The Notch Signaling System Is Present in the Postnatal Pituitary: Marked Expression and Regulatory Activity in the Newly Discovered Side Population

Abstract

Recently, we discovered in the adult anterior pituitary a subset of cells with side population (SP) phenotype, enriched for expression of stem/progenitor cell-associated factors like Sca1, and of Notch1 and Hes (hairy and enhancer of split) 1, components of the classically developmental Notch pathway. In the present study, we elaborated the expression of the Notch signaling system in the postnatal pituitary, and examined its functional significance within the SP compartment. Using RT-PCR, we detected in the anterior pituitary of adult mouse the expression of all four vertebrate Notch receptors, as well as of Hes1, 5, and 6, key downstream targets and effectors of Notch. All Notch receptors, Hes1 and Hes5 were measured at higher mRNA levels in the Sca1(high) SP than in the main population (MP) of differentiated hormonal cells. In contrast, Hes6, known as an inhibitor of Hes1, was more abundant in the MP. Cells with SP phenotype, enriched for Sca1(high) expression, were detected throughout postnatal life. Their proportion was higher in immature mice, but did not change from adult (8 wk old) to much older age (1 yr old). Notch pathway expression was higher in the Sca1(high) SP than in the MP at all postnatal ages analyzed. Functional implication of Notch signaling in the SP was investigated in reaggregate cultures of adult mouse anterior pituitary cells. Treatment with the gamma-secretase inhibitor DAPT down-regulated Notch activity and reduced the proportion of SP cells. Activation of Notch signaling with the conserved DSL motif of Notch ligands, or with a soluble ligand, caused a rise in SP cell number, at least in part due to a proliferative effect. The SP also expanded in proportion when aggregates were treated with leukemia-inhibitory factor, basic fibroblast growth factor, and epidermal growth factor, again at least partly accounted for by a mitogenic action. These intrapituitary growth factors all activated Notch signaling, and DAPT abrogated the expansion of the SP by basic fibroblast growth factor and leukemia-inhibitory factor, thus exposing a possible cross talk. In conclusion, we show that the Notch pathway, typically situated in embryogenesis, is also present and active in the postnatal pituitary, that it is particularly expressed within the SP independent of age, and that it plays a role in the regulation of SP abundance. Whether our data indicate that Notch regulates renewal and fate decisions of putative stem/progenitor cells within the pituitary SP as found in other tissues, remains open for further exploration.

Pituitary Stem Cells: Quest for Hidden Functions

“…Activation of FGF and EGF can lead to increased pituitary stem cell numbers, as has been demonstrated in AP cell aggregate cultures (Chen et al 2006).…”

“…In general, there is no co-localization of PROP1 or SOX2 with hormones (Chen et al 2009;Fauquier et al 2008;Fu et al 2012a,b;Garcia-Lavandeira et al 2009;Gremeaux et al 2012;Yoshida et al 2009), supporting the general notion that stem cell conservators must be downregulated before differentiation starts. The NOTCH pathway is another essential regulator of pituitary embryogenesis (Kita et al 2007;Monahan et al 2009;Raetzman et al 2004;Zhu et al 2005Zhu et al , 2007, of which several components are upregulated in the adult (mouse) pituitary stem cell fraction (Chen et al 2006(Chen et al , 2009Vankelecom 2010) and are found expressed in some of the MZ and parenchymal S100 þ cells of the (rat) AP (Tando et al 2013). Some other transcriptional regulators that play an important role in pituitary embryonic development (such as Hesx1, the earliest known gene expressed in the pituitary primordium, Lhx4, Pax6, Otx2, Ascl1 and the Six/Eya/ Dach genes; Kelberman et al 2009;Zhu et al 2005Zhu et al , 2007 are also higher transcribed in the adult pituitary stem cell fraction (Chen et al 2006(Chen et al , 2009reviewed in Vankelecom 2010reviewed in Vankelecom , 2012, as well as the cyclin-dependent kinase inhibitor p57 that, during embryogenesis, emerges in the RP progenitor cells that stop cycling to embark on differentiation (Bilodeau et al, 2009;Vankelecom 2010;Vankelecom and Chen 2014).…”

“…The NOTCH pathway is another essential regulator of pituitary embryogenesis (Kita et al 2007;Monahan et al 2009;Raetzman et al 2004;Zhu et al 2005Zhu et al , 2007, of which several components are upregulated in the adult (mouse) pituitary stem cell fraction (Chen et al 2006(Chen et al , 2009Vankelecom 2010) and are found expressed in some of the MZ and parenchymal S100 þ cells of the (rat) AP (Tando et al 2013). Some other transcriptional regulators that play an important role in pituitary embryonic development (such as Hesx1, the earliest known gene expressed in the pituitary primordium, Lhx4, Pax6, Otx2, Ascl1 and the Six/Eya/ Dach genes; Kelberman et al 2009;Zhu et al 2005Zhu et al , 2007 are also higher transcribed in the adult pituitary stem cell fraction (Chen et al 2006(Chen et al , 2009reviewed in Vankelecom 2010reviewed in Vankelecom , 2012, as well as the cyclin-dependent kinase inhibitor p57 that, during embryogenesis, emerges in the RP progenitor cells that stop cycling to embark on differentiation (Bilodeau et al, 2009;Vankelecom 2010;Vankelecom and Chen 2014). Finally, components of the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) pathways, having important roles in the specification and proliferation of RP progenitor cells (Ericson et al 1998;Zhu et al 2005Zhu et al , 2007, are also upregulated in the adult stem cells (Vankelecom 2010;Vankelecom and Chen 2014).…”

“…NOTCH signaling is likely also involved in the control of stem cell proliferation in the adult gland, since NOTCH downregulation in the embryonic pituitary coincides with cell cycle exit of the RP progenitor cells and genetic ablation of NOTCH signaling in the developing pituitary results in severe AP hypoplasia due to reduced proliferative capacity of RP progenitors (Kita et al 2007;Monahan et al 2009;Raetzman et al 2004;Tando et al 2013;Zhu et al 2005Zhu et al , 2007. In addition, stem cells increase in number in AP cell cultures upon NOTCH activation (Chen et al 2006;Tando et al 2013). SOX2 and NOTCH (together with PROP1) may form an interacting signaling network within the stem cell compartment (reviewed in Willems and Vankelecom 2014).…”

Pituitary Stem Cells During Normal Physiology and Disease

“…This is a typical property of many cell types that possess properties of stem or progenitor cells. This side population has since been described for pituitaries of other vertebrates (Chen et al 2005(Chen et al , 2006van Rijn et al 2012). In time, additional markers of cells with this in vitro self-renewal capacity have been put forward to refine the characterisation of PSCs, including Nestin, PROP1, SOX9, GFRα2 and PRX1/2 (Gleiberman et al 2008;Yoshida et al 2009;Rizzoti et al 2013;GarciaLavandeira et al 2009;Higuchi et al 2014).…”