Mary P. Gillam

Functional Characterization of Pendrin in a Polarized Cell System EVIDENCE FOR PENDRIN-MEDIATED APICAL IODIDE EFFLUX

Pendreds syndrome is an autosomal recessive disorder characterized by sensorineural deafness, goiter, and impaired iodide organification. It is caused by mutations in the PDS/SLC26A4 gene that encodes pendrin. Functionally, pendrin is a transporter of chloride and iodide in Xenopus oocytes and heterologous mammalian cells and a chloride/base exchanger in β-intercalated cells of the renal cortical collecting duct. The partially impaired thyroidal iodide organification in Pendreds syndrome suggests a possible role of pendrin in iodide transport at the apical membrane of thyroid follicular cells, but experimental evidence for this concept is lacking. The iodide transport properties of pendrin were determined in polarized Madin-Darby canine kidney cells expressing the sodium iodide symporter (NIS), pendrin, or NIS and pendrin using a bicameral system-permitting measurement of iodide content in the basal, intracellular, and apical compartments. Moreover, we determined the functional consequences of two naturally occurring mutations (L676Q and FS306>309X). In polarized Madin-Darby canine kidney cells, NIS mediates uptake at the basolateral membrane. Only minimal amounts of iodide reach the apical compartment in the absence of pendrin. In cells expressing NIS and pendrin, pendrin mediates transport of iodide into the apical chamber. Wild type pendrin also mediates iodide efflux in transiently transfected cells. In contrast, both pendrin mutants lose the ability to promote iodide efflux. These results provide evidence that pendrin mediates apical iodide efflux from polarized mammalian cells loaded with iodide. Consistent with the partial organification defect observed in patients with Pendreds syndrome, naturally occurring mutations of pendrin lead to impaired transport of iodide.

Genetic regulation of thyroid development.

Normal thyroid function is essential for development, growth, and metabolic homeostasis. The prerequisites for an euthyroid metabolic state include a normally developed thyroid gland, a properly functioning system for thyroid hormone synthesis, and sufficient iodine intake. Defects in any of the essential steps in thyroid development or thyroid hormone synthesis may result in morphologic abnormalities and impaired hormonogenesis. These defects can be partial or complete, leading to varying degrees of hypothyroidism. Morphologic alterations associated with congenital hypothyroidism include the absence of detectable thyroid tissue, ectopic tissue, thyroid hypoplasia, or a goitrous thyroid. However, in some patients with hypothyroidism, the thyroid is of normal size. This article focuses on defects in thyroid development. Recent insights into the developmental regulation of the calcitonin-producing C cells will not be discussed, and defects in hormone synthesis are discussed in an accompanying article.

Genetic defects in thyroid hormone synthesis

Thyroid hormone synthesis requires a normally developed thyroid gland, a properly functioning hypothalamic-pituitary-thyroid axis, and sufficient iodine intake. This article focuses on genetic defects in this axis. Defects that are primarily of developmental origin are discussed in our associated article in this issue [1]. Defects in hormone synthesis usually are associated with the development of a goiter, provided that the bioactivity and action of thyrotropin (TSH) are not impaired. In contrast, hypoplasia of the gland may be caused by developmental defects, bioinactive TSH, or resistance to TSH at the level of the receptor or its signaling pathway. At the other end of the spectrum, hyperthyroidism may result from gain of function mutations in genes regulating growth and function.

Aggressive metastatic follicular thyroid carcinoma with anaplastic transformation arising from a long-standing goiter in a patient with Pendred’s syndrome

