Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
J Biol Chem. 2010 Mar 19;285(12):8830-9. doi: 10.1074/jbc.M109.074344. Epub 2010 Jan 20.
Expression of gp78, an E3 ubiquitin ligase in endoplasmic reticulum-associated degradation, is associated with tumor malignancy. To study gp78 overexpression in mammary gland development and tumorigenicity, we generated murine mammary tumor virus (MMTV) long terminal repeat-driven gp78 transgenic mice. Embryos carrying the gp78 transgene cassette were implanted in FVB surrogate mothers, and two founders with high copy integration showed elevated gp78 expression at both transcript and protein levels at the virgin stage and at 12 days gestation. Transgenic mammary glands showed increased ductal branching, dense alveolar lobule formation, and secondary terminal end bud development. Bromodeoxyuridine staining showed increased proliferation in hyperplastic ductal regions at the virgin stage and at 12 days gestation compared with wild type mice. Reduced expression of the metastasis suppressor KAI1, a gp78 endoplasmic reticulum-associated degradation substrate, demonstrates that gp78 ubiquitin ligase activity is increased in MMTV-gp78 mammary gland. Similarly, metastatic MDA-435 cells exhibit increased gp78 expression, decreased KAI1 expression, and elevated proliferation compared with nonmetastatic MCF7 cells whose proliferation was enhanced upon knockdown of KAI1. Importantly, stable gp78 knockdown HEK293 cells showed increased KAI1 expression and reduced proliferation that was rescued upon KAI1 knockdown, demonstrating that gp78 regulation of cell proliferation is mediated by KAI1. Mammary tumorigenesis was not observed in repeatedly pregnant MMTV-long terminal repeat-gp78 transgenic mice over a period of 18 months post-birth. Elevated gp78 ubiquitin ligase activity is therefore not sufficient for mammary tumorigenesis. However, the hyperplastic phenotype observed in mammary glands of MMTV-gp78 transgenic mice identifies a novel role for gp78 expression in enhancing mammary epithelial cell proliferation and nontumorigenic ductal outgrowth.
gp78 是内质网相关降解途径中的一种 E3 泛素连接酶，其表达与肿瘤恶性程度相关。为了研究 gp78 在乳腺发育和致瘤性中的过表达情况，我们构建了受鼠乳腺肿瘤病毒（MMTV）长末端重复序列驱动的 gp78 转基因小鼠。携带 gp78 转基因盒的胚胎被植入 FVB 代孕鼠体内，两个具有高拷贝整合的启动子小鼠在处女期和 12 天妊娠时均表现出 gp78 在转录和蛋白水平上的高表达。转基因乳腺表现出导管分支增加、肺泡小叶形成密集和次级终末芽发育。溴脱氧尿苷（BrdU）染色显示，与野生型小鼠相比，在处女期和 12 天妊娠时，增生的导管区域增殖增加。转移抑制因子 KAI1 的表达降低，作为 gp78 内质网相关降解途径的底物，表明 MMTV-gp78 乳腺中的 gp78 泛素连接酶活性增加。同样，转移性 MDA-435 细胞表现出 gp78 表达增加、KAI1 表达降低和增殖增加，而非转移性 MCF7 细胞的增殖在 KAI1 敲低后增强。重要的是，稳定的 gp78 敲低 HEK293 细胞表现出 KAI1 表达增加和增殖减少，而 KAI1 敲低后得到挽救，表明 gp78 对细胞增殖的调节是通过 KAI1 介导的。在出生后 18 个月的反复妊娠中，未观察到 MMTV-长末端重复序列-gp78 转基因小鼠的乳腺肿瘤发生。因此，gp78 泛素连接酶活性的升高不足以引起乳腺肿瘤发生。然而，在 MMTV-gp78 转基因小鼠的乳腺中观察到的增生表型，确定了 gp78 表达在增强乳腺上皮细胞增殖和非肿瘤性导管生长中的新作用。
