1995;270:21639C21644

1995;270:21639C21644. They bind to extracellular matrix (ECM) proteins via their extracellular domain and interact with cytoskeletal and signaling molecules via their cytoplasmic domain. Integrins function as signaling receptors, stimulating various intracellular signaling cascades. This enables them to modulate important cellular functions such as proliferation, survival, and gene expression (for review, see Giancotti and Ruoslahti, 1999 ). To study the mechanisms of 1-integrin function in vivo, we have generated transgenic mice expressing a dominant negative mutant of 1-integrin under the control of the MMTV promoter (MMTV-1-cyto) in the mammary gland epithelium (Faraldo reporter construct (Promega, Madison, WI), and 650 ng of -casein-luciferase reporter containing the ?344 to ?1 sequences of the rat -casein promoter or 650 ng of NFB-luciferase reporter, containing a NFB binding site (kindly provided by Dr. W.Doppler, Universit?t Innsbruck, Innsbruck, Austria). In addition, when mentioned, cells were cotransfected with 1.2 g BMS-927711 of the MMTV-1-cyto-CD4 plasmid (Faraldo luciferase activities, cells were scrapped in 100 l of Pasive lysis buffer (Promega) and after two freeze/thaw cycles, lysates were cleared by centrifugation at 10,000 substrates (Promega). Values obtained for firefly luciferase were normalized to luciferase activity. At least three independent experiments were performed in each case. RESULTS Mammary Glands of MMTV-1-cyto Mice Achieve Fully Differentiated Phenotype at Peak Lactation In previous studies, we found that the mammary glands of MMTV-1-cyto animals were less developed than those of their wild-type littermates during the first days of lactation and that the mammary epithelium presented differentiation defects, as revealed by low milk protein mRNA levels (Faraldo (1999) carried out with cultured mammary epithelial cells reported that the signals from both 1 and 4-integrins are required for -casein expression and suggested that these integrins may act in concert in the control of the mammary epithelial cell differentiation. Furthermore, two recent reports have demonstrated that conditional ablation of 1-integrin in the epidermis resulted in a reduced expression of 64 integrin in basal keratinocytes (Brakebusch em et al. /em , 2000 ; Raghavan em et al. /em , 2000 ). These results suggest that the perturbation of 1-integrin function may subsequently alter the function and signaling events associated with 64. Accordingly, in the MMTV-1-cyto lactating glands, we have observed an abnormal localization of the 4-integrin chain at the lateral surfaces of the alveolar epithelial cells (Faraldo em et al. /em , 1998 ). Experiments performed with cultured cells suggested that the chimeric proteins similar to that used in this study, i.e., containing 1-integrin cytoplasmic domain, may also interfere with 3 and 5-integrin function (Lukashev em et al. /em , 1994 ). These -chains interact with v to form an integrin dimer. Although the v chain is expressed by many mammary epithelial cell lines, in mammary gland, this integrin chain was not found in luminal epithelial cells, and only a small amount of it was detected on the basal surface of myoepithelial cells (Zutter em et al. /em , 1990 ; Clezardin em et al. /em , 1993 ; Anbazhagan em et al. /em , 1995 ). Similar to 1-integrin, v3 was implicated in the control of cell survival (Brooks em et al. /em , 1994 ). However, using immunofluorescence methods, we did not observe any compensatory increase of v3 or other v-containing integrins in MMTV-1-cyto glands (unpublished data). Briefly, in this study, we show that expression of a 1-integrin dominant negative mutant in involuting mammary gland did not alter the kinetics of apoptosis but resulted in the premature dedifferentiation of secretory epithelium; affected the prolactin/STAT5 signaling pathway, essential for mammary development; and induced the precocious activation of NFB transcription element. These results reveal an important part of cellCECM relationships mediated by 1-integrins in the control of milk gene manifestation and in maintenance of the mammary epithelial cell differentiated BMS-927711 state in vivo. ACKNOWLEDGMENTS We particularly say thanks to Drs. I. Cerutti and C. Gouget, and the staff of the Services d’Exprimentation Animale et de Transgense, Villejuif, especially R. Duchateau and A. Loeuillet, for taking care of the transgenic mice. We also greatly appreciate suggestions of Drs. W. Doppler, P. Furth, D. Medina, and J. Teulire, and donation BMS-927711 of reagents by Drs. W. Doppler, R..W.Doppler, Universit?t