Generation of immortal cell lines from the adult pituitary: role of cAMP on differentiation of SOX2-expressing progenitor cells to mature gonadotropes

Abstract

The pituitary is a complex endocrine tissue composed of a number of unique cell types distinguished by the expression and secretion of specific hormones, which in turn control critical components of overall physiology. The basic function of these cells is understood; however, the molecular events involved in their hormonal regulation are not yet fully defined. While previously established cell lines have provided much insight into these regulatory mechanisms, the availability of representative cell lines from each cell lineage is limited, and currently none are derived from adult pituitary. We have therefore used retroviral transfer of SV40 T-antigen to mass immortalize primary pituitary cell culture from an adult mouse. We have generated 19 mixed cell cultures that contain cells from pituitary cell lineages, as determined by RT-PCR analysis and immunocytochemistry for specific hormones. Some lines expressed markers associated with multipotent adult progenitor cells or transit-amplifying cells, including SOX2, nestin, S100, and SOX9. The progenitor lines were exposed to an adenylate cyclase activator, forskolin, over 7 days and were induced to differentiate to a more mature gonadotrope cell, expressing significant levels of α-subunit, LHβ, and FSHβ mRNAs. Additionally, clonal populations of differentiated gonadotropes were exposed to 30 nM gonadotropin-releasing hormone and responded appropriately with a significant increase in α-subunit and LHβ transcription. Further, exposure of the lines to a pulse paradigm of GnRH, in combination with 17β-estradiol and dexamethasone, significantly increased GnRH receptor mRNA levels. This array of adult-derived pituitary cell models will be valuable for both studies of progenitor cell characteristics and modulation, and the molecular analysis of individual pituitary cell lineages.

Figure 1. Characterization of endocrine markers in immortalized, pituitary cell mixtures.

Representative immunocytochemistry images using either FITC-conjugated (shown in green) or Texas Red-conjugated (shown in red) antibodies, and counter-stained with DAPI. ACTH, adrenocorticotropic hormone; ADH, anti-diuretic hormone; GH, growth hormone; OXY, oxytocin; LH, luteinizing hormone; and PRL, prolactin.

Figure 2. Analysis of pituitary cell markers in subcloned cell lines.

(A) RT-PCR was performed on twelve of the subcloned cell lines for the indicated cellular markers. (B) Immunocytochemical analysis was performed using FITC-conjugated (shown in green) or Texas Red-conjugated (shown in red) antibodies, and counter-stained with DAPI. (C) Representative gels for LHβ (amplicon size = 196 bp) and α-subunit (amplicon size = 168 bp) are shown. M = marker; NTC = non-template control. (D) Immunocytochemical analysis using an antibody against LH was performed in the mPitA-12/3 and -12/4 cell lines.

Figure 3. Expression of gonadotropin and progenitor cell markers in clonal cell lines.

The mPitA-1, 3 and 12 cell lines were subcloned into a single cell population. (A) The cell lines were screened for gonadotropin and progenitor cell markers by RT-PCR, and results are summarized in (B). (C) Previously established progenitor or gonadotroph cell lines (αT1, αT3 and LβT2), generated by targeted tumorogenesis in transgenic mice, were screened for gonadotropin and progenitor cell markers by RT-PCR as a comparison, and results are summarized in (D).

Figure 4. Serum deprivation does not change LHβ, SOX2 or SOX9 mRNA expression in mPitA-1/3, -1/6 or -3/1 cells.

mPitA-1/3, -1/6, and -3/1 cells were exposed to 1% or 10% fetal bovine serum daily for 7 days. LHβ, SOX2 and SOX9 mRNA expression were determined using real-time RT-PCR and levels were normalized to histone. Results are expressed as mean ± SEM (n = 3-4 independent experiments).

Figure 5. Forskolin increases LHβ, FSHβ, α-subunit and SOX9 mRNA expression in mPitA-1/3 cells.

mPitA-1/3 cells were treated with forskolin (100 nM, 1 uM or 10 uM) daily for 7 days. LHβ, FSHβ, α-subunit and SOX9 mRNA expression were determined using real-time RT-PCR and levels were normalized to histone. Results are expressed as mean ± SEM (n = 3-4 independent experiments). *p<0.05, **p<0.01.

Figure 6. Forskolin increases LHβ and SOX9 mRNA expression in mPitA-3/1 cells.

mPitA-3/1 cells were treated with forskolin (100 nM, 1 uM or 10 uM) daily for 7 days. LHβ, FSHβ, α-subunit and SOX9 mRNA expression were determined using real-time RT-PCR and levels were normalized to histone. Results are expressed as mean ± SEM (n = 3-4 independent experiments). *p<0.05, **p<0.001.

Figure 7. Characterization and functional analysis of gonadotrope cell lines.

(A) mPitA-12/3 or mPitA-12/4 cells were screened for the presence on GnRH receptor (GnRH R) mRNA by RT-PCR. Both lines were treated with GnRH (30 nM) for 6 and 24 h. LHβ and α-subunit mRNA expression were determined using real-time RT-PCR and levels were normalized to histone. (B) mPitA-12/3 cells were treated with GnRH (10 nM) over a four-day pulse paradigm (GnRH pulse) or with a single daily treatement (no GnRH pulse), in combination with 10 nM 17β-estradiol -/+ 20 nM dexamethasone. GnRH receptor mRNA expression was determined using semi-quantitative RT-PCR and levels were normalized to histone. A representative RT-PCR gel is shown. Data are expressed as mean ± SEM, n = 3-8 independent experiments. *p<0.05.