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1.
Proc Natl Acad Sci U S A ; 117(11): 6067-6074, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123064

RESUMO

Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.


Assuntos
Células Epiteliais/patologia , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Transdução de Sinais/imunologia , Uveíte/patologia , Animais , Linhagem Celular , Corpo Ciliar/citologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Janus Quinase 2/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Ratos , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/imunologia , Receptores de Hormônios Reguladores de Hormônio Hipofisário/antagonistas & inibidores , Receptores de Hormônios Reguladores de Hormônio Hipofisário/imunologia , Fator de Transcrição STAT3/metabolismo , Sermorelina/análogos & derivados , Sermorelina/farmacologia , Sermorelina/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Uveíte/tratamento farmacológico , Uveíte/imunologia
2.
Endocrinology ; 160(11): 2673-2691, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31436800

RESUMO

Isolated growth hormone deficiency type II (IGHD2) is mainly caused by heterozygous splice-site mutations in intron 3 of the GH1 gene. A dominant-negative effect of the mutant GH lacking exon 3 on wild-type GH secretion has been proposed; however, the molecular mechanisms involved are elusive. To uncover the molecular systems underlying GH deficiency in IGHD2, we established IGHD2 model mice, which carry both wild-type and mutant copies of the human GH1 gene, replacing each of the endogenous mouse Gh loci. Our IGHD2 model mice exhibited growth retardation along with intact cellular architecture and mildly activated endoplasmic reticulum stress in the pituitary gland, caused by decreased GH-releasing hormone receptor (Ghrhr) and Gh gene promoter activities. Decreased Ghrhr and Gh promoter activities were likely caused by reduced levels of nuclear CREB3L2, which was demonstrated to stimulate Ghrhr and Gh promoter activity. To our knowledge, this is the first in vivo study to reveal a novel molecular mechanism of GH deficiency in IGHD2, representing a new paradigm that differs from widely accepted models.


Assuntos
Nanismo Hipofisário/etiologia , Hormônio do Crescimento/metabolismo , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Nanismo Hipofisário/patologia , Feminino , Hormônio do Crescimento/genética , Humanos , Masculino , Camundongos , Hipófise/metabolismo , Hipófise/ultraestrutura , Regiões Promotoras Genéticas , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética
3.
J Dairy Res ; 86(3): 331-336, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31288873

RESUMO

We investigated whether variation of the sheep Growth Hormone Receptor (GHR), Growth Hormone Releasing Hormone Receptor (GHRHR) and Insulin-Like Growth Factor 1 (IGF1) genes were associated with milk coagulation properties (MCP) in sheep. The GHR, GHRHR and IGF1 genes are part of the GH system, which is known to modulate metabolism, growth and reproduction as well as mammogenesis and galactopoiesis in dairy species. A total of 380 dairy Sarda sheep were genotyped for 36 SNPs mapping to these three genes. Traditional MCP were measured as rennet coagulation time (RCT), curd-firming time (k20) and curd firmness at 30 m (a30). Modeling of curd firming over time (CFt) was based on a 60 m lactodynamographic test, generating a total of 240 records of curd firmness (mm) for each milk sample. The model parameters obtained included: the rennet coagulation time as a result of modeling all data available (RCTeq, min); the asymptotic potential value of curd firmness (CFP, mm) at an infinite time; the CF instant rate constant (kCF, %/min); the syneresis instant rate constant (kSR, %/min); the maximum value of CF (CFmax, mm) and the time at achievement of CFmax (tmax, min). Statistical analysis revealed that variation of the GHR gene was significantly associated with RCT, kSR and CFP (P < 0.05). No other significant associations were detected. These findings may be useful for the dairy industry, as well as for selection programs.


