THE JEREMIAH METZGER LECTURE: A BRIEF HISTORY OF EUGENICS IN AMERICA: IMPLICATIONS FOR MEDICINE IN THE 21ST CENTURY.

In the first half of the 20th century, the US was swept up in a multifaceted movement to enhance the genetic makeup of the country's population. This eugenics movement, based on flawed scientific principles promulgated by Galton in the UK and Davenport in the US included legally mandated compulsory sterilization in 27 states in the US and sharply restricted immigration from many parts of the world. Compulsory sterilization legislation was upheld by the Supreme Court in 1927. The American eugenics movement was a model for the compulsory sterilization implemented by the Nazis after they took power in Germany in 1933. The movement waned in America only following World War II when the US public became aware of the full extent of the Nazi Aryan racial superiority program. With the advent of major advances in molecular and cellular biology that are already being applied to clinical medicine in the 21st century, we have entered a new eugenics era. It is critical that we learn the lessons of our earlier eugenics movement if we are to avoid making the same flawed decisions now.

Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders.

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca(2+) i) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα11 mutations mediate uveal melanoma development by constitutively up-regulating MAPK signaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstream Gα11 protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca(2+) i alterations associated with FHH2- and ADH2-causing Gα11 mutations, and evaluated the influence of germline gain-of-function Gα11 mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα11 mutant. WT and mutant Gα11 proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca(2+) i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca(2+) (Ca(2+) o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca(2+) i responses of FHH2- and ADH2-associated Gα11 loss- and gain-of-function mutations, respectively. ADH2-causing Gα11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca(2+) o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic Gα11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα11 mutations induce non-constitutive alterations in MAPK signaling.

The stem cell wars: a dispatch from the front.

The development of human embryonic stem cell (hESC) lines in 1998 offered the prospect of a new era of regenerative medicine in which cell therapy might cure intractable diseases such as type 1 diabetes, Parkinson's disease, and spinal cord injury. The Bush Administration decision in 2001 to restrict federal funding of hESC research touched off a controversy that continues to the present. One response to the Bush policy was establishment of state stem cell research funding programs, notably the California Institute of Regenerative Medicine (CIRM). As Director of the National Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK) and Vice Chair of the National Institutes of Health (NIH) Stem Cell Task Force, and now as a member of the Empire State Stem Cell Funding Board and member of an Institute of Medicine (IOM) committee charged with evaluation of the CIRM, I have had the opportunity to gain a first-hand perspective of the field. Here I present my impressions of the legal and science policy debates and selectively summarize research progress toward the hoped-for cures.

Genome-wide analysis of menin binding provides insights into MEN1 tumorigenesis.

Multiple endocrine neoplasia type I (MEN1) is a familial cancer syndrome characterized primarily by tumors of multiple endocrine glands. The gene for MEN1 encodes a ubiquitously expressed tumor suppressor protein called menin. Menin was recently shown to interact with several components of a trithorax family histone methyltransferase complex including ASH2, Rbbp5, WDR5, and the leukemia proto-oncoprotein MLL. To elucidate menin's role as a tumor suppressor and gain insights into the endocrine-specific tumor phenotype in MEN1, we mapped the genomic binding sites of menin, MLL1, and Rbbp5, to approximately 20,000 promoters in HeLa S3, HepG2, and pancreatic islet cells using the strategy of chromatin-immunoprecipitation coupled with microarray analysis. We found that menin, MLL1, and Rbbp5 localize to the promoters of thousands of human genes but do not always bind together. These data suggest that menin functions as a general regulator of transcription. We also found that factor occupancy generally correlates with high gene expression but that the loss of menin does not result in significant changes in most transcript levels. One exception is the developmentally programmed transcription factor, HLXB9, which is overexpressed in islets in the absence of menin. Our findings expand the realm of menin-targeted genes several hundred-fold beyond that previously described and provide potential insights to the endocrine tumor bias observed in MEN1 patients.

Parathyroid tumor development involves deregulation of homeobox genes.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome caused by mutations in the MEN1 tumor suppressor gene. Loss of the functional second copy of the MEN1 gene causes individuals to develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and pancreas. While it is clear that the protein encoded by MEN1, menin, suppresses endocrine tumors, its biochemical functions and direct downstream targets remain unclear. Recent studies have suggested that menin may act as a scaffold protein to coordinate gene transcription, and that menin is an oncogenic cofactor for homeobox (HOX) gene expression in hematopoietic cancer. The role of HOX genes in adult cell differentiation is still obscure, but growing evidence suggests that they may play important roles in the development of cancer. Therefore, we hypothesized that specific HOX genes were regulated by menin in parathyroid tumor development. Utilizing quantitative TaqMan RT-PCR, we compared expression profiles of the 39 HOX genes in human familial MEN1 (fMEN1) parathyroid tumors and sporadic parathyroid adenomas with normal samples. We identified a large set of 23 HOX genes whose deregulation is specific for fMEN1 parathyroid tumors, and only 5 HOX genes whose misexpression are specific for sporadic parathyroid tumor development. These findings provide the first evidence that loss of the MEN1 tumor suppressor gene is associated with deregulation of specific HOX gene expression in the development of familial human parathyroid tumors. Our results strongly reinforce the idea that abnormal expression of developmental HOX genes can be critical in human cancer progression.

