RESUMEN
There is increasing evidence that anterior pituitary hormones, traditionally thought to have unitary functions in regulating single endocrine targets, act on multiple somatic tissues, such as bone, fat, and liver. There is also emerging evidence for anterior pituitary hormone action on brain receptors in mediating central neural and peripheral somatic functions. Here, we have created the most comprehensive neuroanatomical atlas on the expression of TSHR, LHCGR, and FSHR. We have used RNAscope, a technology that allows the detection of mRNA at single-transcript level, together with protein level validation, to document Tshr expression in 173 and Fshr expression in 353 brain regions, nuclei and subnuclei identified using the Atlas for the Mouse Brain in Stereotaxic Coordinates. We also identified Lhcgr transcripts in 401 brain regions, nuclei and subnuclei. Complementarily, we used ViewRNA, another single-transcript detection technology, to establish the expression of FSHR in human brain samples, where transcripts were co-localized in MALAT1-positive neurons. In addition, we show high expression for all three receptors in the ventricular region-with yet unknown functions. Intriguingly, Tshr and Fshr expression in the ependymal layer of the third ventricle was similar to that of the thyroid follicular cells and testicular Sertoli cells, respectively. In contrast, Fshr was localized to NeuN-positive neurons in the granular layer of the dentate gyrus in murine and human brain-both are Alzheimer's disease-vulnerable regions. Our atlas thus provides a vital resource for scientists to explore the link between the stimulation or inactivation of brain glycoprotein hormone receptors on somatic function. New actionable pathways for human disease may be unmasked through further studies.
Asunto(s)
Glicoproteínas , Células de Sertoli , Animales , Encéfalo , Hormonas , Humanos , Masculino , Ratones , Testículo/fisiologíaRESUMEN
Pharmacological and genetic studies over the past decade have established the follicle-stimulating hormone (FSH) as an actionable target for diseases affecting millions, namely osteoporosis, obesity, and Alzheimer's disease. Blocking FSH action prevents bone loss, fat gain, and neurodegeneration in mice. We recently developed a first-in-class, humanized, epitope-specific FSH-blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a Good Laboratory Practice (GLP)-compliant platform, we now report the efficacy of MS-Hu6 in preventing and treating osteoporosis in mice and parameters of acute safety in monkeys. Biodistribution studies using 89Zr-labeled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone and bone marrow. The MS-Hu6 displayed a ß phase t½ of 7.5 days (180 hr) in humanized Tg32 mice. We tested 217 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze-thaw and at different temperatures, with minimal aggregation, and without self-, cross-, or hydrophobic interactions or appreciable binding to relevant human antigens. The MS-Hu6 showed the same level of "humanness" as human IgG1 in silico and was non-immunogenic in ELISpot assays for IL-2 and IFN-γ in human peripheral blood mononuclear cell cultures. We conclude that MS-Hu6 is efficacious, durable, and manufacturable, and is therefore poised for future human testing.
Asunto(s)
Hormona Folículo Estimulante , Osteoporosis , Animales , Epítopos/metabolismo , Excipientes , Hormona Folículo Estimulante/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Interleucina-2/metabolismo , Leucocitos Mononucleares/metabolismo , Ratones , Osteoporosis/tratamiento farmacológico , Distribución TisularRESUMEN
Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a KD of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH-FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHß subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.
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Tejido Adiposo/metabolismo , Anticuerpos Bloqueadores/inmunología , Huesos/metabolismo , Epítopos , Hormona Folículo Estimulante/inmunología , Animales , Anticuerpos Bloqueadores/química , Anticuerpos Monoclonales , Densidad Ósea , Femenino , Hormona Folículo Estimulante/química , Hormona Folículo Estimulante de Subunidad beta/inmunología , Humanos , Hipercolesterolemia , Ratones , Ratones Endogámicos C57BL , Simulación de Dinámica Molecular , Obesidad , Osteoporosis , Receptores de HFE/metabolismoRESUMEN
Today, research in biomedicine often requires the knowledge and technologies in diverse fields. Therefore, there is an increasing need for collaborative team science that crosses traditional disciplines. Here, we discuss our own lessons from both interdisciplinary and transdisciplinary teams, which ultimately ushered us to expand our research realm beyond bone biology.
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Huesos/metabolismo , Difosfonatos/uso terapéutico , Hormona Folículo Estimulante/metabolismo , Investigación Interdisciplinaria , Neoplasias/tratamiento farmacológico , Tejido Adiposo/metabolismo , Animales , Hormona Folículo Estimulante/antagonistas & inhibidores , Genes erbB-1 , Humanos , Neoplasias/genéticaRESUMEN
We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine ß-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.
