Michael T. McDermott

Pit-1 and GATA-2 Interact and Functionally Cooperate to Activate the Thyrotropin β-Subunit Promoter

The molecular determinants governing cell-specific expression of the thyrotropin (TSH) β-subunit gene in pituitary thyrotropes are not well understood. The P1 region of the mouse TSHβ promoter (−133 to −88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. Northern and Western blot assays demonstrated the presence of GATA-2 transcripts and protein in TtT-97 thyrotropic tumors. In electrophoretic mobility shift assays, a comigrating complex was observed with both TtT-97 nuclear extracts and GATA-2 expressed in COS cells. The complex demonstrated binding specificity to the P1 region DNA probe and could be disrupted by a GATA-2 antibody. When both Pit-1 and GATA-2 were combined, a slower migrating complex, indicative of a ternary protein-DNA interaction was observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 cells synergistically stimulated mouse TSHβ promoter activity 8.5-fold, while each factor alone had a minimal effect. Mutations that abrogated this functional stimulatory effect mapped to the P1 region. Finally, we show that GATA-2 directly interacts with Pit-1 in solution. In summary, these data demonstrate functional synergy and physical interaction between homeobox and zinc finger factors and provide insights into the transcriptional mechanisms of thyrotrope-specific gene expression.

Lack of Diagnosis and Treatment of Osteoporosis in Men and Women After Hip Fracture

Study Objective. To determine whether men and women admitted to a university teaching hospital for a low‐trauma hip fracture were diagnosed, evaluated, or treated for osteoporosis during admission or for up to 1 year after admission.

Towards improving the utility of fine‐needle aspiration biopsy for the diagnosis of thyroid tumours

Thyroid nodules are quite common with approximately 5% of the population, and approximately 10% of older individuals, having palpable thyroid nodules (Mazzaferri, 1993). Between 30 and 50% of people living in the United States and Europe have thyroid nodules noted on ultrasound examination. Fine-needle aspiration biopsy (FNAB) has become the mainstay of thyroid nodule evaluation. While the overall accuracy of FNAB is excellent, an indeterminate or suspicious biopsy can pose a diagnostic and management dilemma. Some of these issues are illustrated in the following patient vignettes.

Determinants of thyrotrope-specific thyrotropin beta promoter activation. Cooperation of Pit-1 with another factor.

Thyrotropin (TSH) β is a subunit of TSH, the expression of which is limited to the thyrotrope cells of the anterior pituitary gland. We have utilized the thyrotrope-derived TtT-97 thyrotropic tumors to investigate tissue-specific expression of the TSHβ promoter. TSHβ promoter activity in thyrotropes is conferred by sequences between −270 and −80 of the 5′-flanking region. We have recently reported that the proximal region from −133 to −100 (P1) is required for promoter expression in thyrotropes. This region interacts with the pituitary-specific transcription factor Pit-1. While Pit-1 appears necessary for TSHβ promoter activity in thyrotropes, this transcription factor is not alone sufficient for promoter activity in pituitary-derived cells. In this report, we have generated a series of promoter mutations in the P1 region to identify additional protein-DNA interactions and determine their functional significance. We have found that Pit-1 interacts with the distal portion of the P1 region, and a second protein interacts with the proximal segment of this region. Each protein is able to independently interact with the TSHβ promoter, but neither alone can maintain promoter activity. Both proteins appear to be necessary for full promoter activity in thyrotropes. Southwestern analysis with the proximal segment of the P1 region (−117 to −88) reveals interaction with a 50-kDa protein. Interestingly, this protein is not found in the pituitary-derived GH3 cells and may represent a thyrotrope-specific transcription factor. Further characterization of this newly identified DNA-binding protein will further our understanding of the tissue-specific expression of the TSHβ gene.

