RESUMO
Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study, we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. Using an immunohistochemical approach with antibodies specific for each Notch receptor, we show that Notch proteins are temporally regulated in mammary epithelial cells during normal mammary gland development in the FVB/N mouse. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. These data define expression of the Notch ligand/receptor system throughout development of the mouse mammary gland and help set the stage for genetic analysis of Notch in this context.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glândulas Mamárias Animais/metabolismo , Receptores Notch/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Feminino , Hormônios Esteroides Gonadais/metabolismo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Jagged-1 , Lactação/genética , Ligantes , Glândulas Mamárias Animais/crescimento & desenvolvimento , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Ovariectomia , Gravidez , Interferência de RNA , RNA Mensageiro/metabolismo , Receptor Notch2/genética , Receptor Notch3 , Receptores Notch/metabolismo , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Serrate-Jagged , TransfecçãoRESUMO
We have identified the transforming acidic coiled-coil protein-3 (Tacc3) as a binding partner for Notch4/Int3 and were able to show that it binds to the intracellular domain (ICD) of all members of the Notch receptor family. Members of the Tacc family reside at the centrosomes and associates with microtubules. Recent studies suggest that Tacc3 also contributes to the regulation of gene transcription. Tacc3 specifically interacts with the Notch4/Int3 CDC10/Ankyrin repeats and to a lesser extent, with residues C-terminal to these repeats in the ICD. Dual label immunofluorescence of mouse mammary tissue shows Tacc3 co-localizes with the Notch3 ICD. Co-immunoprecipitation of endogenous Notch and Tacc3 proteins from NIH3T3 cell extracts, lung and mammary gland confirms that these two proteins interact under physiological conditions. In addition, knock down of Tacc3 in NIH3T3 cells leads to the up-regulation of Hey2, a target gene for Notch signaling. The affinity of Tacc3 binding to Notch4/Int3 ICD is similar to that between Rbpj and Notch4/Int3 ICD. Notch4/Int3 ICD-Tacc3 interaction results in the inhibition of transcription from a Hes1-Luciferase reporter vector in COS-1 cells. The inhibition was reversed in these cells by increasing the levels of Rbpj. Taken together, these results suggest that Tacc3 is a negative regulator of the Notch signaling pathway.