Alterations in Interacting Signaling Pathways

相互作用信号通路的改变

• 批准号: 6949778
• 负责人: STUART H. YUSPA
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6949778
• 关键词: Caenorhabditis elegans; animal tissue; biological signal transduction; carcinogenesis; cell transformation; chemokine; enzyme activity; gene expression; genetic mapping; genetically modified animals; human tissue; inflammation; keratinocyte; laboratory mouse; oncogenes; phosphorylation; polymerase chain reaction; protein isoforms; protein kinase C; skin neoplasms; squamous cell carcinoma; tissue /cell culture

Ras oncogene mediated transformation of mouse keratinocytes to a benign squamous papilloma phenotype is associated with activation of PKC-alpha. Tumors produced by ras transformation of keratinocytes derived from skin targeted PKC-alpha transgenic mice produced in our laboratory grow faster and achieve a larger size, but do not convert to malignancy at a higher rate. Activation of a PKC-alpha transgene by TPA in mouse epidermis induced a marked intraepidermal neutrophilic infiltrate that mimics the inflammatory response in several human cutaneous diseases including psoriasis. Multiple chemokines are upregulated detected by RT-PCR and RPA assays, and supernatants from cultured transgenic keratinocytes contain chemotactic factors for murine neutrophils. In vitro chemotaxis assays indicate that relevant chemokines are downstream of TNF-alpha. Activation of PKC-alpha in transgenic skin causes epidermal apoptosis, and this is prevented in cultured transgenic keratinocytes by inhibiting AP-1 activity. Microarray analysis of TPA treated transgenic keratinocytes has revealed a spectrum of PKC-alpha regulated genes, including differentiation markers, chemokines/cytokines and proteases and these can be selectively altered by specific inhibitors of the AP-1 and NFkappaB pathways. This model continues to reveal unique insights into the regulation of cutaneous inflammation, the regulation of pro-inflammatory and pro-apoptotic pathways in skin and the contribution of inflammation to cutaneous carcinogenesis. Activation of PKC inactivates the AKT kinase in mouse keratinocytes through prevention or reversal of AKT phosphorylation on serine 473. Inhibition of PKC activity enhances IGF-1- mediated protection from UV induced apoptosis, presumably by enhancing AKT activity as detected by AKT phosphorylation. The specific PKC isoforms targeting AKT appear to be delta and/or epsilon. The genomic structures of C. elegans, mouse, rat and human PKCdelta were analyzed after sequencing a genomic fragment of mouse PKC. Overall exon-intron genomic structure was highly conserved among mammals while significantly diverged in C. elegans. A 1.7 kb region of the PKC-delta promoter revealed a hitherto unknown NFkappaB site, and TNF-alpha was shown to upregulate the expression of the gene. Disruption of the nuclear tumor suppressor gene PML function in skin by targeting the PML-RAR-alpha fusion oncogene to the skin of transgenic mice causes spontaneous skin tumor induction. This is associated with cutaneous retinoid deficiency. Tumor development is independent of ras gene mutations. When PML is targeted to the epidermis in transgenic mice, transgenic keratinocytes in vitro senesce more rapidly, adhere more tightly to collagen substrates and have altered proteolytic activity. In vivo, PML transgenic mice undergo spontaneous alopecia, and have delayed skin tumor formation, smaller tumors and a longer latency to carcinoma formation when treated topically with carcinogens.

Ras 癌基因介导的小鼠角质形成细胞向良性鳞状乳头状瘤表型的转化与 PKC-α 的激活相关。我们实验室生产的皮肤靶向 PKC-α 转基因小鼠的角化细胞通过 ras 转化产生的肿瘤生长速度更快，尺寸更大，但转化为恶性肿瘤的比率并不高。 TPA 在小鼠表皮中激活 PKC-α 转基因，诱导明显的表皮内中性粒细胞浸润，模拟了包括牛皮癣在内的多种人类皮肤疾病的炎症反应。 RT-PCR 和 RPA 测定检测到多种趋化因子上调，培养的转基因角质形成细胞的上清液含有鼠中性粒细胞的趋化因子。体外趋化性测定表明相关趋化因子位于 TNF-α 的下游。转基因皮肤中 PKC-α 的激活会导致表皮细胞凋亡，而在培养的转基因角质形成细胞中，可以通过抑制 AP-1 活性来防止表皮细胞凋亡。 TPA 处理的转基因角质形成细胞的微阵列分析揭示了一系列 PKC-α 调节基因，包括分化标记物、趋化因子/细胞因子和蛋白酶，并且这些基因可以通过 AP-1 和 NFkappaB 途径的特定抑制剂进行选择性改变。该模型继续揭示对皮肤炎症调节、皮肤促炎和促凋亡途径的调节以及炎症对皮肤癌发生的影响的独特见解。 PKC 的激活通过阻止或逆转丝氨酸 473 上的 AKT 磷酸化使小鼠角质形成细胞中的 AKT 激酶失活。抑制 PKC 活性可增强 IGF-1 介导的对 UV 诱导细胞凋亡的保护，大概是通过增强 AKT 磷酸化检测到的 AKT 活性来实现。靶向 AKT 的特定 PKC 同工型似乎是 delta 和/或 epsilon。 对小鼠 PKC 的基因组片段进行测序后，对秀丽隐杆线虫、小鼠、大鼠和人类 PKCdelta 的基因组结构进行了分析。整体外显子-内含子基因组结构在哺乳动物中高度保守，而在秀丽隐杆线虫中存在显着差异。 PKC-delta 启动子的 1.7 kb 区域揭示了一个迄今为止未知的 NFkappaB 位点，TNF-α 被证明可以上调该基因的表达。通过将 PML-RAR-α 融合癌基因靶向转基因小鼠的皮肤，破坏皮肤中核肿瘤抑制基因 PML 的功能，导致自发性皮肤肿瘤诱导。这与皮肤类维生素A缺乏有关。肿瘤的发展与ras基因突变无关。当 PML 靶向转基因小鼠的表皮时，转基因角质形成细胞在体外衰老得更快，与胶原蛋白基质的粘附更紧密，并改变了蛋白水解活性。在体内，PML转基因小鼠会出现自发性脱发，并且当局部用致癌物治疗时，皮肤肿瘤形成延迟，肿瘤更小，癌形成潜伏期更长。