Abstract
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.
Highlights
Substrates of gp78 E3 ubiquitin ligase activity include CD3-␦, the T cell receptor, apoB lipoprotein, hydroxymethylglutarylCoA reductase, cystic fibrosis transmembrane conductance regulator, and the metastasis suppressor KAI1 (9 –13)
B, 10 mg of tissue from mammary glands of wild type and gp78 transgenic mice was homogenized in 1ϫ SDS-PAGE loading buffer, boiled for 5 min, and centrifuged, and 15 l of lysates were loaded on 10% gel and subjected to Western blotting. gp78 expression levels probed with anti-gp78 monoclonal antibody or anti-gp78 3F3A rat IgM are increased in both the pubertal virgin and 12-day mid-gestation mammary glands of 235RLF transgenic mouse compared with age-matched wild type
When assessed for the gp78 protein level by Western blotting with the 3F3A anti-gp78 antibody [18], dexamethasone-induced cells showed elevated gp78 protein levels (Fig. 1C). These results suggest that in expressing cells, the mammary tumor virus (MMTV)-LTR-gp78 plasmid is responsive to the GC analogue dexamethasone and that gp78 overexpression is induced in a controlled fashion
Summary
Antibodies, Reagents, and Animals—Rat anti-gp78 3F3A monoclonal antibody was as described previously [18] and mouse anti-gp (ab54787-100) was purchased from Abcam. Genomic DNA obtained from the wild type FVB/N mouse was used as a negative control, and the MMTV-LTR-gp plasmid was used as a positive control. Tail genomic DNA of the obtained seven founders was assessed by PCR for transgene presence (bottom panels), and only six had complete integration of the cassette (right panel compared with left panel). The right panels show Southern blot genotyping where ϳ10 g of tail-genomic DNA from six founders was digested with KpnI and separated on 0.7% agarose gel along with controls (FVB genomic and MMTV-LTR-gp DNAs), capillary-transferred on Zeta probe (Bio-Rad) nylon membrane, and subjected to hybridization and development. Prehybridization, hybridization, Genotyping by PCR and Southern Blotting—Genomic DNA and immune probing of membrane were performed using the was extracted from mouse tails following standard protocols. Ethanol-precipitated mice were injected with 100 g/g of body weight BrdUrd in PBS gp Overexpression Induces Hyperplasia ding and sections (5 m) were prepared by University of British
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