Novel Management of Insulin Autoimmune Syndrome with Rituximab and Continuous Glucose Monitoring

CONTEXT Insulin autoimmune syndrome (IAS), or Hiratas disease, is a rare hypoglycemic disorder characterized by spontaneous hypoglycemia without evidence of exogenous insulin administration, a high serum concentration of total immunoreactive insulin, and the presence of insulin autoantibodies in high titer. The majority of cases occur in the Asian population, and treatment is generally successful with watchful waiting or steroids. CASE DESCRIPTION We report the case of a 71-year-old Caucasian man with severe hypoglycemia due to IAS that was refractory to a prolonged course of high-dose steroids. Type 1 diabetes prevention trials using rituximab have shown selective suppression of insulin autoantibodies, which are the pathogenic antibodies in IAS, and therefore we provided this therapy. Treatment with two doses of rituximab and daily use of a continuous glucose monitor resulted in disease remission. CONCLUSION We present the first case where potentially life-threatening hypoglycemia due to IAS was successfully managed with rituximab and a continuous glucose monitor. We conclude that these treatment modalities are effective for the management of severe, refractory IAS.

Functional interactions of an upstream enhancer of the mouse glycoprotein hormone α-subunit gene with proximal promoter sequences

Transcription of the glycoprotein hormone alpha-subunit gene in the pituitary is governed by different promoter elements in thyrotropes and gonadotropes. We recently identified an upstream enhancer that directs a high level of cell type specific expression in transgenic mice and stimulates proximal promoter activity in cultured alphaTSH and alphaT3 cells. To assess the contribution of promoter sequences that functionally interact with the enhancer, we mutated two proximal elements shown to be important in both thyrotrope and gonadotrope cells. Disruption of the pituitary glycoprotein hormone basal element (PGBE), which binds a LIM homeodomain protein, resulted in a decrease in basal promoter activity in both alphaTSH and alphaT3 cells. Enhancer function was completely abolished by the PGBE site mutation in alphaT3 gonadotropes, whereas some stimulatory activity remained in alphaTSH thyrotropes. Mutation of the gonadotrope specific element (GSE), which binds SF1 and is important for basal activity in gonadotropes and TRH response in thyrotropes, resulted in declines in basal and enhanced promoter activity only in alphaT3 cells and not in alphaTSH cells. Despite this decrease in enhanced activity, the GSE mutated promoter still retained some enhancer stimulated activity, suggesting that the PGBE site still functionally interacts in the absence of an intact GSE. This mutation had no effect in alphaTSH cells. These data suggest that although the enhancer works in both cell types it exhibits cell type specific functional characteristics.

Reverse Transcription Polymerase Chain Reaction Analysis of Pituitary Hormone, Pit-1 and Steroidogenic Factor-1 Messenger RNA Expression in Pituitary Tumors

Pit-1 is a transcription factor that appears early in embryonic pituitary gland formation and is necessary for the development of somatotropes, lactotropes and thyrotropes. Steroidogenic factor-1 (SF-1) is another early appearing transcription factor that is involved in the development of gonadotropes. In this study we have compared RT-PCR analysis of hormone mRNA with traditional IHC for classification of 27 pituitary tumors and have evaluated the correlation of Pit-1 and SF-1 mRNA with hormone mRNA.RT-PCR detected concordant hormone mRNA in 100% of GH IHC positive, 100% of PRL IHC positive, 33% of TSH IHC positive, and 93% of gonadotropin IHC positive tumors. IHC, however, was concordant in only 71% of GH mRNA positive, 78% of PRL mRNA positive, 17% of TSHβ mRNA positive, and 76% of FSHβ mRNA positive tumors.Pit-1 mRNA was positive in 87% of tumors in which mRNA for GH, PRL or TSHβ was detected and in only 17% of GH, PRL and TSHβ mRNA negative tumors. SF-1 mRNA was positive in 94% of tumors in which mRNA for FSHβ was present and in no FSHβ mRNA negative tumors.We conclude that RT-PCR analysis of hormone mRNA may be more sensitive than traditional hormone IHC for classification of pituitary tumors. Furthermore, tumor Pit-1 mRNA positively correlates with GH, PRL and TSHβ mRNA while tumor SF-1 mRNA correlates well with FSHβ mRNA. Combined analysis of hormone and transcription factor mRNA in pituitary tumor tissue may therefore be a more meaningful approach to pituitary tumor characterization.