RESUMO
PURPOSE: Impaired function of gonadotropin-releasing hormone (GnRH) neurons can cause a phenotypic spectrum ranging from delayed puberty to isolated hypogonadotropic hypogonadism (IHH). We sought to identify a new genetic etiology for these conditions. METHODS: Exome sequencing was performed in an extended family with autosomal dominant, markedly delayed puberty. The effects of the variant were studied in a GnRH neuronal cell line. Variants in the same gene were sought in a large cohort of individuals with IHH. RESULTS: We identified a rare missense variant (F900V) in DLG2 (which encodes PSD-93) that cosegregated with the delayed puberty. The variant decreased GnRH expression in vitro. PSD-93 is an anchoring protein of NMDA receptors, a type of glutamate receptor that has been implicated in the control of puberty in laboratory animals. The F900V variant impaired the interaction between PSD-93 and a known binding partner, Fyn, which phosphorylates NMDA receptors. Variants in DLG2 that also decreased GnRH expression were identified in three unrelated families with IHH. CONCLUSION: The findings indicate that variants in DLG2/PSD-93 cause autosomal dominant delayed puberty and may also contribute to IHH. The findings also suggest that the pathogenesis involves impaired NMDA receptor signaling and consequently decreased GnRH secretion.
Assuntos
Hormônio Liberador de Gonadotropina , Hipogonadismo , Hormônio Liberador de Gonadotropina/genética , Guanilato Quinases , Humanos , Hipogonadismo/genética , Proteínas , Transdução de Sinais , Proteínas Supressoras de Tumor , Sequenciamento do ExomaRESUMO
Recombinant human growth hormone (GH) is commonly used to treat short stature in children. However, GH treatment has limited efficacy, particularly in severe, non-GH-deficient conditions such as chondrodysplasias, and potential off-target effects. Because short stature results from decreased growth plate chondrogenesis, we developed a cartilage-targeting single-chain human antibody fragment (CaAb) aiming to deliver therapeutic molecules to the growth plate, thereby increasing treatment efficacy while minimizing adverse effects on other tissues. To this end, we created fusion proteins of these CaAbs conjugated with insulin-like growth factor 1 (IGF-1), an endocrine and/or paracrine factor that positively regulates chondrogenesis. These CaAb-IGF-1 fusion proteins retained both cartilage binding and IGF-1 biological activity, and they were able to stimulate bone growth in an organ culture system. Using a GH-deficient (lit) mouse model, we found that subcutaneous injections of these CaAb-IGF-1 fusion proteins increased overall growth plate height without increasing proliferation in kidney cortical cells, suggesting on-target efficacy at the growth plate and less off-target effect on the kidney than IGF-1 alone. Alternate-day injections of these fusion proteins, unlike IGF-1 alone, were sufficient to produce a therapeutic effect. Our findings provide proof of principle that targeting therapeutics to growth plate cartilage can potentially improve treatment for childhood growth disorders.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Animais , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Condrogênese/efeitos dos fármacos , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/metabolismo , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genéticaRESUMO
OBJECTIVE: Antiandrogen, aromatase inhibitor, and gonadotropin-releasing hormone analog (GnRHa) treatment normalizes growth rate and bone maturation and increases predicted adult height (AH) in boys with familial male-limited precocious puberty (FMPP). To evaluate the effect of long-term antiandrogen, aromatase inhibitor, and GnRHa on AH, boys with FMPP who were treated were followed to AH. STUDY DESIGN: Twenty-eight boys with FMPP, referred to the National Institutes of Health, were started on antiandrogen and aromatase inhibitor at 4.9 ± 1.5 years of age; GnRHa was added at 6.9 ± 1.5 years of age. Treatment was discontinued at 12.2 ± 0.5 years of age (bone age, 14.4 ± 1.3). AH was assessed at 16.4 ± 1.3 years of age (bone age, 18.5 ± 0.6). RESULTS: AH (mean ± SD) for all treated subjects was 173.6 ± 6.8 cm (-0.4 ± 1.0 SD relative to adult US males). For 25 subjects with pretreatment predicted AH, AH significantly exceeded predicted AH at treatment onset (173.8 ± 6.9 vs 164.9 ± 10.7 cm; P < .001), but fell short of predicted AH at treatment discontinuation (177.3 ± 9.0 cm; P < .001). For 11 subjects with maternal or sporadic inheritance, the mean AH was 3.1 cm (0.4 SD score) below sex-adjusted midparental height (175.4 ± 5.8 vs 178.5 ± 3.1 cm [midparental height]; P = .10). For 16 subjects with affected and untreated fathers, AH was significantly greater than fathers' AH (172.8 ± 7.4 vs 168.8 ± 7.2 cm; P < .05). CONCLUSIONS: Long-term treatment with antiandrogen, aromatase inhibitor, and GnRHa in boys with FMPP results in AH modestly below sex-adjusted midparental height and within the range for adult males in the general population.