Two tandemly linked interferon-gamma-activated sequence elements in the promoter of glycosylation-dependent cell adhesion molecule 1 gene synergistically respond to prolactin in mouse mammary epithelial cells.
Previously, we reported that glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) was a novel target for prolactin (PRL) in the mouse mammary gland. However, the signaling pathway by which PRL regulates GlyCAM 1 expression has not been specified. In the present study, we showed that PRL induced GlyCAM 1 expression in primary mammary epithelial cells of mice through the Janus kinase 2/signal transducer and activator of transcription 5 (Stat5) pathway. Deletion and site-directed mutagenesis analyses of the GlyCAM 1 promoter demonstrated that the two tandemly linked Stat5 binding sites [interferon-gamma-activated sequence 1 and -2 (GAS1 and GAS2)] in the proximal promoter region were crucial and synergistically responded to PRL. GAS2, a consensus GAS site, was essential and, by itself, weakly responded to PRL, whereas GAS1, a nonconsensus site, failed to respond to PRL but was indispensable for the maximal activity of the GlyCAM 1 promoter. Gel shift assays showed that probe containing GAS1 and GAS2 bound two Stat5 complexes, which represent Stat5 dimer and tetramer, respectively, while GAS2, by itself, bound Stat5 as a dimer only, and GAS1 showed no apparent binding activity. Interruption of tetramer formation by mutation of a tryptophan to alanine (W37A), and a leucine to serine (L83S) in the N terminus of Stat5A attenuated the synergistic effect between the two tandemly linked GAS sites. Overexpression of W37A and L83S mutants in primary mammary epithelial cells suppressed endogenous GlyCAM 1 expression.
Hou
Zhaoyuan
Z
Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267-0576, USA.
Srivastava
Sunil
S
Mistry
Meenakshi J
MJ
Herbst
Matthew P
MP
Bailey
Jason P
JP
Horseman
Nelson D
ND
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells; D005786:Gene Expression Regulation; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism*; D053614:Janus Kinase 2; D008156:Luciferases; D042361:Mammary Glands, Human / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D008894:Milk Proteins; D009077:Mucins / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011388:Prolactin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D025521:Tumor Suppressor Proteins
D004268:DNA-Binding Proteins; D008894:Milk Proteins; D009077:Mucins; D011518:Proto-Oncogene Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494093:Stat5a protein, mouse; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; C075185:sulfated glycoprotein p50; D012313:RNA; D007371:Interferon-gamma; D011388:Prolactin; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2