Pituitary-specific knockout of the Carney complex gene Prkar1a leads to pituitary tumorigenesis

Abstract

Carney complex (CNC) is an inherited neoplasia syndrome characterized by spotty skin pigmentation, myxomas, endocrine tumors, and schwannomas. Among the endocrine tumors that comprise the syndrome, GH-producing pituitary tumors are seen in approximately 10% of patients, although biochemical abnormalities of the GH axis are much more common. To explore the role of loss of the CNC gene PRKAR1A on pituitary tumorigenesis, we produced a tissue-specific knockout (KO) of this gene in the mouse. For these studies, we generated a mouse line expressing the cre recombinase in pituitary cells using the rat GHRH receptor promoter. These mice were then crossed with Prkar1a conditional null animals to produce tissue-specific KOs. Although prolactinomas were observed in KO and control mice, the KO mice exhibited a significantly increased frequency of pituitary tumors compared with wild-type or conventional Prkar1a(+/-) mice. Characterization of the tumors demonstrated they were composed of cells of the Pit1 lineage that stained for GH, prolactin, and TSH. At the biochemical level, levels of GH in the serum of KO animals were markedly elevated compared with controls, regardless of the presence of a frank tumor. These data indicate that complete loss of Prkar1a is sufficient to allow the formation of pituitary tumors and abnormalities of the GH axis, in close analogy to human patients with CNC.

Figure 1

rGHRHR-cre Mice Express Cre Recombinase Activity in the Anterior Pituitary A, Diagram of the rGHRHR-cre transgene. See Materials and Methods for details of the construction of the transgene; B, rGHRHR-cre mice express cre activity in the anterior pituitary. Left, H&E staining of a frozen section of the pituitary of an rGHRHR-cre(3242) mouse; right, alkaline phosphatase staining (purple) from a Z/AP cross reveals cre activity in isolated clusters of cells in the anterior pituitary. A, Anterior lobe; I, intermediate lobe; P, posterior lobe.

Figure 2

Cre Activity Is Limited to Cells that Produce GH, Prl, and TSH Pituitary glands from lacZ reporter crosses were stained in whole mount for β-galactosidase (blue), postfixed, and then analyzed by immunohistochemistry for hormone subunit production. Staining for each of the hormones is indicated by the brown diaminobenzidine color. Each panel shows sections of the same pituitary gland stained for the hormones indicated in the upper right of each picture. Note the colocalization of blue and brown colors in the GH, Prl, and TSH panels, whereas no colocalization is observed for ACTH, LH, or FSH. A scale bar, applicable to all panels in the figure, is shown at lower right.

Figure 3

Distribution of Hormone Staining and Cre Activity in rGHRHRcre Mice Hormone staining data from pituitaries (as shown in Fig. 2) were quantitated, and the summary data are shown to indicate the relative distribution of the individual hormone-producing pituitary cell types. A, The distribution of cell subtypes for the entire pituitary gland (note that gonadotroph cells producing LH and FSH are shown as a single cell type); B, distribution of cell subtypes for those cells expressing cre recombinase, as determined by lacZ staining. Percentages for each slice of the pie chart are indicated.

Figure 4

PitKO Mice Exhibit Pituitary Tumorigenesis A, H&E-stained section of a pitKO gland showing the presence of a small adenoma, indicated with the dashed line; B, section of the same tumor shows loss of the normal reticulin staining pattern in the region of the tumor (dashed circle); C, higher-power view of the same tumor showing the presence of multiple mitotic figures (black arrows) and dysplastic nuclei (white arrows); D, H&E-stained section of another pitKO pituitary showing the presence of two small adenomas (dashed lines) in the gland. A scale bar applicable to panels A, B, and D is shown at lower right. A separate scale bar for panel C is included in that panel.

Figure 5

Immunohistochemical Characterization of PitKO Tumors Demonstrates Proliferation of Pit1-Derived Cell Types The tumor from Fig. 3, A–C, was stained for Pit1 and pituitary hormones. Note that the tumor stains clearly for the Pit-1 transcription (upper left) and strongly for GH (upper right). There is also focal TSH staining, indicated by the arrows (lower right). Note the absence of ACTH staining in the tumor, although this marker strongly stains the intermediate lobe of the pituitary, as expected (lower left). This tumor also exhibited staining for Prl but not for FSH or LH.