Assuntos
Fator de Crescimento Insulin-Like I/genética , Leite/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores da Somatotropina/genética , Ovinos/genética , Animais , Quimosina/metabolismo , Feminino , Genótipo , Itália , Lactação/genética , Leite/química , Especificidade da Espécie
4.
Hum Mutat ; 40(11): 2033-2043, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31231873

RESUMO

Isolated growth hormone deficiency (IGHD) is a rare condition mainly caused by mutations in GH1. The aim of this study was to assess the contribution of GHRHR mutations to IGHD in an unusually large group of patients. All GHRHR coding exons and flanking intronic regions were sequenced in 312 unrelated patients with nonsyndromic IGHD. Functional consequences of all newly identified missense variants were assessed in vitro (i.e., study of the expression of recombinant GHRHRs and their ability to activate the cyclic adenosine monophosphate (cAMP) signaling pathway). Genotype-phenotype correlation analyses were performed according to the nature of the identified mutation. We identified 20 different disease-causing GHRHR mutations (truncating and missense loss-of-function mutations), among which 15 are novel, in 24 unrelated patients. Of note, about half (13/24) of those patients represent sporadic cases. The clinical phenotype of patients with at least one missense GHRHR mutation was found to be indistinguishable from that of patients with bi-allelic truncating mutations. This study, which unveils disease-causing GHRHR mutations in 8% (24/312) of IGHD cases, identifies GHRHR as the second IGHD gene most frequently involved after GH1. The finding that 8% of IGHD cases without GH1 mutations are explained by GHRHR molecular defects (including missense mutations), together with the high proportion of sporadic cases among those patients, has important implications for genetic counseling.


Assuntos
Nanismo Hipofisário/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , AMP Cíclico , Análise Mutacional de DNA , Nanismo Hipofisário/diagnóstico , Feminino , Genótipo , Hormônio do Crescimento Humano/genética , Humanos , Masculino , Linhagem , Receptores de Neuropeptídeos/química , Receptores de Hormônios Reguladores de Hormônio Hipofisário/química
5.
Endocrinology ; 160(7): 1600-1612, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31070727

RESUMO

In this article, we briefly review the identification of GHRH, provide an abridged overview of GHRH antagonists, and focus on studies with GHRH agonists. Potent GHRH agonists of JI and MR class were synthesized and evaluated biologically. Besides the induction of the release of pituitary GH, GHRH analogs promote cell proliferation and exert stimulatory effects on various tissues, which express GHRH receptors (GHRH-Rs). A large body of work shows that GHRH agonists, such as MR-409, improve pancreatic ß-cell proliferation and metabolic functions and facilitate engraftment of islets after transplantation in rodents. Accordingly, GHRH agonists offer a new therapeutic approach to treating diabetes. Various studies demonstrate that GHRH agonists promote repair of cardiac tissue, producing improvement of ejection fraction and reduction of infarct size in rats, reduction of infarct scar in swine, and attenuation of cardiac hypertrophy in mice, suggesting clinical applications. The presence of GHRH-Rs in ocular tissues and neuroprotective effects of GHRH analogs in experimental diabetic retinopathy indicates their possible therapeutic applications for eye diseases. Other effects of GHRH agonists, include acceleration of wound healing, activation of immune cells, and action on the central nervous system. As GHRH might function as a growth factor, we examined effects of GHRH agonists on tumors. In vitro, GHRH agonists stimulate growth of human cancer cells and upregulate GHRH-Rs. However, in vivo, GHRH agonists inhibit growth of human cancers xenografted into nude mice and downregulate pituitary and tumoral GHRH-Rs. Therapeutic applications of GHRH analogs are discussed. The development of GHRH analogs should lead to their clinical use.


Assuntos
Diabetes Mellitus/tratamento farmacológico , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Neoplasias/tratamento farmacológico , Receptores de Neuropeptídeos/agonistas , Receptores de Hormônios Reguladores de Hormônio Hipofisário/agonistas , Animais , Linhagem Celular Tumoral , Humanos , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo
6.
Exp Eye Res ; 181: 277-284, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30831084

RESUMO

The receptor for growth hormone-releasing hormone (GHRH-R) has been shown to upregulate specifically in the ciliary and iris epithelial cells and infiltrating cells in the aqueous humor in a rat model of acute anterior uveitis. Treatment with GHRHR-R antagonist alleviates significantly these inflammatory responses. Herein we investigated whether the ciliary and iris epithelial cells can respond directly to lipopolysaccharide (LPS) without the influences of circulating leukocytes to produce inflammatory mediators through a GHRH-R mediated mechanism. In explant cultures of rat ciliary body and iris, LPS caused a substantial increase of GHRH-R in 24 h. Immunohistochemistry showed a localization of TLR4, the receptor for LPS, and an elevated expression of IL-6 and IL-1ß in ciliary and iris epithelial cells after LPS treatment. LPS also elevated the level of IL-1ß, IL-6, and iNOS and increased secretion of IL-1ß and IL-6 from the explants. The GHRH-R antagonist, MIA-602, suppressed the elevated expression of IL-1ß and IL-6, and reduced the release of IL-6. Such effects were not seen for the GHRHR agonist, MR-409. When co-cultured with leukocytes, expression of GHRH-R in the ocular explants was further enhanced during LPS treatment. Our results demonstrate a direct action of LPS on ciliary and iris epithelial cells to produce pro-inflammatory factors through a GHRH-R mediated mechanism, and suggest a role of these epithelial cells, in addition to the resident antigen presenting cells, in immune surveillance of the eye. Infiltrating leukocytes may enhance these inflammatory responses by regulating GHRH-R in ciliary and iris epithelial cells, in addition to their functions of synthesizing proinflammatory cytokines.