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Inibidores da Aromatase/uso terapêutico , Estatura/efeitos dos fármacos , Leuprolida/uso terapêutico , Puberdade Precoce/tratamento farmacológico , Pamoato de Triptorrelina/análogos & derivados , Adulto , Anastrozol , Criança , Pré-Escolar , Esquema de Medicação , Quimioterapia Combinada , Seguimentos , Hormônio Liberador de Gonadotropina/análogos & derivados , Humanos , Masculino , Nitrilas/uso terapêutico , Puberdade Precoce/fisiopatologia , Espironolactona/uso terapêutico , Testolactona/uso terapêutico , Resultado do Tratamento , Triazóis/uso terapêutico , Pamoato de Triptorrelina/uso terapêuticoRESUMO
Histone methyltransferases EZH1 and EZH2 catalyse the trimethylation of histone H3 at lysine 27 (H3K27), which serves as an epigenetic signal for chromatin condensation and transcriptional repression. Genome-wide associated studies have implicated EZH2 in the control of height and mutations in EZH2 cause Weaver syndrome, which includes skeletal overgrowth. Here we show that the combined loss of Ezh1 and Ezh2 in chondrocytes severely impairs skeletal growth in mice. Both of the principal processes underlying growth plate chondrogenesis, chondrocyte proliferation and hypertrophy, are compromised. The decrease in chondrocyte proliferation is due in part to derepression of cyclin-dependent kinase inhibitors Ink4a/b, while ineffective chondrocyte hypertrophy is due to the suppression of IGF signalling by the increased expression of IGF-binding proteins. Collectively, our findings reveal a critical role for H3K27 methylation in the regulation of chondrocyte proliferation and hypertrophy in the growth plate, which are the central determinants of skeletal growth.
Assuntos
Desenvolvimento Ósseo , Condrócitos/metabolismo , Condrócitos/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Animais Recém-Nascidos , Desenvolvimento Ósseo/genética , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Lâmina de Crescimento/metabolismo , Hipertrofia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Tíbia/metabolismo , Regulação para CimaRESUMO
With age, growth plate cartilage undergoes programmed senescence, eventually causing cessation of bone elongation and epiphyseal fusion. Estrogen accelerates this developmental process. We hypothesized that senescence occurs because progenitor cells in the resting zone are depleted in number and that estrogen acts by accelerating this depletion. To test this hypothesis, juvenile ovariectomized rabbits received injections of estradiol cypionate or vehicle for 5 weeks, and then were left untreated for an additional 5 weeks. Exposure to estrogen accelerated the normal decline in growth plate height and in the number of proliferative and hypertrophic chondrocytes. Five weeks after discontinuation of estrogen treatment, these structural parameters remained advanced, indicating an irreversible advancement in structural senescence. Similarly, transient estrogen exposure hastened epiphyseal fusion. Estrogen also caused a more rapid decline in functional parameters of growth plate senescence, including growth rate, proliferation rate, and hypertrophic cell size. However, in contrast to the structural parameters, once the estrogen treatment was discontinued, the growth rate, chondrocyte proliferation rate, and hypertrophic cell size all normalized, suggesting that estrogen has a reversible, suppressive effect on growth plate function. In addition, estrogen accelerated the normal loss of resting zone chondrocytes with age. This decrease in resting zone cell number did not appear to be due to apoptosis. However, it was maintained after the estrogen treatment stopped, suggesting that it represents irreversible depletion. The findings are consistent with the hypothesis that estrogen causes irreversible depletion of progenitor cells in the resting zone, thus irreversibly accelerating structural senescence and hastening epiphyseal fusion. In addition, estrogen reversibly suppresses growth plate function.