Asunto(s)
Disfunción Eréctil/tratamiento farmacológico , Osteogénesis/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Inhibidores de Fosfodiesterasa 5/farmacología , Envejecimiento/fisiología , Animales , Densidad Ósea/efectos de los fármacos , Densidad Ósea/fisiología , Huesos/citología , Huesos/efectos de los fármacos , Huesos/metabolismo , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Diferenciación Celular/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Reposicionamiento de Medicamentos , Disfunción Eréctil/complicaciones , Humanos , Masculino , Ratones , Persona de Mediana Edad , Modelos Animales , Modelos Moleculares , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Osteoporosis/complicaciones , Fracturas Osteoporóticas/etiología , Fracturas Osteoporóticas/prevención & control , Inhibidores de Fosfodiesterasa 5/química , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Cultivo Primario de Células , Tadalafilo/química , Tadalafilo/farmacología , Tadalafilo/uso terapéutico , Diclorhidrato de Vardenafil/química , Diclorhidrato de Vardenafil/farmacología , Diclorhidrato de Vardenafil/uso terapéuticoRESUMEN
The primitive neurohypophyseal nonapeptide oxytocin (OXT) has established functions in parturition, lactation, appetite, and social behavior. We have shown that OXT has direct actions on the mammalian skeleton, stimulating bone formation by osteoblasts and modulating the genesis and function of bone-resorbing osteoclasts. We deleted OXT receptors (OXTRs) selectively in osteoblasts and osteoclasts using Col2.3Cre and Acp5Cre mice, respectively. Both male and female Col2.3Cre+:Oxtrfl/fl mice recapitulate the low-bone mass phenotype of Oxtr+/- mice, suggesting that OXT has a prominent osteoblastic action in vivo. Furthermore, abolishment of the anabolic effect of estrogen in Col2.3Cre+:Oxtrfl/fl mice suggests that osteoblastic OXTRs are necessary for estrogen action. In addition, the high bone mass in Acp5Cre+:Oxtrfl/fl mice indicates a prominent action of OXT in stimulating osteoclastogenesis. In contrast, we found that in pregnant and lactating Col2.3Cre+:Oxtrfl/fl mice, elevated OXT inhibits bone resorption and rescues the bone loss otherwise noted during pregnancy and lactation. However, OXT does not contribute to ovariectomy-induced bone loss. Finally, we show that OXT acts directly on OXTRs on adipocytes to suppress the white-to-beige transition gene program. Despite this direct antibeiging action, injected OXT reduces total body fat, likely through an action on OXT-ergic neurons. Consistent with an antiobesity action of OXT, Oxt-/- and Oxtr-/- mice display increased total body fat. Overall, the actions of OXT on bone mass and body composition provide the framework for future therapies for osteoporosis and obesity.
RESUMEN
The lysosomal enzyme glucocerebrosidase-1 (GCase) catalyzes the cleavage of a major glycolipid glucosylceramide into glucose and ceramide. The absence of fully functional GCase leads to the accumulation of its lipid substrates in lysosomes, causing Gaucher disease, an autosomal recessive disorder that displays profound genotype-phenotype nonconcordance. More than 250 disease-causing mutations in GBA1, the gene encoding GCase, have been discovered, although only one of these, N370S, causes 70% of disease. Here, we have used a knowledge-based docking protocol that considers experimental data of protein-protein binding to generate a complex between GCase and its known facilitator protein saposin C (SAPC). Multiscale molecular-dynamics simulations were used to study lipid self-assembly, membrane insertion, and the dynamics of the interactions between different components of the complex. Deep learning was applied to propose a model that explains the mechanism of GCase activation, which requires SAPC. Notably, we find that conformational changes in the loops at the entrance of the substrate-binding site are stabilized by direct interactions with SAPC and that the loss of such interactions induced by N370S and another common mutation, L444P, result in destabilization of the complex and reduced GCase activation. Our findings provide an atomistic-level explanation for GCase activation and the precise mechanism through which N370S and L444P cause Gaucher disease.