Parathyroid-specific double knockout of Gq and G11 alpha-subunits leads to a phenotype resembling germline knockout of the extracellular Ca2+ -sensing receptor.

Germline knockout of the extracellular Ca2+ -sensing receptor (CaR) leads to a phenotype that includes severe hypercalcemia, hyperparathyroidism, relative hypocalciuria, skeletal abnormalities, retarded growth, and early postnatal death. To investigate the role of heterotrimeric G proteins in CaR signaling, we used cre/lox technology to delete the respective alpha-subunits of Gq and G11 selectively in parathyroid cells. Mice that were PTH-Cre(+/-); Gnaq(flox/flox); Gna11(-/-) (PTH-Galphaq/Galpha11 -double knockouts) were viable, but showed all the features of germline knockout of the CaR except hypocalcuria. Our results demonstrate the critical role of both Gq and G11 in mediating inhibition of PTH secretion by extracellular Ca2+.

A double negative: inhibition of hepatic Gi signaling improves glucose homeostasis.

Hepatic glucose production (HGP) is a key determinant of glucose homeostasis. Glucagon binding to its cognate seven-transmembrane Gs-coupled receptor in hepatocytes stimulates cAMP production, resulting in increased HGP. In this issue of the JCI, Rossi and colleagues tested the hypothesis that activation of hepatic Gi-coupled receptors, which should inhibit cAMP production, would oppose the cAMP-inducing action of glucagon and thereby decrease HGP. Surprisingly, however, the opposite occurred: activation of Gi signaling increased HGP via a novel mechanism, while inhibition of Gi signaling reduced HGP. These results define a new physiologic role for hepatic Gi signaling and identify a potential therapeutic target for HGP regulation.

Functional effects of monoclonal antibodies to the purified amino-terminal extracellular domain of the human Ca(2+) receptor.

UNLABELLED: We generated three functionally unique monoclonal antibodies to the purified human CaR extracellular domain. Flow cytometry studies of chimeric receptors localized their epitopes to lobe 2 of the VFT domain. These results lead us to propose a mechanism for the functional effects of these antibodies. INTRODUCTION: The human Ca(2+) receptor (CaR), which plays a central role in the regulation of [Ca(2+)](0) homeostasis, has a distinctively large extracellular domain that consists of a bilobed Venus flytrap (VFT) domain, involved in agonist binding, and a cysteine-rich domain. Functional antibodies that specifically bind to this domain would have therapeutic potential and could be used as a tool to gain insights into receptor activation as well. MATERIALS AND METHODS: We generated three monoclonal antibodies (mAbs), 7F8, 5C8, and 1A8, to the purified human CaR extracellular domain. Functional characterization of these antibodies included Ca(2+) stimulation of phosphoinositide hydrolysis to examine effects of intact or protease digested antibodies on sensitivity of the receptor to extracellular Ca(2+) and flow cytometry assay of binding of the antibodies to HEK-293 cells expressing chimeric receptors to map antibody epitopes. RESULTS: We found these mAbs specifically recognize native but not denatured human CaR or homologous native Fugu CaR. Sensitivity of the human CaR to extracellular calcium was increased by binding of 5C8 but decreased by binding of 1A8. A chimeric receptor FCFCF, with lobe 2 region of the human CaR VFT domain in the Fugu CaR backbone, bound all three mAbs, and the sensitivity of this chimeric CaR to extracellular Ca(2+) was also increased by binding of 5C8 and decreased by binding of 1A8. CONCLUSIONS: The epitopes of these mAbs reside in the lobe 2 region of the human CaR VFT domain. 5C8 might activate the receptor by facilitating closure and/or rotation of the VFT domains on agonist binding, whereas 1A8 might inhibit the receptor by impeding such agonist-induced conformational changes. Recombinant antibodies with antigen binding domains of 5C8 and 1A8 could be useful in the treatment of hyperparathyroidism and osteoporosis, respectively.

The parathyroid/pituitary variant of multiple endocrine neoplasia type 1 usually has causes other than p27Kip1 mutations.