Assuntos
Envelhecimento/fisiologia , Desenvolvimento Ósseo , Estradiol/fisiologia , Estrogênios/fisiologia , Lâmina de Crescimento/fisiologia , Animais , Proliferação de Células , Tamanho Celular , Condrócitos/citologia , Feminino , Lâmina de Crescimento/citologia , Coelhos , Tíbia/crescimento & desenvolvimentoRESUMO
INTRODUCTION: In many normal tissues, proliferation rates decline postnatally, causing somatic growth to slow. Previous evidence suggests that this decline is due, in part, to decline in the expression of growth-promoting imprinted genes including Mest, Plagl1, Peg3, Dlk1, and Igf2. Embryonal cancers are composed of cells that maintain embryonic characteristics and proliferate rapidly in childhood. We hypothesized that the abnormal persistent rapid proliferation in embryonal cancers occurs in part because of abnormal persistent high expression of growth-promoting imprinted genes. RESULTS: Analysis of microarray data showed elevated expression of MEST, PLAGL1, PEG3, DLK1, and IGF2 in various embryonal cancers, especially rhabdomyosarcoma, as compared to nonembryonal cancers and normal tissues. Similarly, mRNA expression, assessed by real-time PCR, of MEST, PEG3, and IGF2 in rhabdomyosarcoma cell lines was increased as compared to nonembryonal cancer cell lines. Furthermore, siRNA-mediated knockdown of MEST, PLAGL1, PEG3, and IGF2 expression inhibited proliferation in Rh30 rhabdomyosarcoma cells. DISCUSSION: These findings suggest that the normal postnatal downregulation of growth-promoting imprinted genes fails to occur in some embryonal cancers, particularly rhabdomyosarcoma, and contributes to the persistent rapid proliferation of rhabdomyosarcoma cells and, more generally, that failure of the mechanisms responsible for normal somatic growth deceleration can promote tumorigenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Impressão Genômica , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Criança , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Análise em Microsséries , Proteínas/genética , Proteínas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
With age, the growth plate undergoes senescent changes that cause linear bone growth to slow and finally cease. Based on previous indirect evidence, we hypothesized that this senescent decline occurs because growth plate stem-like cells, located in the resting zone, have a finite proliferative capacity that is gradually depleted. Consistent with this hypothesis, we found that the proliferation rate in rabbit resting zone chondrocytes (assessed by continuous 5-bromo-2'-deoxy-uridine labeling) decreases with age, as does the number of resting zone chondrocytes per area of growth plate. Glucocorticoid excess slows growth plate senescence. To explain this effect, we hypothesized that glucocorticoid inhibits resting zone chondrocyte proliferation, thus conserving their proliferative capacity. Consistent with this hypothesis, we found that dexamethasone treatment decreased the proliferation rate of rabbit resting zone chondrocytes and slowed the numerical depletion of these cells. Estrogen is known to accelerate growth plate senescence. However, we found that estradiol cypionate treatment slowed resting zone chondrocyte proliferation. Our findings support the hypotheses that growth plate senescence is caused by qualitative and quantitative depletion of stem-like cells in the resting zone and that growth-inhibiting conditions, such as glucocorticoid excess, slow senescence by slowing resting zone chondrocyte proliferation and slowing the numerical depletion of these cells, thereby conserving the proliferative capacity of the growth plate. We speculate that estrogen might accelerate senescence by a proliferation-independent mechanism, or by increasing the loss of proliferative capacity per cell cycle.