Asunto(s)
Aprendizaje Profundo , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/fisiopatología , Glucosilceramidasa/metabolismo , Simulación de Dinámica Molecular , Dominio Catalítico , Activación Enzimática , Glucosilceramidasa/química , Humanos , Enlace de Hidrógeno , Proteínas Mutantes/química , Mapas de Interacción de Proteínas , Estructura Secundaria de Proteína , Saposinas/metabolismoRESUMEN
Nonclassical congenital adrenal hyperplasia (NC-CAH) is by far a subtler and milder enzymatic defect to the classical form of the disease. A nuanced understanding of NC-CAH will lead to increased detection of the disorder in those initially misdiagnosed as having polycystic ovary syndrome, will assist in the detection of pregnancies at risk for severe genetic steroid disorders, and will facilitate appropriate ovulation induction and reduction in the hyperandrogenic symptoms which are a cornerstone of the disease. We describe the history of the disease as well as elucidate the pathophysiology, diagnosis, and treatment of the disorder.
Asunto(s)
Hiperplasia Suprarrenal Congénita/enzimología , Hiperplasia Suprarrenal Congénita/genética , Fertilidad/fisiología , Hiperplasia Suprarrenal Congénita/terapia , Femenino , Humanos , Masculino , Esteroide 21-Hidroxilasa/genética , Esteroide 21-Hidroxilasa/metabolismoRESUMEN
Congenital adrenal hyperplasia, an endocrine autosomal recessive disorder caused by several deficiencies of enzymes and/or proteins involved in adrenal cortisol biosynthesis, is often associated with reproductive dysfunction. While the most common disorder is due to 21-hydroxylase deficiency, several other enzymes in the steroidogenesis pathway have been described, all of which can result in a range of reproductive disorders in both males and females. Although for many enzymes the phenotypic presentation is associated with a particular genotype, the severity of disease cannot always be predicted.
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Hiperplasia Suprarrenal Congénita/enzimología , Hiperplasia Suprarrenal Congénita/genética , Reproducción/fisiología , Hiperplasia Suprarrenal Congénita/diagnóstico , Femenino , Humanos , Masculino , Mutación/fisiologíaRESUMEN
Fibrous sheath interacting protein 1 (FSIP1) is a cancer antigen expressed in the majority of breast cancer tissues and is associated with poor prognosis. However, the role of FSIP1 in the progression and drug sensitivity of triple-negative breast cancer (TNBC) has not been explored. Here, we show that FSIP1 deficiency by shRNA-mediated knockdown or CRISPR-Cas9-mediated knockout significantly inhibits the proliferation and invasion of TNBC cells and impairs chemotherapy-induced growth inhibition in vivo. Computational modeling predicted that FSIP1 binds to ULK1, and this was established by coimmunoprecipitation. FSIP1 deficiency promoted autophagy, enhanced AMP-activated protein kinase (AMPK) signaling, and decreased mechanistic target of rapamycin (mTOR) and Wnt/ß-catenin activity. In contrast, knockdown of AMPK or inhibition of autophagy restored the sensitivity to chemotherapy drugs in TNBC cells. Our findings uncover a role of FSIP1 as well as mechanisms underlying FSIP1 action in drug sensitivity and may, therefore, aid in design of TNBC therapies.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Proteínas Portadoras/metabolismo , Resistencia a Antineoplásicos , Proteínas de Plasma Seminal/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Proteínas Quinasas Activadas por AMP/genética , Antineoplásicos/farmacología , Proteínas Portadoras/genética , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Invasividad Neoplásica , Proteínas de Plasma Seminal/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Células Tumorales CultivadasRESUMEN
Mutations in the gene NR0B1 have been associated with several clinical phenotypes of X-linked adrenal hypoplasia congenita (AHC). The degree and onset of adrenal insufficiency and involvement of hypogonadotropic hypogonadism is variable and may not be concordant with the identified mutation. We review a patient with AHC in which prenatal estriol levels were low, presenting with early-onset mineralocorticoid deficiency in the newborn period followed by glucocorticoid deficiency 2 years later. The reported child is hemizygous for a novel mutation that is deemed de novo in the ligand-binding site of the protein (DAX1) expressed by NR0B1. The identified frameshift mutation results in a T407N/fs protein change. Low prenatal estriol levels may represent a sensitive marker of potentially fatal disorders associated with adrenal insufficiency and should be utilized more frequently. Additionally, accurate reporting of mutations in NR0B1 and the associated phenotype are important to eventually establish a genotype-phenotype correlation that may help anticipate guidance in AHC.
