Induction of the endogenous whey acidic protein (Wap) gene and a Wap-myc hybrid gene in primary murine mammary organoids.
In rodents, the whey acidic protein (Wap) is the major whey protein expressed in mammary glands in response to lactogenic hormones. The regulation of the Wap gene differs from that of other milk protein genes, with one consequence being that little or no Wap expression is detectable in cell culture. Here we describe the efficient in vitro induction of the Wap gene in mammary organoids isolated from midpregnant mice. Mammary organoids were isolated as intact epithelial subcomponents which retained the glandular microarchitecture. If organoids were cultured in contact with a monolayer of 3T3-L1 adipocytes, significant levels of Wap mRNA were induced upon hormonal stimulation, with the highest level of Wap mRNA being induced by a combination of hydrocortisone, prolactin, and insulin. Dissociation of the three-dimensional organization abrogated Wap inducibility. Organoids cultured on plastic or hydrated type I collagen did not transcribe Wap mRNA even after hormonal stimulation. Addition of hormones was required to maintain low levels of Wap mRNA in organoids cultured on reconstituted basement membrane, however, Wap mRNA was not induced. Organoid-adipocyte interactions as well as cell-cell interactions inherent in the structure of organoids promote hormone-dependent Wap mRNA expression. In order to study the Wap promoter region in vitro, we cocultured organoids from transgenic mice harboring a chimeric Wap-myc gene with 3T3-L1 adipocytes. Lactogenic hormones induced the Wap-myc transgene in vitro. The kinetics of induction were similar for both the transgene and the endogenous Wap gene indicating that the 2.5-kb regulatory Wap region present in the hybrid gene contains the sequence elements required for hormone-induced gene expression in vitro.
Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115.
C A
CA
Schoenenberger
A
A
Zuk
B
B
Groner
W
W
Jones
A C
AC
Andres
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000502:physiology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D005796:Genes; D006854:Hydrocortisone / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D008894:Milk Proteins / Q000096:biosynthesis / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D009940:Organoids / Q000378:metabolism / Q000648:ultrastructure; D011247:Pregnancy; D011388:Prolactin / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc; D011519:Proto-Oncogenes; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic
D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; C024471:whey acidic proteins; D011388:Prolactin; D011505:Protein-Tyrosine Kinases; D006854:Hydrocortisone