Assuntos
Envelhecimento/fisiologia , Condrócitos/fisiologia , Lâmina de Crescimento/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Condrócitos/efeitos dos fármacos , Dexametasona/farmacologia , Estradiol/farmacologia , Estrogênios/fisiologia , Glucocorticoides/farmacologia , Imuno-Histoquímica/métodos , Masculino , Coelhos , Células-Tronco/fisiologiaRESUMO
In children with precocious puberty (PP), treatment with GnRH analogs (GnRHa) often decreases height velocity below normal. Based on previous animal studies, we hypothesized that this impaired growth is due to excessive advancement in growth plate senescence induced by the prior estrogen exposure. This hypothesis predicts that the height velocity during treatment will be inversely related to the severity of prior estrogen exposure. We analyzed data from 100 girls (age, 5.8 +/- 2.1 yr; mean +/- SD) with central PP who were treated with GnRHa. During GnRHa therapy, height velocity was low for age (-1.6 +/- 1.7 SD score; mean +/- SD). The absolute height velocity correlated most strongly with the bone age (BA), which we used as a surrogate marker for growth plate senescence (r = -0.727, P < 0.001). The severity of the growth abnormality (height velocity SD score for age) correlated inversely with markers of the severity of prior estrogen exposure, including duration of PP (r = -0.375, P < 0.001), Tanner breast stage (r = -0.220, P < 0.05), and BA advancement (r = -0.283, P < 0.01). Stepwise regression confirmed that BA was the best independent predictor of growth during GnRHa therapy. The findings are consistent with our hypothesis that impaired growth during GnRHa therapy is due, at least in part, to premature growth plate senescence induced by the prior estrogen exposure.
Assuntos
Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/efeitos adversos , Transtornos do Crescimento/induzido quimicamente , Puberdade Precoce/tratamento farmacológico , Puberdade Precoce/fisiopatologia , Pamoato de Triptorrelina/análogos & derivados , Pamoato de Triptorrelina/efeitos adversos , Determinação da Idade pelo Esqueleto , Estatura , Criança , Pré-Escolar , Estrogênios/fisiologia , Feminino , Hormônio Liberador de Gonadotropina/uso terapêutico , Lâmina de Crescimento/fisiopatologia , Humanos , Puberdade , Análise de Regressão , Fatores de Tempo , Pamoato de Triptorrelina/uso terapêuticoRESUMO
BACKGROUND: The purpose of the present study was to determine whether longitudinal growth of the cortex occurs through intramembranous bone formation involving the periosteum or through endochondral bone formation involving the growth plate and to explore the cellular and biochemical mechanisms responsible for this process. METHODS: Cortical bone formation was studied in the metaphyses of growing New Zealand White rabbits by means of (1) oxytetracycline labeling and fluorescence microscopy, (2) computer-assisted histomorphometry, (3) osteoblast culture and [(3) H]-thymidine incorporation in the presence of periosteum or periosteum-conditioned medium, and (4) surgical insertion of membranes between the periosteum and the underlying spongiosa. RESULTS: Within the metaphyseal cortex, oxytetracycline labeling produced fluorescent closed curves outlining enlarging trabeculae derived from coalescing endochondral trabecular bone. In this region of coalescing trabeculae close to the periosteum, osteoblast surface was increased compared with trabeculae farther from the periosteum (p < 0.001). The osteoclast surface did not differ. In vitro, osteoblast proliferation was increased in the presence of periosteum (p < 0.001) or periosteum-conditioned medium (p < 0.001). Surgical insertion of permeable or impermeable membranes between the periosteum and the spongiosa did not prevent cortex formation. CONCLUSIONS: These observations demonstrate that metaphyseal cortical bone is formed by coalescence of endochondral trabecular bone. This coalescence is associated with increased osteoblast surface in the peripheral spongiosa. The increased osteoblast surface could be due to inductive effects of periosteum; in the present study, periosteum stimulated osteoblast proliferation in vitro but was not required for metaphyseal cortical bone formation in vivo. CLINICAL RELEVANCE: Understanding metaphyseal cortical growth may help to elucidate the pathophysiology of osseous growth disorders in children.