Lineage-specific restraint of pituitary gonadotroph cell adenoma growth

Abstract

Although pituitary adenomas are usually benign, unique trophic mechanisms restraining cell proliferation are unclear. As GH-secreting adenomas are associated with p53/p21-dependent senescence, we tested mechanisms constraining non-functioning pituitary adenoma growth. Thirty six gonadotroph-derived non-functioning pituitary adenomas all exhibited DNA damage, but undetectable p21 expression. However, these adenomas all expressed p16, and >90% abundantly expressed cytoplasmic clusterin associated with induction of the Cdk inhibitor p15 in 70% of gonadotroph and in 26% of somatotroph lineage adenomas (p = 0.006). Murine LβT2 and αT3 gonadotroph pituitary cells, and αGSU.PTTG transgenic mice with targeted gonadotroph cell adenomas also abundantly expressed clusterin and exhibited features of oncogene-induced senescence as evidenced by C/EBPβ and C/EBPδ induction. In turn, C/EBPs activated the clusterin promoter ∼5 fold, and elevated clusterin subsequently elicited p15 and p16 expression, acting to arrest murine gonadotroph cell proliferation. In contrast, specific clusterin suppression by RNAis enhanced gonadotroph proliferation. FOXL2, a tissue-specific gonadotroph lineage factor, also induced the clusterin promoter ∼3 fold in αT3 pituitary cells. As nine of 12 pituitary carcinomas were devoid of clusterin expression, this protein may limit proliferation of benign adenomatous pituitary cells. These results point to lineage-specific pathways restricting uncontrolled murine and human pituitary gonadotroph adenoma cell growth.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. PTTG and DNA damage in human pituitary adenomas.

A) PTTG immunoreactivity (brown signal, both intranuclear and cytoplasmic)) in non-tumorous pituitary and human pituitary gonadotroph adenomas (T-1,2); B) DNA damage in human pituitary adenoma. Confocal image of human pituitary adenoma and non-tumorous pituitary tissue specimens labeled with γH2.AX or pATM antibodies (green). Both proteins are expressed in the nucleus. Specimens here and shown below were counterstained with DNA-specific dye ToPro3 (blue).

Figure 2. Cdk inhibitor expression in human pituitary adenomas.

Confocal image of human gonadotroph pituitary adenoma specimens labeled with p21, p16 or p15 antibodies (green, intranucelar).

Figure 3. Clusterin in human pituitary adenomas.

Confocal image of A) human pituitary adenoma and non-tumorous pituitary tissue specimens showing clusterin (green) expressed exclusively in the cytoplasm; B) Co-localization of clusterin with GH, PRL and αGSU in respective human pituitary adenoma specimens (clusterin green, respective hormones red).

Figure 4. Pituitary proliferation, DNA damage and senescence markers in the αGSU.PTTG pituitary gland.

A) In vivo BrdU incorporation. Mice were injected with BrdU (50 µg/g BW), and pituitary sections stained for BrdU. One thousand cells/section, 3 sections/animal, n = 3 animals/genotype were analyzed. *, p<0.05; Western blot analysis of B) proliferation markers; C) DNA damage, DNA repair and p53-dependent senescence markers, and D) oncogene-induced senescence markers; E) Confocal image showing immunofluorescent cytoplasmic clusterin, and intranuclear p15 and p16 expression (green) in WT and in pre-tumorous αGSU.PTTG pituitary glands, and in αGSU.PTTG pituitary adenomas; F) Pituitary SA-β-galactosidase enzymatic activity (blue) in WT and in pre-tumorous αGSU.PTTG pituitary gland. Three pituitary cryosections/animal were analyzed from 3 animals/genotype, and a representative image shown. Western blots here and elsewhere were repeated 3 times with similar results and representative blots shown.

Figure 5. Senescence markers in gonadotroph-derived LβT2 cells transfected with mPttg.

Western blot analysis of senescence markers in A) LβT2 cells transiently transfected with mPttg; B) in LβT2 cells stably transfected with mPttg; C) Percent BrdU positive cells in two selected clones stably transfected with mPttg. Duplicate samples were pulsed with BrdU for 30 min and analyzed by flow cytometry; D) Senescent morphology of LβT2 cells stably transfected with mPttg. Brown dots depict incorporated BrdU; E) Percent apoptotic cells stably transfected with mPttg. Cells were fixed, and one thousand cells/field counted in three randomly chosen visual fields; F) Percent SA-β-galactosidase positivity in cells stably transfected with Pttg was assessed in 6-well plates in triplicate. One thousand cells/field were counted in three fields/well. G) SA-β-galactosidase enzymatic activity (blue) in cells stably transfected with mPttg. *, p<0.05.

