Gpr48在皮肤鳞状细胞癌发生中的功能及机制

G protein-coupled receptor 48 (Gpr48/Lgr4) is an orphan receptor that plays important roles in the development of myriad tissues including the male reproductive system, eyelid, hair follicle, and bone. It is also involved in tumorigenesis. Little is known about the molecular mechanism(s) underlying the function of Gpr48 in these biological processes. Our preliminary experiments have demonstrated that Gpr48 knockout mice are resistant to DMBA/TPA-induced formation of skin squamous cell carcinoma (SCC), and show significantly decreased proliferative activity in the basal layer of the skin epidermis. We have also found Gpr48 depletion to result in reduced phosphorylation of Protein Kinase C (PKC) and Extracellular Signal-regulated Kinases (ERK) and Glycogen Synthase Kinase 3β (GSK3β) proteins. We propose to build on these findings as well as the established mouse and cell culture models to investigate the cellular function of Gpr48 and the underlying molecular mechanism in SCC formation. Through the proposed analysis of whether and how Gpr48 regulates PKC-ERK and Wnt signaling pathways and their functions in normal and cancerous skin stem cells, our work promises to uncover the critical downstream molecular pathways and effectors that mediate the function of Gpr48 in SCC. This work will also play a solid foundation for future effort in developing novel therapeutics for treating SCC and other cancers that require Gpr48.

G蛋白偶联受体48(Gpr48/Lgr4)是一种孤儿受体，在男性生殖道、眼睑、头发和骨骼的发育中起重要作用，与肿癌的发生也密切相关，但Gpr48作用的分子机制还不十分清楚。我们的前期实验结果发现，Gpr48基因敲除小鼠能显著降低DMBA/TPA诱导皮肤鳞状细胞癌（SCC）的发生率，其小鼠表皮基底层细胞的分裂增殖水平也明显降低。初步的分子机制揭示，Gpr48缺失会导致表皮PKC、ERK、GSK3β蛋白磷酸化水平的降低。本课题将利用小鼠皮肤SCC模型和Gpr48基因敲除细胞模型，从整体、细胞和分子等方面深入探索Gpr48在皮肤SCC发生中的功能及调控机制。我们将通过研究Gpr48对PKC-ERK、Wnt信号通路以及它们在表皮干细胞和SCC干细胞中功能的影响，揭示并寻找Gpr48在皮肤SCC发生中的关键信号通路和重要效应分子，为今后通过抑制Gpr48活性来治疗SCC提供理论和实验依据。

G蛋白偶联受体48 (Gpr48)又名G蛋白偶联受体Lgr4（Leucine-rich repeat containing G-protein coupled receptor 4）在男性生殖道、眼睑、头发和骨骼的发育中起重要作用，它与肿瘤的生长和转移也密切相关。Lgr4在皮肤组织中有高表达，并影响着皮肤的发育过程，但Lgr4在皮肤癌的发生发展中是否起着重要的作用，还未见任何报道。我们通过研究发现Lgr4缺失能够明显抑制由DMBA/TPA诱导小鼠皮肤癌的发生。我们的研究还发现在野生型小鼠中TPA能够明显地激活MEK1/2，ERK1/2，β-catenin的表达，但当Lgr4缺失后，这些蛋白的激活受到明显地抑制。我们通过抑制剂的实验证明了当MEK1/2-ERK1/2激活受到抑制后，β-catenin的入核现象也受到了明显抑制，这就提示我们，ERK1/2的激活可能激活了β-catenin的入核，从而激活了下游的靶基因例如cyclinD1的活性。我们的研究证明了Lgr4可以通过PKC/ERK和Wnt/β-catenin两条信号通路来调控皮肤癌的发生和生长，那么这两条信号通路是否存在Crosstalk的现象呢，于是我们利用抑制剂实验，将U0126与FH535加入鳞癌细胞A431和小鼠原代培养的角质形成细胞中检测这两种信号通路相关蛋白的表达，利用shRNA干扰实验成功敲低了ERK1/2的表达，结果发现ERK1/2受到抑制或表达下调，能够引起β-catenin表达的降低和下调,这足以证明ERK1/2能够直接调控β-catenin的表达，从而促进皮肤癌的产生。而P-ERK1/2的激活一方面可以激活p-gsk3β，使蛋白质降解复合体解体，从而促进β-catenin的稳定性，入核增加使得下游的c-myc，cyclinD1等癌基因表达。另一方面，p-ERK1/2可以激活下游AP-1的入核促进下游靶基因的表达，从而调控细胞的增殖。因此当Lgr4敲除后，它能够降低PKC的活性，从而阻断了下游ERK1/2和Wnt信号通路的激活，因此抑制了小鼠皮肤鳞状细胞癌的产生，这些研究为进一步探讨Lgr4可能成为治疗皮肤鳞状细胞癌的新靶点提供了理论和实验依据。