Asunto(s)
Receptor Nuclear Huérfano DAX-1/genética , Estriol/sangre , Mutación del Sistema de Lectura , Insuficiencia Corticosuprarrenal Familiar/genética , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Biomarcadores/sangre , Preescolar , Cromosomas Humanos X/genética , Femenino , Estudios de Asociación Genética , Glucocorticoides/deficiencia , Humanos , Insuficiencia Corticosuprarrenal Familiar/sangre , Masculino , Intercambio Materno-Fetal , Persona de Mediana Edad , Embarazo , Alineación de SecuenciaRESUMEN
The Study of Women's Health Across the Nation has taught us that impending ovarian failure during late perimenopause is associated with a sharp rise in serum FSH, which coincides with the most rapid rate of bone loss and the onset of visceral adiposity. At this time in a woman's life, serum estrogen levels are largely unaltered, so the hypothesis that hypoestrogenemia is the sole cause of bone loss and visceral obesity does not offer a full explanation. An alternative explanation, arising from animal models and human data, is that both physiologic aberrations, obesity and osteoporosis, arise at least in part from rising FSH levels. Here, we discuss recent findings on the mechanism through which FSH exerts biological actions on bone and fat and review clinical data that support a role for FSH in causing osteoporosis and obesity. We will also provide a conceptual framework for using a single anti-FSH agent to prevent and treat both osteoporosis and obesity in women across the menopausal transition.
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Tejido Adiposo/metabolismo , Envejecimiento , Huesos/metabolismo , Hormona Folículo Estimulante/metabolismo , Índice de Masa Corporal , Densidad Ósea , Estrógenos/sangre , Femenino , Hormona Folículo Estimulante/sangre , Humanos , Osteoporosis/sangreRESUMEN
Hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway, catalyzes the head-to-tail condensation of four molecules of porphobilinogen (PBG) to form the linear tetrapyrrole 1-hydroxymethylbilane (HMB). Mutations in human HMBS (hHMBS) cause acute intermittent porphyria (AIP), an autosomal-dominant disorder characterized by life-threatening neurovisceral attacks. Although the 3D structure of hHMBS has been reported, the mechanism of the stepwise polymerization of four PBG molecules to form HMB remains unknown. Moreover, the specific roles of each of the critical active-site residues in the stepwise enzymatic mechanism and the dynamic behavior of hHMBS during catalysis have not been investigated. Here, we report atomistic studies of HMB stepwise synthesis by using molecular dynamics (MD) simulations, mutagenesis, and in vitro expression analyses. These studies revealed that the hHMBS active-site loop movement and cofactor turn created space for the elongating pyrrole chain. Twenty-seven residues around the active site and water molecules interacted to stabilize the large, negatively charged, elongating polypyrrole. Mutagenesis of these active-site residues altered the binding site, hindered cofactor binding, decreased catalysis, impaired ligand exit, and/or destabilized the enzyme. Based on intermediate stages of chain elongation, R26 and R167 were the strongest candidates for proton transfer to deaminate the incoming PBG molecules. Unbiased random acceleration MD simulations identified R167 as a gatekeeper and facilitator of HMB egress through the space between the enzyme's domains and the active-site loop. These studies identified the specific active-site residues involved in each step of pyrrole elongation, thereby providing the molecular bases of the active-site mutations causing AIP.
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Hidroximetilbilano Sintasa/química , Simulación de Dinámica Molecular , Mutación Missense , Porfiria Intermitente Aguda/enzimología , Pirroles/química , Sustitución de Aminoácidos , Humanos , Hidroximetilbilano Sintasa/genética , Hidroximetilbilano Sintasa/metabolismo , Porfiria Intermitente Aguda/genética , Estructura Secundaria de Proteína , Pirroles/metabolismoRESUMEN
Studies over the past decade have challenged the long-held belief that pituitary hormones have singular functions in regulating specific target tissues, including master hormone secretion. Our discovery of the action of thyroid-stimulating hormone (TSH) on bone provided the first glimpse into the non-traditional functions of pituitary hormones. Here we discuss evolving experimental and clinical evidence that growth hormone (GH), follicle-stimulating hormone (FSH), adrenocorticotrophic hormone (ACTH), prolactin, oxytocin and arginine vasopressin (AVP) regulate bone and other target tissues, such as fat. Notably, genetic and pharmacologic FSH suppression increases bone mass and reduces body fat, laying the framework for targeting the FSH axis for treating obesity and osteoporosis simultaneously with a single agent. Certain 'pituitary' hormones, such as TSH and oxytocin, are also expressed in bone cells, providing local paracrine and autocrine networks for the regulation of bone mass. Overall, the continuing identification of new roles for pituitary hormones in biology provides an entirely new layer of physiologic circuitry, while unmasking new therapeutic targets.