In this article we describe detailed pathological and molecular genetics studies in a consanguineous kindred with Pendreds syndrome. The index patient was a 53-year-old female patient with congenital deafness and goiter. Her parents were first-degree cousins. She had a large goiter (150 g) that had been present since childhood. One of her sisters and a niece are also deaf and have goiter as well. The presence of Pendreds syndrome was confirmed by a positive perchlorate test and the demonstration of a Mondini malformation. Thyroid function tests (under levothyroxine [LT4] therapy) were in the euthyroid range with a thyrotropin [TSH] level of 2.8 microU/mL (0.2-3.2), a serum total thyroxine (T4) of 90 nmol/L (54-142), and a serum total triiodothyronine (T3) of 2.7 nmol/L (0.8-2.4). Total thyroidectomy was performed, and the mass in the right lobe was found to have invaded adjacent tissues. The histopathological findings were consistent with a follicular carcinoma with areas of anaplastic transformation and lung metastasis. The patient was treated twice with 100 mCi 131iodine (3,700 MBq) and received suppressive doses of LT4. Postoperatively, the serum thyroglobulin (Tg) levels remained markedly elevated (2,352 to 41,336 ng/mL). The patient died of a sudden severe episode of hemoptysis. Sequence analysis of the PDS gene performed with DNA from the two relatives with Pendreds syndrome revealed the presence of a deletion of thymidine 279 in exon 3, a point mutation that results in a frameshift and a premature stop codon at codon 96 in the pendrin molecule. We concluded that prolonged TSH stimulation because of iodine deficiency or dyshormonogenesis in combination with mutations of oncogenes and/or tumor suppressor genes, may result in the development of follicular thyroid carcinomas that undergo transformation into anaplastic cancers. It is likely that these pathogenetic mechanisms have been involved in the development of aggressive metastatic thyroid cancer in this unusual patient with Pendreds syndrome.

MEN1 tumorigenesis in the pituitary and pancreatic islet requires Cdk4 but not Cdk2.

Recent studies suggest that physiological and tumorigenic proliferation of mammalian cells is controlled by multiple cyclin-dependent kinases (CDKs) largely in tissue-specific manners. We and others previously demonstrated that adult mice deficient for the Cyclin D partner CDK4 (Cdk4−/− mice) exhibit hypoplasia in the pituitary and pancreatic islet due to primary postnatal defects in proliferation. Intriguingly, those neuroendocrine tissues affected in Cdk4−/− mice are the primary targets of tumorigenesis in the syndrome of multiple endocrine neoplasia type-1 (MEN1). Mice with heterozygous disruption of the tumor suppressor Men1 gene (Men1+/−) develop tumors in the pituitary, pancreatic islets and other neuroendocrine tissues, which is analogous to humans with MEN1 mutations. To explore the genetic interactions between loss of Men1 and activation of CDKs, we examined the impact of Cdk4 or Cdk2 disruption on tumorigenesis in Men1+/− mice. A majority of Men1+/− mice with wild-type CDKs developed pituitary and islet tumors by 15 months of age. Strikingly, Men1+/−; Cdk4−/− mice did not develop any tumors, and their islets and pituitaries remained hypoplastic with decreased proliferation. In contrast, Men1+/−; Cdk2−/− mice showed pituitary and islet tumorigenesis comparable to those in Men1+/− mice. Pituitaries of Men1+/−; Cdk4−/− mice showed no signs of loss of heterozygosity (LOH) in the Men1 locus, whereas tumors in Men1+/− mice and Men1+/−; Cdk2−/− mice exhibited LOH. Consistently, CDK4 knockdown in INS-1 insulinoma cells inhibited glucose-stimulated cell cycle progression with a significant decrease in phosphorylation of retinoblastoma protein (RB) at specific sites including Ser780. CDK2 knockdown had minimum effects on RB phosphorylation and cell cycle progression. These data suggest that CDK4 is a critical downstream target of MEN1-dependent tumor suppression and is required for tumorigenic proliferation in the pituitary and pancreatic islet, whereas CDK2 is dispensable for tumorigenesis in these neuroendocrine cell types.

HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells

Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the beta-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms.

Chapter 15 – Prolactinoma

Publisher Summary Prolactinomas are classified on a clinical basis by size—microadenomas are defined as less than 10 mm in diameter; macroadenomas are greater than 10 mm in diameter. Giant prolactinomas are defined as greater than 4 cm in diameter and those with more than 2 cm of suprasellar extension. Supra-, infra- and parasellar extension occur when adenomas grow beyond the immediate sellar region. Coincident with the initial characterization of prolactin (PRL) in the early 1930s by Riddle and his colleagues were the first clinical reports of a syndrome of amenorrhea coupled with galactorrhea. However, it was not until 1972 that Friesen et al. first demonstrated elevated PRL levels in the serum of a patient with a prolactinoma, the decline in such levels with partial adenomectomy, and the production of PRL by the tumor in vitro. Over the ensuing years, improvements in diagnostic radiologic procedures, advances in surgical techniques and the development of dopamine agonists as medical therapy have resulted in highly successful rates of treatment for the majority of prolactinomas. Prolactinomas are the most common subtype of the hormone-secreting pituitary tumours according to autopsy, epidemiological and surgical series.

