Differential effects of androgens and estrogens over cellular GH sensitivity in HEPG2 cells.

Testosterone and estrogen concentrations progressively increase during puberty, and in association with growth hormone (GH), lead to the increase in height velocity known as the pubertal growth spurt. Very limited information is available however, regarding the possible effects of sex steroids over GH cellular sensitivity. OBJECTIVE: To investigate the effects of different concentrations of testosterone, estradiol and dihydrotestosterone over the GH intracellular signaling pathway. METHODS: We evaluated the effects of these sex steroids on the nuclear phosphorylation of STAT5b and IGF-1 expression, in HEPG2 human hepatoma cells. In addition, we studied whether Tamoxifen (TAM), can modulate these effects. RESULTS: The highest concentration of T tested (10 ng/mL) co-incubated with a fixed concentration of GH (40 ng/mL) increased nuclear STAT5b phosphorylation compared with GH alone (1.34 ± 0.2 vs 0.6 ± 0.09 AU; *p < 0.05), as well as IGF-1 expression (0.6 ± 0.03 vs 0.32 ± 0.05 AU; *p < 0.05). This effect was not observed with lower concentrations of T tested (1 and 5 ng/mL). A similar increase in nuclear STAT5b phosphorylation was observed with the lowest concentration of E2 tested (20 pg/mL), co-incubated with the same fixed concentration of GH (3.6 ± 0.5 vs 1.28 ± 0.33 AU; *p < 0.05). This effect was also associated with an increase in IGF-1 expression (0.73 ± 0.02 vs 0.39 ± 0.04 AU; *p < 0.05). These results were not observed with higher concentrations of E2 tested (75 and 200 pg/mL). DHT at concentrations of 0.1, 0.25 and 0.5 ng/mL, co-stimulated with GH, did not change cytoplasmic STAT5b phosphorylation, nuclear STAT5b or IGF-1 expression. In addition, the co-incubation of TAM with the highest concentration of T tested (10 ng/mL) and GH (40 ng/mL) did not change cytoplasmic, nuclear pSTAT5 levels or IGF-1 expression. CONCLUSIONS: T and E2 potentiate the GH signaling pathway in a concentration-dependent fashion. The observation that the non-aromatizable androgen dihydrotestosterone does not stimulate this pathway, and that the effects of T are blocked with TAM, suggests that the effects of T over the GH signaling pathway appear to be mediated by estrogen.

Intrauterine Twin Discordancy and Partial Postnatal Catch-up Growth in a Girl with a Pathogenic IGF1R Mutation

Objective: Insulin like growth factors-1 (IGF-1) is essential for normal in utero and postnatal human growth. It mediates its effects through the IGF-1 receptor (IGF1R), a widely expressed cell surface tyrosine kinase receptor. The aim of the study was to analyze pre- and post-natal growth, clinical features and laboratory findings in a small for gestational age (SGA) girl in whom discordant postnatal growth persisted and her appropriate for gestational age (AGA) brother. Methods: A girl born with a low weight and length [-2.3 and -2.4 standard deviation (SD) score (SDS), respectively] but borderline low head circumference (-1.6 SD) presented with a height of -1.7 SDS, in contrast to a normal height twin brother (0.0 SDS). IGF-1 resistance was suspected because of elevated serum IGF-1 levels. Results: Sequencing revealed the presence of a previously described pathogenic heterozygous mutation (p.Glu1050Lys) in the SGA girl which was not present in the parents nor in the AGA twin brother. Conclusion: The pathogenic IGF1R mutation in this girl led to intrauterine growth retardation followed by partial postnatal catch-up growth. Height in mid-childhood was in the lower half of the reference range, but still 1.7 SD shorter than her twin brother.

Efecto de Estradiol y Testosterona sobre la activación de la vía JAK2/STAT5 inducida por hormona de crecimiento / Estradiol and Testosterone effect on the activation of the JAK2/STAT5 pathway induced by growth hormone

Abstract: Sex hormones play a major role during pubertal growth. Estradiol (E2) and testosterone (T) levels progressively increase during puberty and in the presence of growth hormone (GH), growth velocity increases. Understanding the interactions between sex hormones and GH, may optimize the treatment of pubertal children with growth disorders. The aim of our study was to investigate possible molecular mechanisms which might potentiate longitudinal growth during puberty due to E 2or T combined with GH. We evaluated the GH/JAK2/STAT5 signaling pathway in the human hepatoma cell line HEPG2. Our results suggest that sex hormones potentiate the GH signaling pathway in a dose dependent fashion. Relatively low concentrations of E 2associated with GH induce a substantial activation of the GH pathway, whereas relatively high concentrations of T associated with GH produce a similar effect. These findings are concordant with the physiology of the pubertal growth spurt, which is an early event in girls (when E 2 circulating levels are low), and a late event in boys (when T circulating levels are high).