Asunto(s)
Tejido Adiposo/metabolismo , Huesos/metabolismo , Hormonas Hipofisarias/fisiología , Tejido Adiposo/efectos de los fármacos , Hormona Adrenocorticotrópica/farmacología , Hormona Adrenocorticotrópica/fisiología , Animales , Arginina Vasopresina/farmacología , Arginina Vasopresina/fisiología , Huesos/efectos de los fármacos , Hormona Folículo Estimulante/farmacología , Hormona Folículo Estimulante/fisiología , Hormona del Crecimiento/farmacología , Hormona del Crecimiento/fisiología , Humanos , Oxitocina/farmacología , Oxitocina/fisiología , Hormonas Hipofisarias/farmacología , Prolactina/farmacología , Prolactina/fisiología , Tirotropina/farmacología , Tirotropina/fisiologíaRESUMEN
Pituitary hormones have long been thought solely to regulate single targets. Challenging this paradigm, we discovered that both anterior and posterior pituitary hormones, including FSH, had other functions in physiology. We have shown that FSH regulates skeletal integrity, and, more recently, find that FSH inhibition reduces body fat and induces thermogenic adipose tissue. A polyclonal antibody raised against a short, receptor-binding epitope of FSHß was found not only to rescue bone loss postovariectomy, but also to display marked antiobesity and probeiging actions. Questioning whether a single agent could be used to treat two medical conditions of public health importance--osteoporosis and obesity--we developed two further monoclonal antibodies, Hf2 and Mf4, against computationally defined receptor-binding epitopes of FSHß. Hf2 has already been shown to reduce body weight and fat mass and cause beiging in mice on a high-fat diet. Here, we show that Hf2, which binds mouse Fsh in immunoprecipitation assays, also increases cortical thickness and trabecular bone volume, and microstructural parameters, in sham-operated and ovariectomized mice, noted on microcomputed tomography. This effect was largely recapitulated with Mf4, which inhibited bone resorption by osteoclasts and stimulated new bone formation by osteoblasts. These effects were exerted in the absence of alterations in serum estrogen in wild-type mice. We also reconfirm the existence of Fshrs in bone by documenting the specific binding of fluorescently labeled FSH, FSH-CH, in vivo. Our study provides the framework for the future development of an FSH-based therapeutic that could potentially target both bone and fat.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Epítopos , Hormona Folículo Estimulante de Subunidad beta/inmunología , Animales , Especificidad de Anticuerpos , Densidad Ósea , Resorción Ósea , Dominio Catalítico , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Ovariectomía , Unión Proteica , Conformación ProteicaRESUMEN
The risk of intersex-related stigma often serves as social indication for "corrective" genital surgery, but has not been comprehensively documented. In preparation for the development of an intersex-specific stigma assessment tool, this qualitative project aimed to explore stigma in girls and women with classical congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. As part of a comprehensive follow-up project, 62 adult women with classical CAH (age range 18-51 years) took part in an open-ended retrospective interview focusing on the impact of CAH and its treatment on various aspects of girls' and women's lives. Deductive qualitative content analysis (Patton, 2014) of de-identified transcripts involved categorization of three types of stigma: experienced, anticipated, and internalized. Two-fifths of the participants reported CAH-related stigma in romantic/sexual situations. Stigma enactment by romantic partners occurred in reaction to both genital and non-genital sex-atypical features of CAH and sometimes included explicit questioning of the women's true gender. Stigma anticipation by the women and their related avoidance of nudity, genital exposure, and romantic involvement altogether were frequent. Internalization of stigma occurred as well. In conclusion, the data suggest that many women with CAH experience, anticipate, and/or internalize intersex-related stigma in the context of their romantic/sexual lives.