Assuntos
Humor Aquoso/metabolismo , Corpo Ciliar/metabolismo , Citocinas/biossíntese , Infecções Oculares Bacterianas/genética , Regulação da Expressão Gênica , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Uveíte Anterior/genética , Animais , Corpo Ciliar/patologia , Modelos Animais de Doenças , Infecções Oculares Bacterianas/metabolismo , Infecções Oculares Bacterianas/patologia , Imuno-Histoquímica , Iris/metabolismo , Masculino , RNA/genética , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeos/biossíntese , Receptores de Hormônios Reguladores de Hormônio Hipofisário/biossíntese , Uveíte Anterior/metabolismo , Uveíte Anterior/patologia
7.
Proc Natl Acad Sci U S A ; 116(6): 2226-2231, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30659154

RESUMO

Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated with exposure to asbestos, with poor prognosis and no effective therapies. The strong inhibitory activities of growth hormone-releasing hormone (GHRH) antagonists have been demonstrated in different experimental human cancers, including lung cancer; however, their role in MPM remains unknown. We assessed the effects of the GHRH antagonists MIA-602 and MIA-690 in vitro in MPM cell lines and in primary MPM cells, and in vivo in MPM xenografts. GHRH, GHRH receptor, and its main splice variant SV1 were found in all the MPM cell types examined. In vitro, MIA-602 and MIA-690 reduced survival and proliferation in both MPM cell lines and primary cells and showed synergistic inhibitory activity with the chemotherapy drug pemetrexed. In MPM cells, GHRH antagonists also regulated activity and expression of apoptotic molecules, inhibited cell migration, and reduced the expression of matrix metalloproteinases. These effects were accompanied by impairment of mitochondrial activity and increased production of reactive oxygen species. In vivo, s.c. administration of MIA-602 and MIA-690 at the dose of 5 µg/d for 4 wk strongly inhibited the growth of MPM xenografts in mice, along with reduction of tumor insulin-like growth factor-I and vascular endothelial growth factor. Overall, these results suggest that treatment with GHRH antagonists, alone or in association with chemotherapy, may offer an approach for the treatment of MPM.


Assuntos
Antineoplásicos/farmacologia , Hormônio Liberador de Hormônio do Crescimento/antagonistas & inibidores , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mesotelioma/metabolismo , Mesotelioma/patologia , Neoplasias Pleurais/metabolismo , Neoplasias Pleurais/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias Pleurais/tratamento farmacológico , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Growth Horm IGF Res ; 42-43: 94-101, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30390533

RESUMO

OBJECTIVE: Characterization of a deletion in the exon 1 and 5' regulatory region of the GHRHR gene in a proband with isolated growth hormone deficiency. METHODS: Multiple ligation dependent probe amplification (MLPA) assay was carried out to confirm the homozygous deletion which was suspected during screening of the GHRHR gene by single strand conformation polymorphism. A series of short range PCR amplifications were carried out to map the approximate location of the break points of the deletion. Sanger sequencing was carried out to locate the break points and to identify the length of the deletion. Long range PCR amplification was carried out to confirm the length of the deletion and to screen the parents of the proband for the deletion. RESULTS: A homozygous deletion was confirmed via MLPA assay. Zones of sequence similarity between upstream intergenic region and intron 1 of the GHRHR gene were identified. Break points of the deletion were identified within perfectly matching 32 bp repeat sequences ie: microhomologies in the specified zones. The novel deletion may have arisen via Alu specific microhomology mediated non-recurrent rearrangement in the maternal lineage of the proband. The deletion being reported in this study include, last 3118 bp from the upstream intergenic region and complete exon 1 and first 2620 bp from intron 1 and one of the 32 bp microhomologies. The total length of the deleted segment was 5875 bp. As the deleted region contained significant elements essential for gene expression, the identified deletion is being reported as likely pathogenic. The same deletion was identified in the mother in heterozygous state. CONCLUSION: We have characterized a novel deletion that seems to have arisen via Alu specific microhomology mediated non-recurrent rearrangement at GHRHR gene locus. HGVS nomenclature of the deletion is c.-3166_58-2057del. This novel structural variant was identified to be the cause of IGHD of the affected proband.