Innsbruck, Innsbruck, Austria). gene transcription and in the maintenance of the mammary epithelial cell differentiated state. Intro Integrins are adhesive transmembrane heterodimer receptors composed of an and a subunit. They bind to extracellular matrix (ECM) proteins via their extracellular website and interact with cytoskeletal and signaling molecules via their cytoplasmic website. Integrins function as signaling receptors, revitalizing numerous intracellular signaling cascades. This enables them to modulate important cellular functions such as proliferation, survival, and gene manifestation (for review, observe Giancotti and Ruoslahti, 1999 ). To study the mechanisms of 1-integrin function in vivo, we have generated transgenic mice expressing a dominating bad mutant BMS-927711 of 1-integrin under the control of the MMTV promoter (MMTV-1-cyto) in the mammary gland epithelium (Faraldo reporter construct (Promega, Madison, WI), and 650 ng of -casein-luciferase reporter comprising the ?344 to ?1 sequences of the rat -casein promoter or 650 ng of NFB-luciferase reporter, containing a NFB binding site (kindly provided by Dr. W.Doppler, Universit?t Innsbruck, Innsbruck, Austria). In addition, when pointed out, cells were cotransfected with 1.2 g of the MMTV-1-cyto-CD4 plasmid (Faraldo luciferase activities, cells were scrapped in 100 l of Pasive lysis buffer (Promega) and after two freeze/thaw cycles, lysates were cleared by centrifugation at 10,000 substrates (Promega). Ideals acquired for firefly luciferase were normalized to luciferase activity. At least three self-employed experiments were performed in each case. RESULTS Mammary Glands of MMTV-1-cyto Mice Achieve Fully Differentiated Phenotype at Maximum Lactation In earlier studies, we found that the mammary glands of MMTV-1-cyto animals were less developed than those of their wild-type littermates during the 1st days of lactation and that the mammary epithelium offered differentiation problems, as exposed by low milk protein mRNA levels (Faraldo BMS-927711 (1999) carried out with cultured mammary epithelial cells reported the signals from both 1 and 4-integrins are required for -casein manifestation and suggested that these integrins may take action in concert in the control of the mammary epithelial cell differentiation. Furthermore, two recent reports have shown that conditional ablation of 1-integrin in the epidermis resulted in a reduced manifestation of 64 integrin in basal keratinocytes (Brakebusch em et al. /em , 2000 ; Raghavan em et al. /em , 2000 ). These results suggest that the perturbation of 1-integrin function may consequently alter the function and signaling events associated with 64. Accordingly, in the MMTV-1-cyto lactating glands, we have observed an irregular localization of the 4-integrin IGFBP3 chain in the lateral surfaces of the alveolar epithelial cells (Faraldo em et al. /em , 1998 ). Experiments performed with cultured cells suggested the chimeric proteins related to that used in this study, i.e., containing 1-integrin cytoplasmic website, may also interfere with 3 and 5-integrin function (Lukashev em et al. /em , 1994 ). These -chains interact with v to form an integrin dimer. Even though v chain is indicated by many mammary epithelial cell lines, in mammary gland, this integrin chain was not found in luminal epithelial cells, and only a small amount of it was recognized within the basal surface of myoepithelial cells (Zutter em et al. /em , 1990 ; Clezardin em et al. /em , 1993 ; Anbazhagan em et al. /em , 1995 ). Much like 1-integrin, v3 was implicated in the control of cell survival (Brooks em et al. /em , 1994 ). However, using immunofluorescence methods, we did not observe any compensatory increase of v3 or additional v-containing integrins in MMTV-1-cyto glands (unpublished data). Briefly, in this study, we display that manifestation of a 1-integrin dominant bad mutant in involuting mammary gland did not alter the kinetics of apoptosis but resulted in the premature dedifferentiation of secretory epithelium; affected the prolactin/STAT5 signaling pathway, essential for mammary development; and induced the precocious activation of NFB transcription element. These results reveal an important part of cellCECM relationships mediated by 1-integrins in the control of milk gene manifestation and in maintenance of the mammary epithelial cell differentiated state in vivo. ACKNOWLEDGMENTS We particularly say thanks to Drs. I. Cerutti and C. Gouget, and the personnel of the Services d’Exprimentation Animale et de Transgense, Villejuif, especially R. Duchateau and.