CONTEXT: One variant of multiple endocrine neoplasia type 1 (MEN1) is defined by sporadic tumors of both the parathyroids and pituitary. The prevalence of identified MEN1 mutations in this variant is lower than in familial MEN1 (7% vs. 90%), suggesting different causes. Recently, one case of this variant had a germline mutation of p27(Kip1)/CDKN1B. OBJECTIVE: The objective was to test p27 in germline DNA from cases with tumors of both the parathyroids and pituitary. DESIGN: Medical record review and sequence analysis in DNA were performed. SETTING: This study involved an inpatient and outpatient referral program for cases of endocrine tumors. PATIENTS: Sixteen index cases had sporadic tumors of two organs, both the parathyroids and the pituitary. There were 18 additional index cases with related features of familial tumors. Five subjects were normal controls. No case had an identified MEN1 mutation. INTERVENTIONS: Clinical status of endocrine tumors was tabulated. Sequencing of germline DNA from index cases and control cases for the p27 gene was performed by PCR. MAIN OUTCOME MEASURES: Endocrine tumor types and their expressions were measured, as were sequence changes in the p27 gene. RESULTS: Tumor features were documented in index cases and families. One p27 germline single nucleotide change was identified. This predicted a silent substitution of Thr142Thr. Furthermore, there was a normal prevalence of heterozygosity for a common p27 polymorphism, making a large p27 deletion unlikely in all or most of these cases. CONCLUSIONS: The MEN1 variant with sporadic parathyroid tumors, sporadic pituitary tumor, and no identified MEN1 mutation is usually not caused by p27 germline mutations. It is usually caused by as yet unknown process(es).

The 32-kilodalton subunit of replication protein A interacts with menin, the product of the MEN1 tumor suppressor gene.

Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.

Of mice and MEN1: Insulinomas in a conditional mouse knockout.

Patients with multiple endocrine neoplasia type 1 (MEN1) develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and endocrine pancreas, due to the inactivation of the MEN1 gene. A conditional mouse model was developed to evaluate the loss of the mouse homolog, Men1, in the pancreatic beta cell. Men1 in these mice contains exons 3 to 8 flanked by loxP sites, such that, when the mice are crossed to transgenic mice expressing cre from the rat insulin promoter (RIP-cre), exons 3 to 8 are deleted in beta cells. By 60 weeks of age, >80% of mice homozygous for the floxed Men1 gene and expressing RIP-cre develop multiple pancreatic islet adenomas. The formation of adenomas results in elevated serum insulin levels and decreased blood glucose levels. The delay in tumor appearance, even with early loss of both copies of Men1, implies that additional somatic events are required for adenoma formation in beta cells. Comparative genomic hybridization of beta cell tumor DNA from these mice reveals duplication of chromosome 11, potentially revealing regions of interest with respect to tumorigenesis.

Inherited endocrine diseases involving G proteins and G protein-coupled receptors.

Naturally occurring mutations in the G protein Gs-alpha subunit and in a number of G protein-coupled receptors (GPCRs) have been identified in human diseases. Loss-of-function mutations in GPCRs for various hormones lead to hormone resistance manifest as hypofunction of the gland expressing the affected GPCR. Conversely, GPCR gain-of-function mutations lead to hormone-independent activation and hyperfunction of the involved gland. Our laboratory has focused on the extracellular calcium-sensing GPCR (CaR) expressed primarily, but not exclusively, in parathyroid glands and kidney. Loss-of-function CaR mutations lead to a form of hyperparathyroidism, an apparent exception to the general pattern described above, but in fact reflecting resistance to the normal inhibition of parathyroid hormone secretion by the 'hormone' agonist, extracellular Ca2+. CaR gain-of function-mutations cause autosomal dominant hypocalcemia due to activation of the receptor at subphysiologic concentrations of serum Ca2+, leading to 'inappropriate' inhibition of parathyroid hormone secretion. I will describe our recent work that helps inform design of novel therapeutics targeting this important GPCR.

Pancreatic insulinomas in multiple endocrine neoplasia, type I knockout mice can develop in the absence of chromosome instability or microsatellite instability.

Multiple endocrine neoplasia, type I (MEN1) is an inherited cancer syndrome characterized by tumors arising primarily in endocrine tissues. The responsible gene acts as a tumor suppressor, and tumors in affected heterozygous individuals occur after inactivation of the wild-type allele. Previous studies have shown that Men1 knockout mice develop multiple pancreatic insulinomas, but this occurs many months after loss of both copies of the Men1 gene. These studies imply that loss of Men1 is not alone sufficient for tumor formation and that additional somatic genetic changes are most likely essential for tumorigenesis. The usual expectation is that such mutations would arise either by a chromosomal instability or microsatellite instability mechanism. In a study of more then a dozen such tumors, using the techniques of array-based comparative genomic hybridization, fluorescent in situ hybridization, loss of heterozygosity analysis using multiple microsatellite markers across the genome, and real time PCR to assess DNA copy number, it appears that many of these full-blown clonal adenomas remain remarkably euploid. Furthermore, the loss of the wild-type Men1 allele in heterozygous Men1 mice occurs by loss and reduplication of the entire mutant-bearing chromosome. Thus, the somatic genetic changes that are postulated to lead to tumorigenesis in a mouse model of MEN1 must be unusually subtle, occurring at either the nucleotide level or through epigenetic mechanisms.