Assuntos
Elementos Alu , Deleção de Genes , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Hormônio do Crescimento Humano/deficiência , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Sequência de Bases , Pré-Escolar , Feminino , Transtornos do Crescimento/metabolismo , Humanos , Homologia de Sequência , Sri Lanka
9.
Proc Natl Acad Sci U S A ; 115(47): 12028-12033, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30373845

RESUMO

The effects of the growth hormone-releasing hormone (GHRH) agonist MR409 on various human cancer cells were investigated. In H446 small cell lung cancer (SCLC) and HCC827 and H460 (non-SCLC) cells, MR409 promoted cell viability, reduced cell apoptosis, and induced the production of cellular cAMP in vitro. Western blot analyses showed that treatment of cancer cells with MR409 up-regulated the expression of cyclins D1 and D2 and cyclin-dependent kinases 4 and 6, down-regulated p27kip1, and significantly increased the expression of the pituitary-type GHRH receptor (pGHRH-R) and its splice-variant (SV1). Hence, in vitro MR409 exerts agonistic action on lung cancer cells in contrast to GHRH antagonists. However, in vivo, MR409 inhibited growth of lung cancers xenografted into nude mice. MR409 given s.c. at 5 µg/day for 4 to 8 weeks significantly suppressed growth of HCC827, H460, and H446 tumors by 48.2%, 48.7%, and 65.6%, respectively. This inhibition of tumor growth by MR409 was accompanied by the down-regulation of the expression of pGHRH-R and SV1 in the pituitary gland and tumors. Tumor inhibitory effects of MR409 in vivo were also observed in other human cancers, including gastric, pancreatic, urothelial, prostatic, mammary, and colorectal. This inhibition of tumor growth parallel to the down-regulation of GHRH-Rs is similar and comparable to the suppression of sex hormone-dependent cancers after the down-regulation of receptors for luteinizing hormone-releasing hormone (LHRH) by LHRH agonists. Further oncological investigations with GHRH agonists are needed to elucidate the underlying mechanisms.


Assuntos
Receptores de Neuropeptídeos/efeitos dos fármacos , Receptores de Hormônios Reguladores de Hormônio Hipofisário/efeitos dos fármacos , Sermorelina/análogos & derivados , Processamento Alternativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Hormônio Liberador de Hormônio do Crescimento/agonistas , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Humanos , Camundongos , Camundongos Nus , Processamento de RNA/efeitos dos fármacos , Sermorelina/metabolismo , Sermorelina/farmacologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
10.
Invest Ophthalmol Vis Sci ; 59(12): 5060-5066, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30357400

RESUMO

Purpose: The aim of the study was to investigate the signaling of growth hormone-releasing hormone receptor (GHRH-R) in the pathogenesis of pterygium and determine the apoptotic effect of GHRH-R antagonist on pterygium epithelial cells (PECs). Methods: Fourteen samples of primary pterygium of grade T3 with size of corneal invasion ≥ 4 mm were obtained for investigation by histology, immunofluorescence, electron microscopy, explant culture, and flow cytometry. Results: We found that PECs were localized in the basal layer of the epithelium in advancing regions of the head of pterygium. These cells harbored clusters of rough endoplasmic reticulum, ribosomes, and mitochondria, which were consistent with their aggressive proliferation. Immunofluorescence studies and Western blots showed that GHRH-R and the downstream growth hormone receptor (GH-R) were intensively expressed in PECs. Their respective ligands, GHRH and GH, were also elevated in the pterygium tissues as compared to conjunctival cells. Explanted PECs were strongly immunoreactive to GHRH-R and exhibited differentiation and proliferation that led to lump formation. Treatment with GHRH-R antagonist MIA-602 induced apoptosis of PECs in a dose-dependent manner, which was accompanied by a downregulation of ERK1 and upregulation of Caspase 3 expression. Conclusions: Our results revealed that GHRH-R signaling is involved in survival and proliferation of PECs and suggest a potential therapeutic approach for GHRH-R antagonist in the treatment of pterygium.