Resumen: Las hormonas sexuales, modulan el crecimiento durante la pubertad. Los niveles de estradiol (E2) y testosterona (T) aumentan progresivamente durante la pubertad y en combinación con la hormona de crecimiento (GH), producen un incremento en la velocidad de crecimiento en este período conocido como el "estirón puberal". El estudio de la interacción entre las hormonas sexuales y la GH, es de gran importancia para optimizar el tratamiento de niños(as) con alteraciones del crecimiento durante la pubertad. El objetivo de nuestro estudio fue investigar los posibles mecanismos que podrían potenciar el crecimiento longitudinal durante la pubertad, en especial las interacciones entre E 2o T en combinación con GH. Se evaluó la activación de la vía de señalización GH/JAK2/STAT5 frente al estímulo combinado con estas hormonas en cultivos celulares de hepatoma humana HEPG2. Nuestros resultados sugieren que existe un efecto potenciador de las hormonas sexuales sobre la vía de señalización de GH. Observamos que concentraciones relativamente bajas de E2 junto con GH producen una clara activación de la vía de señalización para GH, mientras que concentraciones relativamente altas de T junto con GH producen una activación similar. Estos hallazgos son concordantes con la fisiología del estirón puberal, que es más precoz en niñas (cuando los niveles circulantes de E2 son bajos), y más tardíos en varones (cuando los niveles circulantes de T son altos).

Expanding Genetic and Functional Diagnoses of IGF1R Haploinsufficiencies.

BACKGROUND: The growth-promoting effects of IGF-I is mediated through the IGF-I receptor (IGF1R), a widely expressed cell-surface tyrosine kinase receptor. IGF1R copy number variants (CNV) can cause pre- and postnatal growth restriction or overgrowth. METHODS: Whole exome sequence (WES), chromosomal microarray, and targeted IGF1R gene analyses were performed on 3 unrelated children who share features of small for gestational age, short stature, and elevated serum IGF-I, but otherwise had clinical heterogeneity. Fluorescence-activated cell sorting (FACS) analysis of cell-surface IGF1R was performed on live primary cells derived from the patients. RESULTS: Two novel IGF1R CNV and a heterozygous IGF1R nonsense variant were identified in the 3 patients. One CNV (4.492 Mb) was successfully called from WES, utilizing eXome-Hidden Markov Model (XHMM) analysis. FACS analysis of cell-surface IGF1R on live primary cells derived from the patients demonstrated a ∼50% reduction in IGF1R availability associated with the haploinsufficiency state. CONCLUSION: In addition to conventional methods, IGF1R CNV can be identified from WES data. FACS analysis of live primary cells is a promising method for efficiently evaluating and screening for IGF1R haploinsufficiency. Further investigations are necessary to delineate how comparable IGF1R availability leads to the wide spectrum of clinical phenotypes and variable responsiveness to rhGH therapy.

Growth hormone signaling in fibroblasts from newborn boys and prepubertal boys.

BACKGROUND/AIM: Responsiveness to GH in target cells is mediated by its receptor, which activates the Janus kinase-2 (JAK2) and STAT5 (signal transducers and activators of transcription 5) leading to the expression of IGF-1 and IGFALS. The aim of this study was to compare the GH signaling pathway in newborns and prepubertal boys. SUBJECTS AND METHODS: We determined the GHR protein content and the effect of stimulation with recombinant human GH (rhGH; 200ng/mL) on JAK2 and STAT5 phosphorylation in skin fibroblast cultures obtained from newborns and prepubertal boys. The transcript levels of IGFALS and IGF-I, were also studied and compared after 16h or 24h of stimulation with GH in both study groups. RESULTS: Newborn infants showed less GHR protein than the prepubertal boys. After rhGH stimulation, JAK2 and STAT5 phosphorylation was absent in skin fibroblasts from newborns, but was clearly detectable in prepubertal boys. After 16h of treatment with rhGH, IGFALS and IGF-I transcript levels increased in the prepubertal boys when compared to baseline. In newborns, however, we did not observe a response after 16 and 24h of rhGH stimulation. CONCLUSION: The significant attenuation of the GH signaling pathway observed in fibroblasts from newborn boys appears to be related to a reduction in GHR content and lack of phosphorylation of JAK2 and STAT5 in response to rhGH. This might impair STAT5 dimer formation, leading to a reduction in the transcript levels of IGFALS and IGF-I during the newborn period.

