Gene Expression in Mouse Thyrotrope Adenoma: Transcription Elongation Factor Stimulates Proliferation

Abstract

Thyrotrope hyperplasia and hypertrophy are common responses to primary hypothyroidism. To understand the genetic regulation of these processes, we studied gene expression changes in the pituitaries of Cga(-/-) mice, which are deficient in the common α-subunit of TSH, LH, and FSH. These mice have thyrotrope hypertrophy and hyperplasia and develop thyrotrope adenoma. We report that cell proliferation is increased, but the expression of most stem cell markers is unchanged. The α-subunit is required for secretion of the glycoprotein hormone β-subunits, and mutants exhibit elevated expression of many genes involved in the unfolded protein response, consistent with dilation and stress of the endoplasmic reticulum. Mutants have elevated expression of transcription factors that are important in thyrotrope function, such as Gata2 and Islet 1, and those that stimulate proliferation, including Nupr1, E2f1, and Etv5. We characterized the expression and function of a novel, overexpressed gene, transcription elongation factor A (SII)-like 5 (Tceal5). Stable expression of Tceal5 in a pituitary progenitor cell line is sufficient to increase cell proliferation. Thus, Tceal5 may act as a proto-oncogene. This study provides a rich resource for comparing pituitary transcriptomes and an analysis of gene expression networks.

Figure 1.

Cga^−/− mice show a progressive increase in Tshb mRNA expression from birth to adulthood and present with extremely dilated ER cisternae. A, Quantitative PCR with relative quantification showing the increase of Tshb transcript expression during postnatal life in the Cga^−/− vs wild type. B, Transmission EM showing thyrotropes identified by TSHB antibody staining from a normal and Cga^−/− mouse pituitary at 8 weeks. Top panels show a low-magnification view of the overall difference in the size of a typical thyrotrope shaded with green. Bottom panels depict an area within a typical thyrotrope with the characteristic thin-layered ER structure in wild types and greatly dilated ER in mutants. Also note the decreased number of secretory granules in the mutants, as well as the more electron dense mitochondria. cap, capillary; nuc, nucleus; g, secretory granule; m, mitochondrion; rer, rough ER.

Figure 2.

Thyrotrope hypertrophy and hyperplasia are associated with proliferation of TSHB-negative cells. A, Pituitaries were stained for EdU, which marks proliferating cells in S phase with green and counterstained with DAPI that makes the nuclei blue. B, Pituitary sections stained for TSHB in red, EdU in green, and nuclei in blue showed no costaining of TSHB and EdU in either genotype. Arrows indicate EdU-positive cells. Clusters of thyrotropes are interspersed with TSHB-negative cells. Scale bar, 100 μm (A, B, and D). Mice were 8 weeks of age. C, The proportion of DAPI positive cells that are EdU positive is increased in the Cga^−/− mice. Ratio is compared with wild type. D, Thyrotropes stained with TSHB (red) from representative areas of 8-week-old pituitaries show a mix of POU1F1 positive (green) and negative cells in both genotypes. Nuclei were counterstained with DAPI.

Figure 3.

Characterization of TCEAL5 in the pituitary and in a progenitor cell. A, Tceal5 expression is increased from birth to adulthood in the Cga^−/− pituitary. Absolute quantification with qPCR showed an incremental increase in Tceal5 expression from birth through 4 and 8 weeks of age in 50-ng cDNA; P ≤ .011 for all pairs. B, Tceal5 transcripts are localized in the anterior and intermediate lobes of the pituitary. Tceal5 is expressed in few AL and IL cells in the wild-type, and Cga^−/− pituitaries show an altered distribution within these lobes. Purple stain marks Tceal5, whereas green is nuclear counterstaining. Scale bar, 50 μm in all panels. C, Cga^−/− but not wild-type pituitaries have Tceal5-positive thyrotropes. Combined Tceal5 ISH (purple) with TSHB IHC (brown) and green nuclear counterstaining. In a typical view, yellow arrows mark some Tceal5+/TSHB−, and black arrows mark Tceal5+/TSHB+ cells. Note that single Tceal5+ cells are typically close to thyrotrope clusters. D, A small portion of proliferating cells express Tceal5. Proliferation detected with EdU (green) and Tceal5 mRNA (purple) over nuclear staining with DAPI (blue). Representative views from both genotypes showing cells with arrows as EdU+/Tceal5+ (black), Edu+/Tceal5− (yellow) and EdU−/Tceal5+ (red), respectively. E, Transgenic TCEAL5-EGFP has nuclear expression in the Pit1-triple pituitary progenitor cell line. Individual cells were imaged with confocal microscopy to determine the localization of EGFP in stable, transgenic cell populations. EGFP without TCEAL5 was localized to the cytoplasm, whereas the TCEAL5-GFP fusion localized to the nucleus. Scale bar, 10 μm. F, Stable overexpression of Tceal5 significantly enhances proliferation in a pituitary progenitor cell line. The Pit1-triple line expresses Pou1f1, Gh, Prl, Tshb, and Cga (38). We created heterologous lines stably expressing a Tceal5-Egfp gene fusion or Egfp only and plated the same number of cells on day 0. Six plates with each were monitored and 3 were harvested for cell counting after 4 or 8 days. G, Stable overexpression of Tceal5 does not change the apoptosis rate in a pituitary progenitor line. Nine wells in a cluster plate were assayed for apoptosis with the TUNEL assay heterologous cell lines stably expressing Tceal5-Egfp gene fusion or Egfp only. Nuclei were counterstained with DAPI, and the percentage of TUNEL+ cells was compared. NS, not significant.

Figure 4.

Protein sequence comparison suggests similar and divergent functions for the mouse TCEAL5. A, Conserved domain comparison for mouse TCEAL proteins (NCBI Conserved Domain search, http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). TCEAL proteins share the BEX domain and TCEAL3/6 include an approximately 70-amino acid-long segment similar to the conserved DUF2042 domain, which has an unknown function. B, The mouse TCEAL5 protein is highly conserved among mammals. Q5H9L2, Q8CCT4, E2RDN6, H2R4M4, and F6UAE4 protein sequences from the UniProt database (http://www.uniprot.org/) for human, mouse, dog, chimpanzee, and horse, respectively. Symbols mark the following: asterisk, identical; colon/semicolon, similar; hyphen, missing amino acids. The BEX domain is marked with magenta box. C, Functional domain comparison suggests different functions in proliferation for TCEAL5 compared with related proteins. Alignment was created with K-Align from the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/msa/kalign/) with default settings and coloring of individual amino acid residues followed the Clustal X color scheme (http://www.jalview.org/help/html/colourSchemes/clustal.html). Magenta, translucent blue, and green boxes are BEX domain, proapoptotic, and regulatory functional domain, respectively.