The Pit-1/Pou1f1 transcription factor regulates and correlates with prolactin expression in human breast cell lines and tumors
1Department of Physiology, School of Medicine, University Clinical Hospital, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain
2Department of Obstetrics and Gynecology, School of Medicine, University Clinical Hospital, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain
3Department of Morphological Sciences, School of Medicine, University Clinical Hospital, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain
4Unidad de Investigación del Hospital de Jove, 33920, Gijón, Spain
(Correspondence should be addressed to R Perez-Fernandez, Departamento de Fisiología, Facultad de Medicina, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain; Email:roman.perez.fernandez@usc.es)
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This article has been cited by other articles in PMC.Abstract
The transcription factor Pit-1/Pou1f1 regulates GH and prolactin (PRL) secretion in the pituitary gland. Pit-1 expression and GH regulation by Pit-1 have also been demonstrated in mammary gland. However, no data are available on the role of Pit-1 on breast PRL. To evaluate this role, several human breast cancer cell lines were transfected with either the Pit-1 expression vector or a Pit-1 small interference RNA construct, followed by PRL mRNA and protein evaluation. In addition, transient transfection of MCF-7 cells by a reporter construct containing the proximal PRL promoter, and ChIP assays were performed. Our data indicate that Pit-1 regulates mammary PRL at transcriptional level by binding to the proximal PRL promoter. We also found that Pit-1 raises cyclin D1 expression before increasing PRL levels, suggesting a PRL-independent effect of Pit-1 on cell proliferation. By using immunohistochemistry, we found a significant correlation between Pit-1 and PRL expression in 94 human breast invasive ductal carcinomas. Considering the possible role of PRL in breast cancer disorders, the function of Pit-1 in breast should be the focus of further research.
Introduction
The transcription factor Pit-1/Pou1f1 was first described in the pituitary gland, where it acts in cell differentiation during organogenesis of the anterior pituitary in mammals and as a transcriptional activator for pituitary gene transcription (Lefevre et al. 1987,Nelson et al. 1988). Mice with inactivating mutations or deletions of the Pit-1 gene fail to generate somatotrophs, lactotrophs, and thyrotrophs, and consequently exhibit anterior pituitary hypoplasia and dwarfism (Li et al. 1990), thereby demonstrating the importance of Pit-1 in the ontogeny of the pituitary gland. While its pituitary function has been extensively evaluated, Pit-1 is also expressed in several extrapituitary tissues and cell lines, including the mammary gland, where its role is still unclear (Delhase et al. 1993, Bamberger et al. 1995, Gil-Puig et al. 2002, 2005). Moreover, as in the pituitary gland, Pit-1 regulates GH expression in the MCF-7 human breast adenocarcinoma cell line (Gil-Puig et al. 2005).
Prolactin (PRL) and its corresponding receptor have also been found in extrapituitary tissues, including normal human breast, mammary tumors, and breast cell lines (Clevenger et al. 1995, Ginsburg & Vonderhaar 1995, Mertani et al. 1998), but the regulation of extrapituitary PRL is not yet understood. Human PRL gene is regulated at a transcriptional level by two different promoters (Berwaer et al. 1994, Gellersen et al. 1994). First, the proximal pituitary promoter is located in the exon 1b and contains Pit-1 response elements; the human pituitary PRL gene contains only this promoter. Secondly, the distal or extrapituitary promoter is located at 5.8
kb upstream of the above mentioned proximal PRL promoter and does not contain any Pit-1-binding sites. The distal promoter is present in PRL gene at extrapituitary sites, such as placenta, lymphoid cells, normal and neoplastic human breast, and human prostate (Berwaer et al. 1994, Gellersen et al. 1994, Shaw-Bruha et al. 1997, Dagvadorj et al. 2007). However, both proximal pituitary and distal extrapituitary PRL promoters are expressed in a subset of normal and tumoral breast tissues, breast cell lines, and in prostate tissue (Shaw-Bruha et al. 1997, Dagvadorj et al. 2007). PRL transcripts arising from either the distal or proximal promoter encode identical proteins, which gives the PRL gene a tissue-specific regulation. The effects of PRL on milk production and on mammary growth and differentiation are well known (Freeman et al. 2000). In addition, PRL provides a proliferative stimulus to rodent mammary tumorogenesis (Welsch & Nagasawa 1977). Several researchers have related endocrine and/or autocrine/paracrine PRL production with mammary disorders, including tumorogenesis (Vonderhaar 1999, Wennbo & Törnell 2000, Ben-Jonathanet al. 2002, Clevenger et al. 2003). For instance, recent prospective studies have linked PRL levels with breast cancer etiology in women, particularly in estrogen-positive tumors (Tworoger et al. 2007). The administration of anti-PRL antibodies, PRL antisense oligonucleotides, or PRL antagonists suppresses the mitogenic activity of locally produced PRL and inhibits growth in breast tumor cell lines (Fuh & Wells 1995, Llovera et al. 2000). In addition, transgenic mice that overexpress the PRL gene develop mammary carcinoma, and this tumor development is caused by the activation of PRL receptor (Wennbo et al. 1997).
kb upstream of the above mentioned proximal PRL promoter and does not contain any Pit-1-binding sites. The distal promoter is present in PRL gene at extrapituitary sites, such as placenta, lymphoid cells, normal and neoplastic human breast, and human prostate (Berwaer et al. 1994, Gellersen et al. 1994, Shaw-Bruha et al. 1997, Dagvadorj et al. 2007). However, both proximal pituitary and distal extrapituitary PRL promoters are expressed in a subset of normal and tumoral breast tissues, breast cell lines, and in prostate tissue (Shaw-Bruha et al. 1997, Dagvadorj et al. 2007). PRL transcripts arising from either the distal or proximal promoter encode identical proteins, which gives the PRL gene a tissue-specific regulation. The effects of PRL on milk production and on mammary growth and differentiation are well known (Freeman et al. 2000). In addition, PRL provides a proliferative stimulus to rodent mammary tumorogenesis (Welsch & Nagasawa 1977). Several researchers have related endocrine and/or autocrine/paracrine PRL production with mammary disorders, including tumorogenesis (Vonderhaar 1999, Wennbo & Törnell 2000, Ben-Jonathanet al. 2002, Clevenger et al. 2003). For instance, recent prospective studies have linked PRL levels with breast cancer etiology in women, particularly in estrogen-positive tumors (Tworoger et al. 2007). The administration of anti-PRL antibodies, PRL antisense oligonucleotides, or PRL antagonists suppresses the mitogenic activity of locally produced PRL and inhibits growth in breast tumor cell lines (Fuh & Wells 1995, Llovera et al. 2000). In addition, transgenic mice that overexpress the PRL gene develop mammary carcinoma, and this tumor development is caused by the activation of PRL receptor (Wennbo et al. 1997).To explore the regulation of autocrine breast PRL, in the present study, several human breast cell lines were used to evaluate the effect of Pit-1 overexpression and/or knockdown on PRL expression. In addition, we evaluate the effect of Pit-1 and PRL on cell proliferation. Finally, we evaluate the relationships between Pit-1 and PRL expression in human breast tumors.
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