Effects of levothyroxine on growth hormone (gh) sensitivity in children with idiopathic short stature.

BACKGROUND: The possible relationship between the circulating concentrations of T4 and GH sensitivity has not been elucidated. OBJECTIVE: The aim of this study is to evaluate the effect of levothyroxine supplementation on GH sensitivity in prepubertal boys with idiopathic short stature (ISS). METHODS: We selected 28 prepubertal boys with ISS (mean age 8.2±0.5years) and free T4 (Ft4) concentrations between the 3rd and the 25th percentiles (Ft4: 0.8-1.5ng/dl). They were randomly divided into two groups: Group A received thyroid supplementation (1-3µg/kg/day) for 120days, and Group B received placebo for the same period. To evaluate GH sensitivity, an IGF-I generation test (GH: 33µg/kg/day sc for 3days) was performed in both groups: under basal conditions, and after 120days of levothyroxine supplementation (or placebo). RESULTS: After thyroid supplementation, Group A had higher Ft4 concentrations compared with Group B (2.14±0.06 vs 1.48±0.06ng/dl, p=0.01), their growth velocity was significantly higher (2.3±0.1 vs 1.5±0.2cm/4months), and they exhibited a greater increase in IGF-I after GH administration (Group A: 32.5±3.8% vs Group B 17.3±2.6%). CONCLUSION: Supplementation with levothyroxine for 120days promotes an increase in growth velocity, and a greater IGF-I response to short-term GH administration in prepubertal boys with ISS and low-normal thyroid hormone concentrations.

Effects of thyroid hormone on the GH signal transduction pathway.

BACKGROUND/AIM: The importance of thyroid hormone on growth and development in children is well recognized. In addition, linear growth is highly dependent on the response of peripheral tissues to growth hormone, a process known as GH sensitivity, but little is known about the possible effects of T4 on this process. METHODS: We determined the effect of stimulation with recombinant human GH (rhGH; 200 ng/mL) alone or in combination with two different concentrations of T4 (250 nM and 500 nM for 24 h) on JAK2 and STAT5 activation in skin fibroblast cultures obtained from prepubertal boys with normal height. RESULTS: JAK2 and STAT5 were activated under co-incubation with T4 (at both concentrations) and rhGH in the non-nuclear fraction of the fibroblasts. In addition, after 24h of co-incubation with rhGH and T4 (500 nM), we observed an increase in phospho-STAT5 in the nuclear fraction, when compared to GH and T4 stimulation alone. This effect was not observed when the fibroblasts were co-incubated with GH and the lower concentration of T4 (250 nM). CONCLUSION: Combined stimulation with GH and T4 at a concentration of 500 nM increases synergistically nuclear phospho-STAT5 in skin fibroblasts, which may amplify tissue sensitivity to GH. These findings may help to explain the effect of T4 administration on growth velocity in some children with idiopathic short stature.

Phosphotyrosine phosphatases in GH-stimulated skin fibroblasts from children with idiopathic short stature.