Parathyroid gland-specific deletion of the mouse Men1 gene results in parathyroid neoplasia and hypercalcemic hyperparathyroidism.

The inactivation of the MEN1 tumor suppressor gene in patients leads to a constellation of changes in endocrine tissues, including parathyroid neoplasia, pituitary adenomas, pancreatic neuroendocrine tumors, and carcinoids. To study the pathophysiological consequences of the deletion of the MEN1 gene, we set out to create a mouse model of hyperparathyroidism resulting from the deletion of the Men1 gene in parathyroid tissue. We introduced a Men1 gene flanked by loxP sites into the mouse germ line and then used a parathyroid cell-specific promoter to drive the expression of Cre recombinase, resulting in the deletion of the Men1 gene. Here, we show that loss of Men1 gene function in the parathyroid glands of mice results in histological changes consistent with parathyroid neoplasia as well as systemic hypercalcemia. This model provides a means for dissecting the molecular basis of this familial cancer syndrome and may allow for the development of new strategies to treat related forms of hypercalcemia.

Executive summary of the Strategic Plan for National Institutes of Health Obesity Research.

The Strategic Plan for National Institutes of Health (NIH) Obesity Research is intended to serve as a guide for coordinating obesity research activities across the NIH and for enhancing the development of new efforts based on identification of areas of greatest scientific opportunity and challenge. Developed by the NIH Obesity Research Task Force with critical input from external scientists and the public, the Strategic Plan reflects a dynamic planning process and presents a multidimensional research agenda, with an interrelated set of goals and strategies for achieving the goals. The major scientific themes around which the Strategic Plan is framed include the following: preventing and treating obesity through lifestyle modification; preventing and treating obesity through pharmacologic, surgical, or other medical approaches; breaking the link between obesity and its associated health conditions; and cross-cutting topics, including health disparities, technology, fostering of interdisciplinary research teams, investigator training, translational research, and education/outreach efforts. Through the efforts described in the Strategic Plan for NIH Obesity Research, the NIH will strive to facilitate and accelerate progress in obesity research to improve public health.

Naturally occurring mutations of the extracellular Ca2+-sensing receptor: implications for its structure and function.

The extracellular Ca2+-sensing receptor is a member of the G-protein-coupled receptor family 3 in which agonists bind to a dimeric Venus-flytrap domain in the extracellular portion of the receptor. How agonist binding to this domain leads to activation of the seven-transmembrane domain is a major unresolved question. Information derived from the three-dimensional structure of the Venus-flytrap domain of the related metabotropic glutamate type 1 receptor, and from naturally occurring mutations of the Ca2+-sensing receptor identified in subjects with familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia offers new insights into the mechanism of receptor activation, and into the mechanism of action of allosteric modulators of the receptor.

The Calcilytic Agent NPS 2143 Rectifies Hypocalcemia in a Mouse Model With an Activating Calcium-Sensing Receptor (CaSR) Mutation: Relevance to Autosomal Dominant Hypocalcemia Type 1 (ADH1).

Autosomal dominant hypocalcemia type 1 (ADH1) is caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and may lead to symptomatic hypocalcemia, inappropriately low serum PTH concentrations and hypercalciuria. Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1. However, the effectiveness of calcilytic drugs for the treatment of ADH1-associated hypocalcemia remains to be established. We have investigated NPS 2143, a calcilytic compound, for the treatment of ADH1 by in vitro and in vivo studies involving a mouse model, known as Nuf, which harbors a gain-of-function CaSR mutation, Leu723Gln. Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry. NPS 2143 was also administered as a single ip bolus to wild-type and Nuf mice and plasma concentrations of calcium and PTH, and urinary calcium excretion measured. In vitro administration of NPS 2143 decreased the intracellular calcium responses of HEK293 cells expressing the mutant Gln723 CaSR in a dose-dependent manner, thereby rectifying the gain-of-function associated with the Nuf mouse CaSR mutation. Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion. These studies of a mouse model with an activating CaSR mutation demonstrate NPS 2143 to normalize the gain-of-function causing ADH1 and improve the hypocalcemia associated with this disorder.