The role of ERBB2 and ERBB3 receptors in skin development, homeostasis, and tumor susceptibility

表皮生长因子受体（EGFR/ERBB1）及其配体是皮肤和毛囊稳态的调节因子，它们的失调会迅速导致病理改变。相比之下，尽管酪氨酸激酶受体ERBB2和ERBB3也在皮肤中表达，并与皮肤肿瘤发生有关，但它们在该组织中发挥的功能在很大程度上仍未被探索。我们建议在皮肤中产生特异性缺乏ERBB2或ERBB3的小鼠系。特别是，我们建议将携带Erbb2或Erbb3基因的固定等位基因的小鼠与在牛角蛋白5启动子（K5cre）的控制下表达cre重组酶的小鼠杂交。由此产生的K5cre-Erbb2fl/fl （= K5-Erbb2del）和K5cre-Erbb3fl/fl （= K5-Erbb3del）小鼠将有一个早期缺失（从胚胎日e15.5左右开始），限制在毛囊和毛囊间表皮角质形成细胞的相应受体，使我们能够评估ERBB2在皮肤发育和稳态中的作用。我们将评估皮肤组织学（H&E，增殖，凋亡），通过定位几种分化标志物进行表皮分化，以及毛囊形态发生和周期诱导的动力学。我们还建议通过使用K14creERT2系和他莫昔芬治疗来制造皮肤特异性ERBB2或ERBB3缺失的小鼠，并使它们接受两阶段化学致癌方案。在该方案中，单次应用引发剂DMBA后，反复应用促炎剂TPA，导致良性乳头状瘤退化或进展为鳞状细胞癌。每周记录肿瘤的数量和大小，在实验结束时（应用DMBA后~ 20周），将肿瘤固定用于组织学分析或冷冻用于表达分析。因此，两阶段化学癌变为明确阐明ERBB2和ERBB3在皮肤癌发生和进展的早期和晚期事件中的影响提供了机会。最后，一些研究表明，ERBB2被紫外线照射激活，并增加紫外线诱导的皮肤肿瘤发生。不幸的是，所采用的模型并没有给出ERBB2在这一过程中作用的明确结论。因此，我们建议将erbb2缺陷小鼠和对照组仔鼠暴露于UVA或UVB照射下。24小时后，我们将评估表皮厚度、表皮细胞增殖和凋亡、晒伤细胞数量等参数。最后，为了进行机制研究，我们建议建立缺乏ERBB2或ERBB3的永久角化细胞细胞系以及控制系。在这些细胞系中，我们将评估EGFR/ERBB二聚体的形成和下游信号通路、细胞增殖、粘附和非锚定生长，以及ERBB2和ERBB3与E-cadherin和LRIG1之间的相互作用。

The epidermal growth factor receptor (EGFR/ERBB1) and its ligands are established regulators of skin and hair follicle homeostasis, and their deregulation rapidly results in pathological alterations. In contrast - although they are also expressed in the skin and have been implicated in skin tumorigenesis - the functions exerted by the related tyrosine kinase receptors ERBB2 and ERBB3 in this tissue remain largely unexplored.We propose to generate mouse lines lacking ERBB2 or ERBB3 specifically in the skin. In particular, we propose to cross mice carrying floxed alleles of the Erbb2 or Erbb3 genes with mice expressing cre recombinase under the control of the bovine keratin 5 promoter (K5cre). Resulting K5cre-Erbb2fl/fl (= K5-Erbb2del) and K5cre-Erbb3fl/fl (= K5-Erbb3del) mice will have an early deletion (starting around embryonic day e15.5) of the corresponding receptors restricted to keratinocytes of the hair follicle and interfollicular epidermis, allowing us to evaluate the role of ERBB2 for skin development and homeostasis. We will evaluate the skin histology (H&E, proliferation, apoptosis), the epidermal differentiation by localizing several differentiation markers, and the kinetics of hair follicle morphogenesis and cycle induction.We also propose to create mice with skin-specific loss of ERBB2 or ERBB3 by employing the K14creERT2 line and tamoxifen treatment and subject them to a two-stage chemical carcinogenesis protocol. In this protocol, a single application of the initiator DMBA is followed by repeated applications of the pro-inflammatory agent TPA, resulting in benign papillomas that regress or progress to squamous cell carcinoma. The number and the size of the tumors will be recorded weekly and, at the end of the experiment (~ 20 weeks after DMBA application), tumors will be fixed for histological analysis or frozen for expression analysis. Thus, the two-stage chemical carcinogenesis provides an opportunity to definitively clarify the influence of ERBB2 and ERBB3 in early and late events in skin cancer development and progression. Finally, several studies showed that ERBB2 is activated by UV irradiation and increases UV-induced skin tumorigenesis. Unfortunately, the employed models did not allow definitive conclusions about the role of ERBB2 in this process. Therefore, we propose to expose ERBB2-deficient mice and control littermates to UVA or UVB irradiation. Twenty-four hours later, we will evaluate parameters such as the epidermal thickness, the epidermal cell proliferation and apoptosis, and the number of sunburn cells.Finally, to allow mechanistic studies, we propose the establishment of permanent keratinocyte cell lines lacking ERBB2 or ERBB3 as well as control lines. With these cell lines, we will evaluate the formation of EGFR/ERBB dimers and the downstream signaling pathways, cell proliferation, adhesion, and anchorage-independent growth, as well as the interaction between ERBB2 and ERBB3 with E-cadherin and LRIG1.