AIM: Some cases of idiopathic short stature (ISS) may be caused by defects in the modulation of the negative feedback regulation of the growth hormone receptor (GHR)/ Janus kinase (JAK)2/signal transducers and activators of transcription (STAT)5 signaling pathway. The cytosolic tyrosine phosphatases, protein tyrosine phosphatase 1B (PTP1B) and Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase-1 (SHP-1), the later which translocates to the nucleus after activation, interact with JAK2 in a GH-dependent manner. The possible contribution of PTP1B and SHP-1 to GH signaling in fibroblasts from ISS patients has not been studied. METHODS: We determined the basal protein content of PTP1B and SHP-1 in the presence of recombinant human GH (rhGH) for 24 h in skin fibroblast cultures, obtained from patients with ISS, and were compared with a normal height control children group. JAK2 activation was determined in both groups. RESULTS: JAK2 activation was delayed in fibroblasts from ISS patients compared to controls. Under basal conditions, the protein content of SHP-1 was lower in ISS, and after incubation with rhGH, it decreased in the non-nuclear and nuclear fraction of controls, but not in ISS patients. The protein content of PTP1B, however, increased in a similar fashion in fibroblasts from both ISS and control children. CONCLUSION: The delayed activation of JAK2 and the lack of response of SHP-1 after incubation with GH in fibroblasts from ISS patients, suggests that the growth retardation observed in some of these children may be mediated in part by this phosphotyrosine phosphatase.

Expression of SOCS1, SOCS2, and SOCS3 in growth hormone-stimulated skin fibroblasts from children with idiopathic short stature.

BACKGROUND/AIM: Possible etiologies of idiopathic short stature (ISS) include a range of conditions, some of which may be caused by defects in the modulation of the growth hormone (GH)-signaling pathway. The Janus kinase/signal transducer and activator of transcription pathway is regulated by several mechanisms, including negative feedback regulation by the suppressors of cytokine signaling (SOCS). However, the specific induction of SOCS transcript levels in fibroblasts from ISS patients has not been studied. METHODS: We determined the transcript levels of the SOCS1-3 genes under basal conditions, and in the presence or absence of stimulation with rhGH for 24 h in skin fibroblast cultures obtained from patients with ISS and children with normal height. RESULTS: Under basal conditions, ISS patients express higher SOCS2-3 transcript levels than control children. After incubation with recombinant human GH (rhGH), the transcript levels of SOCS2 increased significantly in ISS patients compared to controls (0.79 +/- 0.06 vs. 0.55 +/- 0.07; p = 0.03), a pattern which did not achieve statistical significance for SOCS3 transcript levels (0.55 +/- 0.08 vs. 0.40 +/- 0.07). CONCLUSION: The higher baseline transcript levels of the SOCS genes, and the increase observed for SOCS2 after rhGH treatment in ISS patients, suggest that growth retardation in some of these children may be mediated, at least in part, by intracellular overexpression of the SOCS genes.

Cytoplasmic and nuclear STAT3 in GH-stimulated fibroblasts of children with idiopathic short stature.

BACKGROUND: STAT5, which plays an important role in GH signal transduction, has been studied extensively in children with growth retardation, but there is scarce information regarding STAT3. AIM: We determined total and phosphorylated STAT3 after GH stimulation in fibroblasts from children with idiopathic short stature (ISS) and control children with normal stature. SUBJECTS AND METHODS: We studied 15 prepubertal children (age 7.6 ± 0.4 years) with short stature (height -2.8 ± 0.2 SDS), decreased growth velocity (10 ng/ml to the clonidine stimulation test and decreased serum IGF-I concentrations (<-1 SDS), and 19 control children with normal stature (age 6.7 ± 0.3 years). We determined the levels of total and phosphorylated STAT3 in the cytoplasmic and nuclear fractions of fibroblast cultures obtained from a skin biopsy, stimulated with GH (200 ng/ml) for 15-60 min. RESULTS: We observed a reduction in nuclear pSTAT3 levels and a lower nuclear/cytoplasmic STAT3 phosphorylated ratio in 3 patients from the study group compared to the control children. CONCLUSION: These results suggest that some children with ISS may exhibit a reduction in the nuclear content of their phosphorylated STAT3.

The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling.

Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-induced MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal alpha-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.

Gene therapy reduces ethanol intake in an animal model of alcohol dependence.