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Hiperplasia Suprarrenal Congénita/psicología , Trastornos del Desarrollo Sexual/psicología , Conducta Sexual/psicología , Estigma Social , Adolescente , Adulto , Femenino , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Adulto JovenRESUMEN
Mutations in 11ß-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) cause an extraordinarily rare autosomal recessive disorder, apparent mineralocorticoid excess (AME). AME is a form of low renin hypertension that is potentially fatal if untreated. Mutations in the HSD11B2 gene result either in severe AME or a milder phenotype (type 2 AME). To date, â¼40 causative mutations have been identified. As part of the International Consortium for Rare Steroid Disorders, we have diagnosed and followed the largest single worldwide cohort of 36 AME patients. Here, we present the genotype and clinical phenotype of these patients, prominently from consanguineous marriages in the Middle East, who display profound hypertension and hypokalemic alkalosis. To correlate mutations with phenotypic severity, we constructed a computational model of the HSD11B2 protein. Having used a similar strategy for the in silico evaluation of 150 mutations of CYP21A2, the disease-causing gene in congenital adrenal hyperplasia, we now provide a full structural explanation for the clinical severity of AME resulting from each known HSD11B2 missense mutation. We find that mutations that allow the formation of an inactive dimer, alter substrate/coenzyme binding, or impair structural stability of HSD11B2 yield severe AME. In contrast, mutations that cause an indirect disruption of substrate binding or mildly alter intramolecular interactions result in type 2 AME. A simple in silico evaluation of novel missense mutations could help predict the often-diverse phenotypes of an extremely rare monogenic disorder.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Genotipo , Síndrome de Exceso Aparente de Mineralocorticoides , Mutación Missense , Multimerización de Proteína/genética , Adolescente , Niño , Preescolar , Simulación por Computador , Estabilidad de Enzimas , Femenino , Humanos , Lactante , Masculino , Síndrome de Exceso Aparente de Mineralocorticoides/enzimología , Síndrome de Exceso Aparente de Mineralocorticoides/genética , Síndrome de Exceso Aparente de Mineralocorticoides/patologíaRESUMEN
Fibrous sheath interacting protein 1 (FSIP1), a spermatogenesis-related testicular antigen, is expressed in abundance in breast cancers, particularly in those overexpressing human epidermal growth factor receptor 2 (HER2); however, little is known about its role in regulating the growth and metastasis of breast cancer cells. We and others have shown previously that FSIP1 expression in breast cancer correlates positively with HER2-positivity, recurrence, and metastases and negatively with survival. Here, using coimmunoprecipitation and microscale thermophoresis, we find that FSIP1 binds to the intracellular domain of HER2 directly. We further show that shRNA-induced FSIP1 knockdown in SKBR3 and MCF-7 breast cancer cells inhibits proliferation, stimulates apoptosis, attenuates epithelial-mesenchymal transition, and impairs migration and invasiveness. Consistent with reduced proliferation and enhanced apoptosis, xenotransplantation of SKBR3 cells stably transfected with sh-FSIP1 into nu/nu mice results in reduced tumor volumes compared with sh-NC transplants. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping using sh-FSIP1 gene signature yielded associations with extracellular matrix protein pathways, and a reduction in SNAI2 protein expression was confirmed on Western blot analysis. Complementarily, interrogation of the Connectivity Map using the same gene signature yielded, as top hits, chemicals known to inhibit epithelial-mesenchymal transition, including rapamycin, 17-N-allylamino-17-demethoxygeldanamycin, and LY294002. These compounds phenocopy the effects of sh-FSIP1 on SKBR3 cell viability. Thus, FSIP1 suppression limits oncogenesis and invasiveness in breast cancer cells and, considering its absence in most other tissues, including normal breast, may become a potential target for breast cancer therapy.
Asunto(s)
Apoptosis , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptor ErbB-2/metabolismo , Proteínas de Plasma Seminal/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Recurrencia Local de Neoplasia/genética , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
Mutations of CYP21A2 variably decrease 21-hydroxylase activity and result in a spectrum of disease expressions in patients with congenital adrenal hyperplasia (CAH). We examined the association between CYP21A2 mutations and virilization (Prader score) in females with CAH. The study population included 187 CAH females with fully characterized CYP21A2 mutations. One hundred fifty-eight patients were sorted into groups by expected enzyme activity (percent of normal activity) of the less severely affected allele: (A) null, 0%; (B) I2G, 1%; (C) I172N, 2%; and (D) V281L, >2%. We observed an inverse relationship between virilization and residual enzyme activity (P < 0.001). Subjects in group A or B had a significantly higher likelihood (unadjusted odds ratio: 16; P < 0.001) of developing severe virilization compared with those in group C. Surprisingly, 24% of group D patients, whose mutation is usually associated with nonclassical (NC) CAH, had severe virilization. Among subjects with the NC P30L mutation, 66% expressed unexpected virilization. Virilization, usually leading to extensive reconstructive surgery, is highly likely in patients with null or I2G mutations; however, NC mutations (P30L/V281L) may also lead to unexpected virilization. These findings have implications for prenatal counseling and highlight the need for additional investigations into other factors that influence virilization in CAH.