Mertk,I型受体酪氨酸激酶和TAM受体家族成员,在生理条件下具有促进细胞增生和解决炎症的重要功能。近年来,Mertk还与癌症的病理生理作用有关,据此,在几种癌症类型中,包括实体癌和白血病/淋巴瘤。Mertk作为致癌酪氨酸激酶有助于增殖和细胞存活的致癌特征。此外,在巨噬细胞上表达的Mertk,包括肿瘤相关的巨噬细胞,促进癌症中的免疫逃避,并被认为类似于骨髓检查点抑制剂,该抑制剂使巨噬细胞偏向抑制宿主T细胞抗肿瘤免疫的抑制性表型。在本研究中,为了更好地了解控制单核细胞/巨噬细胞中Mertk表达的翻译后调控机制,我们使用PMA分化的THP-1细胞模型来询问Mertk表达的调节,并开发了一种新型的Mertk报告细胞系来研究Mertk的细胞内运输。我们表明,PMA处理有效上调Mertk以及胞外域蛋白水解处理平台ADAM17的成分,而PMA差异调节经典Mertk配体Gas6和Pros1(Gas6下调,Pros1上调)。在非刺激稳态条件下,PMA分化的THP1细胞中的Mertk通过ADAM17和早老素/γ-分泌酶复合物的顺序活性显示出活性的组成型蛋白水解裂解,表明Mertk通过ADAM17和γ-分泌酶的组合顺序作用而稳态裂解,之后,Mertk的裂解的细胞内片段以蛋白酶体依赖性机制降解。使用嵌合Flag-Mertk-EGFP-Myc报告受体,我们证实了γ-分泌酶和MG132的抑制剂,其抑制26S蛋白酶体,稳定Mertk的细胞内片段,没有核易位的证据。最后,用活性γ-羧化Gas6处理细胞,而非非活性华法林处理的非γ-羧化Gas6,调节不同的蛋白水解过程涉及的受体清除和溶酶体蛋白水解。一起,这些结果表明,多向和复杂的蛋白水解活性将Mertk胞外域裂解调节为稳态负调控事件,以防止Mertk的过度激活。
Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years,
Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition,
Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel
Mertk reporter cell line to study the intracellular trafficking of
Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of
Mertk.