BACKGROUND: Some gene polymorphisms strongly protect against the development of alcoholism. A large proportion of East Asians carry a protective inactivating mutation in aldehyde dehydrogenase (ALDH2*2). These subjects display high levels of blood acetaldehyde when consuming alcohol, a condition that exerts a 66 to 99% protection against alcohol abuse and alcoholism. Present knowledge allows the incorporation of therapeutic genes that can modify the expression of disease predisposing genes, an effect that can last from months to years. In line with the above, we have tested if inhibiting the expression of the aldehyde dehydrogenase gene (ALDH2) by an anti-Aldh2 antisense gene can curtail the drive of alcohol-dependent animals to consume alcohol. METHODS: Wistar-derived rats bred as high alcohol drinkers (UChB; Universidad de Chile Bibulous) were rendered alcohol dependent by a 2-month period of voluntary ethanol (10%) intake, subjected to a 3-day withdrawal period and further allowed access to 10% ethanol for only 1 hour each day. This condition results in a high ethanol intake (1.2 g/kg/60 min) which is 10 times higher than that of naïve UChB rats. RESULTS: The single intravenous administration of an anti-Aldh2 antisense gene carried by an adenoviral vector reduced liver ALDH2 activity by 85% (p < 0.002) and inhibited voluntary ethanol intake by 50% (ANOVA p < 0.005) for 34 days. CONCLUSIONS: This proof-of-principle study indicates that gene therapy approaches can be employed to achieve a long-term reduction of alcohol intake in alcohol-dependent animals and suggests that gene vectors may be developed as long-lasting therapeutic adjuncts for the treatment of alcoholism.

Aldehyde dehydrogenase (ALDH2) activity in hepatoma cells is reduced by an adenoviral vector coding for an ALDH2 antisense mRNA.

BACKGROUND: Individuals carrying the Glu487Lys coding mutation in the gene for mitochondrial aldehyde dehydrogenase (ALDH2) have a diminished capacity to metabolize acetaldehyde. This deficiency leads to increases in blood acetaldehyde levels when they consume ethanol, which results in an aversion to alcohol and in marked protection against alcoholism. In the present studies, we aimed to mimic the high-acetaldehyde low-ALDH2 activity phenotype in a rat hepatoma cell line by inhibiting Aldh2 gene expression by an Aldh2 antisense-coding gene carried by an adenoviral vector. METHODS: We designed and produced elevated titers of adenoviral vectors (10 virions/ml) carrying Aldh2 cDNA cloned in the reverse orientation preceded by a CMV promoter and followed by a poly-A termination signal. Rat hepatoma cells were infected with these vectors. RESULTS: Studies showed that 1) the antisense gene is actively transcribed in the cells and high levels of antisense mRNA are attained, 2) the antisense gene reduced ALDH2 activity by 65%, and 3) when incubated with 10 mM ethanol, acetaldehyde accumulation by cells increased 8-fold to levels (80-90 microM) known to be aversive to animals and humans. CONCLUSIONS: Data presented show that antialcohol drugs that inhibit Aldh2 gene expression can be generated endogenously in liver cells infected by an adenoviral vector carrying an antisense-coding gene, thus mimicking the high-acetaldehyde phenotype that exists in humans carrying the Glu487Lys mutation who are protected against alcoholism.

Mecanismos moleculares en la apoptosis del cardiomiocito y sus proyecciones patológicas y terapéuticas / Molecular mechanisms of cardiomyocyte apoptosis and their pathological and terapeutical perspectives

La apoptosis junto a la paraptosis y la necrosis constituyen las principales formas de muerte celular conocidas hasta la fecha. La apoptosis se caracteriza por una disminución del volumen celular y a laformación de cuerpos apoptóticos, manteniendo íntegra la membrana plasmática, evitando así el vaciamiento del contenido intracelular y el desarrollo de un proceso inflamatorio. En el cardiomiocito se han descrito dos vías apoptóticas: la tipo I (extrínseca o mediada a través de receptores de muerte) y la tipo II (intrínseca o mitocondrial). Ambas vías convergen en la caspasa 3, que es la responsable de la ejecución final de la apoptosis. Existe apoptosis en varias enfermedades cardíacas, como por ejemplo en la insuficiencia cardíaca de origen isquémico y no isquémico, en el infarto al miocardio y en las arritmias. Debido a que los cardiomiocitos son incapaces de proliferar, su muerte conduce a la pérdida de masa cardíaca, disminución de la capacidad contráctil del miocardio y remodelamiento. Dado que la apoptosis del cardiomiocito contribuye directamente a un deterioro funcional irreversible del corazón y favorece el desarrollo de diversas cardiopatías, el conocimiento de sus mecanismos y blancos moleculares proporcionará novedosas estrategias terapéuticas para la prevención y tratamiento de las diferentes cardiopatías