中 SARS-CoV-2 的进入因子。

• 批准号: 10598134
• 负责人: WEI CHEN
• 金额: $ 19.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10598134
• 关键词: 2019-nCoV; 3-Dimensional; ACE2; Analysis of Variance; Binding; Binding Proteins; Biological Assay; COVID-19; COVID-19 pandemic; COVID-19 patient; CRISPR/Cas technology; Cell Differentiation process; Cell Proliferation; Cell Separation; Cell membrane; Cells; Containment; Data; Ectopic Expression; Epigenetic Process; Epithelial Cells; Epithelium; Functional disorder; Gene Expression; Genes; Genetic Transcription; Gingiva; Glycoproteins; Goals; Green Fluorescent Proteins; Health; Human; In Vitro; Infection; Interferon alpha; Interferons; Knock-out; Mediating; Membrane; Methods; Modeling; Molecular; Mus; Oral; Oral cavity; Oral mucous membrane structure; Organ; Outcome; Pattern; Peptide Hydrolases; Play; Predisposition; Process; Public Health; Publishing; Reporter; Research; Role; Route; SARS-CoV-2 infection; SARS-CoV-2 inhibitor; SARS-CoV-2 spike protein; SARS-CoV-2 transmission; Salivary Glands; Serine Protease; Signal Pathway; Site; TMPRSS2 gene; Testing; Tissues; Transcriptional Regulation; Transduction Gene; Tropism; Vesicular stomatitis Indiana virus; Viral; Virus; Virus Diseases; coronavirus disease; effective therapy; experimental study; gain of function; high risk; high throughput screening; in vivo; inhibitor; keratinocyte; knock-down; knockout gene; loss of function; monolayer; mouse model; new therapeutic target; novel; oral cavity epithelium; promoter; receptor; screening; small molecule inhibitor; transcription factor; translational potential; viral entry inhibitor

Abstract The long-term goal of our research is to develop effective therapies against SARS-CoV-2-elicited disease Covid-19. SARS-CoV-2 spike (S) protein binds to its cognate receptor, angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine proteases (TMPRSSs), promotes cellular entry. Co-expressions of these viral entry factors are found in the epithelial cells of many organs, including oral mucosa. Recently, oral mucosa has been found to be a potentially high-risk route of SARS-CoV-2 infection. GRHL2, as a transcriptional factor, modulates epithelial gene expression and plasticity through multiple signaling pathways. ACE2 and TMPRSS2 are strongly expressed in epithelial cells, coinciding with the pattern of GRHL2 expression in oral mucosa. GRHL2 regulates ACE2 and TMPRSS2 expression in normal human oral keratinocytes (NHOK). Ectopic expression of GRHL2 induces ACE2 and TMPRSSs expression, while knockdown of GRHL2 leads to decreased levels of these entry factors. ACE2 is an interferon-stimulated gene. IFN-α activates ACE2 expression in part through regulating GRHL2. Small-molecule inhibitors of GRHL2 suppress viral entry factor expression in NHOK. ACE2 and TMPRSS2 are widely expressed in oral mucosal epithelium, knockout of GRHL2 leads to suppression of ACE2 and TMPRSS2 expression in vivo. These findings indicate that GRHL2 is required for ACE2 and TMPRSS expression in NHOK cultured in vitro and oral mucosal epithelium in vivo. Based on these data, we hypothesize that targeting GRHL2 inhibits SARS-CoV-2 transmission into oral mucosal epithelium through regulating viral entry factor expression. This novel hypothesis will be tested by examining the molecular mechanism underlying GRHL2 regulates expression of SARS-CoV-2 entry factors (ACE2 and TMPRSSs) in oral mucosal epithelial cells. Analysis of variance methods will be used to compare means of these gene expression levels. We will also generate a pseudotyped virus in which vesicular stomatitis virus (VSV) green fluorescent protein (GFP) reporter virus expressing SARS- CoV-2 S in replacement of native glycoprotein. Utilizing this chimeric virus, a high throughput assay will be developed to screen GRHL2 inhibitors, which possess the potentials to abrogate SARS-CoV-2 virus infection into oral mucosal epithelium.

抽象的 我们研究的长期目标是开发针对SARS-COV-2引起的疾病的有效疗法 新冠肺炎。 SARS-COV-2尖峰（S）蛋白与其同源受体，血管紧张素转换酶2结合 （ACE2），并与宿主蛋白酶一致，主要是跨膜丝氨酸蛋白酶（TMPRSSS）， 促进细胞进入。这些病毒进入因子的共表达在 许多器官，包括口腔粘膜。最近，发现口服粘膜是一种潜在的高风险 SARS-COV-2感染的途径。 GRHL2作为转录因子，调节上皮基因表达 和通过多个信号通路的可塑性。 ACE2和TMPRSS2在 上皮细胞，与口服粘膜中GRHL2表达的模式一致。 GRHL2调节ACE2 正常人口服角质形成细胞（NHOK）中的TMPRSS2表达。 GRHL2的异位表达 诱导ACE2和TMPRSSS表达，而GRHL2的敲低导致这些水平的提高 进入因素。 ACE2是干扰素刺激的基因。 IFN-α通过 调节GRHL2。 GRHL2的小分子抑制剂抑制了NHOK病毒进入因子的表达。 ACE2和TMPRSS2在口服粘膜上皮中广泛表达，GRHL2的敲除导致 在体内抑制ACE2和TMPRSS2表达。这些发现表明需要GRHL2 对于在体内体外和口服粘膜上皮培养的NHOK中的ACE2和TMPRSS表达。基于 在这些数据上，我们假设靶向GRHL2抑制SARS-COV-2传播到口服 粘膜上皮通过调节入口因子表达。这个新颖的假设将是 通过检查GRHL2的分子机制测试，调节SARS-COV-2的表达 口服粘膜上皮细胞中的进入因子（ACE2和TMPRSSS）。方差方法的分析将是 用于比较这些基因表达水平的平均值。我们还将在 哪种囊泡口腔炎病毒（VSV）绿色荧光蛋白（GFP）记者病毒表达SARS- COV-2 S替代天然糖蛋白。利用这种嵌合病毒，高吞吐量测定将 可以开发以筛选GRHL2抑制剂，这可能会消除SARS-COV-2病毒 感染口服粘膜上皮。

项目成果

Orai3 Calcium Channel Contributes to Oral/Oropharyngeal Cancer Stemness through the Elevation of ID1 Expression.

• DOI: 10.3390/cells12182225
• 发表时间: 2023-09-07
• 期刊: CELLS
• 影响因子: 6
• 作者: Nguyen, Anthony;Sung, Youngjae;Lee, Sung Hee;Martin, Charlotte Ellen;Srikanth, Sonal;Chen, Wei;Kang, Mo K.;Kim, Reuben H.;Park, No-Hee;Gwack, Yousang;Kim, Yong;Shin, Ki-Hyuk
• 通讯作者: Shin, Ki-Hyuk