Figure 6. C/EBPs induce clusterin.

A) C/EBPβ is up-regulated in the αGSU.PTTG pituitary. Confocal image showing C/EBPβ co-localization with αGSU-positive, GH-positive and PRL-positive cells in WT and pre-tumorous αGSU.PTTG pituitary glands. (Hormones-green, cytoplsmic, C/EBPβ–red, intranuclear); B) Western blot analysis of C/EBPβ and δ isoforms induced in LβT2 cells stably transfected with mPttg; C) Effects of C/EBPs on the clusterin promoter in LβT2 and αT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-mClu reporter plasmid and 800 ng murine pCDNA3-C/EBPα, β or δ. The ratio of luciferase to co-trasfected β-galactosidase control reporter vector was normalized to pCDNA3-null expression vector. SEM was calculated from triplicate assays, and experiments repeated three times with similar results. Results of a representative experiment are shown.*, p<0.05, **,p<0.01; D) Western blot analysis of clusterin expression in gonadotroph-derived αT3 cells 24 hours after transfection with pCDNA3-C/EBPβ or E) pCDNA3-C/EBPδ; F) Western blot analysis of clusterin expression in LβT2 mPttg cells 48 hours after simultaneous transfection with siC/EBPβ and siC/EBPδ (3 nM each). Two different combinations of siRNAs were used.

Figure 7. Clusterin restrains pituitary cell proliferation by inducing Cdk inhibitors.

Western blot analysis of Cdk inhibitors and proliferation markers A) in LβT2 cells, B) in αT3 cells 48 h after transfection with mClu; C) Confocal images of immunofluoprescence of histone H3 methylation on lysine 9 (H3-K9M) (red) in vector and Clu-expressing αT3 cells 48 hours after transfection; D) Quantification of positive H3-K9M foci. Cells were fixed, stained with H3-K9M antibody, and one thousand cells/field counted in three randomly chosen visual fields; E) Percentage of BrdU positive cells 48 h after transfection with mClu. Triplicate samples were pulsed with BrdU for 30 min and analyzed by flow cytometry, *, p<0.05; F) αT3 cells stably overexpressing mClu or vector were synchronized in 0.1% fetal bovine serum for 18 hours, and then cultured in 10% fetal bovine serum. At the indicated times, duplicate samples were pulsed with BrdU for 30 min, analyzed by flow cytometry, and cells in S-phase identified by staining with BrdU antibodies.

Figure 8. Clusterin attenuation promotes proliferation.

Western blot analysis of Cdk inhibitors and proliferation markers A) in LβT2 cells, B) in αT3 cells; C) Percentage of BrdU positive cells 48 h after transfection with siClu. D) Upper panel, Western blot confirms p15 down-regulation, Lower panel, Percentage of BrdU positive LβT2 cells 48 h after transfection with sip15. E) Upper panel, Western blot confirms p16 down-regulation, Lower panel, Percentage of BrdU positive LβT2 cells 48 h after transfection with sip16. For BrdU detection, cells were fixed, stained with BrdU antibody and one thousand cells/field in three randomly chosen fields counted. *, p<0.05.

Figure 9. FOXL2 stimulates the clusterin promoter.

A) Effects of FOXL2 on the clusterin promoter in αT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-mClu reporter plasmid and indicated amounts of pcDNA3-His-mFoxl2. The ratio of luciferase to co-trasfected β-galactosidase control reporter vector was normalized to pCDNA3-null expression vector. SEM was calculated from triplicate assays, and experiments repeated three times with similar results. Results of a representative experiment are shown; **,p<0.01; B) Western blot analysis of clusterin expression in αT3 cells 24 hours after transfection with pcDNA3-His-mFoxl2; C) ChiP assay was performed in nuclear fractions derived from αT3 cell lysates. Top, schematic of the approximate location of primers used in the PCR reactions. Enrichment of specific clusterin promoter sequences was obtained with primer Set 2. FOXL2, specific antibody, IgG, nonspecific antibody, PCP, positive control primers. The experiment was repeated twice, and results of a representative assay shown.

Figure 10. Proliferation restricting pathways in the pituitary gonadotroph cell lineage.

FOXL2 directly activates the clusterin promoter, while Pttg overexpression results in proliferation, DNA damage and stimulation of C/EBPβ and δ; C/EBPs activate the clusterin promoter. High levels of secretory clusterin trigger expression of Cdk inhibitors p15, p16 and p27, and C/EBPβ also cooperates to induce p15. Up-regulated tumor suppressor proteins likely underlie proliferation restraint preventing uncontrolled growth of benign pituitary adenomas of gonadotroph cell origin.