Somatotroph-Specific Aip-Deficient Mice Display Pretumorigenic Alterations in Cell-Cycle Signaling

Patients with familial isolated pituitary adenoma are predisposed to pituitary adenomas, which in a subset of cases is due to germline inactivating mutations of the aryl hydrocarbon receptor–interacting protein (AIP) gene. Using Cre/lox and Flp/Frt technology, a conditional mouse model was generated to examine the loss of the mouse homolog, Aip, in pituitary somatotrophs. By 40 weeks of age, >80% of somatotroph specific Aip knockout mice develop growth hormone (GH) secreting adenomas. The formation of adenomas results in physiologic effects recapitulating the human syndrome of acromegaly, including increased body size, elevated serum GH and insulin-like growth factor 1 levels, and glucose intolerance. The pretumorigenic Aip-deficient somatotrophs secrete excess GH and exhibit pathologic hyperplasia associated with cytosolic compartmentalization of the cyclin-dependent kinase (CDK) inhibitor p27kip1 and perinuclear accentuation of CDK-4. Following tumor formation, the Aip-deficient somatotrophs display reduced expression of somatostatin receptor subtype 5 with impaired response to octreotide. The delayed tumor emergence, even with loss of both copies of Aip, implies that additional somatic events are required for adenoma formation. These findings suggest that pituitary hyperplasia precedes adenomatous transformation in somatotroph-specific Aip-deficient mice and reveal potential mechanisms involved in the pretumorigenic state that ultimately contribute to transformation.

Chapter 5 – Prolactin

Publisher Summary In the late 1920s and early 1930s, it was found that pituitary extracts could induce milk secretion. Riddle and coworkers found that this substance, which they named prolactin (PRL), could be differentiated from the known growth- and gonad-stimulating substances. In these experiments, they showed that PRL stimulated milk production by guinea pig mammary glands and a milk-like substance from the crop sacs of pigeons and doves, giving rise to the pigeon crop sac bioassay for PRL. Over the ensuing years, PRL was characterized, sequenced and specific radioimmunoassays (RIAs) developed for PRL from a number of species. Because of the high lactogenic activity of even very highly purified preparations of human growth hormone (GH), however, it was impossible to separate human PRL from GH using the relatively crude pigeon crop assay. Finally, in 1970, Frantz and Kleinberg developed a sensitive in vitro bioassay which involved staining milk produced by cultured, lactating mouse mammary tissue in response to PRL that was capable of measuring PRL levels as low as 5 ng/ml. In this assay, they added excess antibody to GH to neutralize any potential lactogenic effects it had and, for the first time, were able to demonstrate measurable PRL levels in women with puerperal and nonpuerperal galactorrhea, but not in most normal men and women. Shortly thereafter, an RIA for human RL was developed which could finally measure PRL levels in the sera of normal individuals, permitting the eventual sequencing of human PRL and determination of its cDNA sequence.

OSTEOPOROSIS: Vitamin D inadequacy in women treated for osteoporosis

Men were younger than age 75 years at study entry; median age was 64 years. About 75% of men had T2lesions and 60% had Gleason grade 6 or less. No data are available on PSA velocity before randomization. Radical prostatectomy was done in 34 7 men and a watchand-wait philosophy was applied to 348 men. After 8 years of follow-up, 83 men on the surgery arm had died and 106 men on watch-and-wait had died. Deaths from causes other than prostate cancer were similar in the two arms (53 for surgery; 56 for watch-and-wait); however, 50 men on the watch-and-wait arm died of prostate cancer compared to 30 men in the surgery arm (p = .01). This difference projects to a 5.3% difference at 10 years in favor of surgery. When results were analyzed by age, the biggest difference favoring surgery was in men