Assuntos
Apoptose/efeitos dos fármacos , Pterígio/patologia , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Hormônios Reguladores de Hormônio Hipofisário/antagonistas & inibidores , Sermorelina/análogos & derivados , Western Blotting , Caspase 3/metabolismo , Contagem de Células , Proliferação de Células , Sobrevivência Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Técnica Indireta de Fluorescência para Anticorpo , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Humanos , Microscopia Eletrônica de Transmissão , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Pterígio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Receptores da Somatotropina/metabolismo , Sermorelina/farmacologia , Transdução de Sinais
12.
J Dairy Sci ; 101(11): 9978-9986, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30146276

RESUMO

The growth hormone receptor (GHR), the growth hormone releasing hormone receptor (GHRHR), and the insulin-like growth factor 1 (IGF1) genes are known to modulate growth, reproduction, and lactation traits in livestock. The aim of the current work was to investigate if the variation of the sheep GHR, GHRHR, and IGF1 genes is associated with milk yield and quality traits. Three hundred eighty dairy Sarda sheep were genotyped for 36 single nucleotide polymorphisms (SNP) mapping to these 3 loci, and records for milk yield and daily fat and protein yield, as well as for fat, protein, casein, lactose, and milk urea contents, pH, somatic cell score, logarithmic bacterial count, and milk energy were obtained. The linkage disequilibrium analysis was performed only for GHR, as both GHRHR and IGF1 had only 1 polymorphic SNP. Haplotype analysis revealed the existence of 7 haplotype blocks in GHR. Two haplotype blocks, including part of the intron 1 and the upstream region, were clearly separated from the remaining 5 blocks by SNP rs412986330, which may be a recombination hotspot. The latter 5 blocks were contiguous, spanning from intron 2 to exon 10. Statistical analysis revealed that the GHR polymorphism is significantly associated with milk traits for daily fat and protein yield and fat, milk urea, and lactose content. Moreover, variation in IGF1 was associated with milk protein and casein content. Data generated in this research provide new insights into the allelic effects of the ovine GHRHR, GHR, and IGF1 genes on milk production and quality traits, information that may be useful in gene-assisted selection programs.


Assuntos
Fator de Crescimento Insulin-Like I/genética , Leite/química , Polimorfismo de Nucleotídeo Único/genética , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores da Somatotropina/genética , Ovinos/genética , Animais , Caseínas/metabolismo , Gorduras/análise , Feminino , Genótipo , Haplótipos/genética , Lactação/genética , Lactose/análise , Desequilíbrio de Ligação , Proteínas do Leite/análise , Característica Quantitativa Herdável , Ovinos/fisiologia
13.
Artigo em Inglês | MEDLINE | ID: mdl-30114526

RESUMO

Phoenixin (Pnx) is an endogenous peptide known to be involved in reproduction and food intake in rats, with two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleost. Here, pnx was cloned and was detected in all tissues of both male and female in spotted scat (Scatophagus argus), including growth axis, hypothalamus, pituitary, and liver. Real-time PCR analysis showed that pnx in the hypothalamus increased significantly after 2 d and 7 d fasting, while reduced significantly after re-feeding (P < 0.05). When pituitary and liver fragments were cultured in vitro with Pnx-14 and Pnx-20 (10 nM and 100 nM) for 6 h, the expression of ghrhr (growth hormone-releasing hormone receptor) and gh (growth hormone) in the pituitary, and ghr1 (growth hormone receptor 1) in the liver increased significantly, except ghr2 (growth hormone receptor 2) incubated with 10 nM and 100 nM Pnx-20 and ghr1 incubated with 10 nM Pnx-20. Similarly, the expression of ghrhr and gh in the pituitary, as well as ghr1 and ghr2 in the liver, increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10 ng/g and 100 ng/g body weight). These results indicate that Pnx is likely to be involved in the regulation of food intake, and also regulates the growth of S. argus by increasing ghrhr and gh expression in the pituitary, ghr1 and ghr2 in the liver, and ghr1 directly in the liver.


Assuntos
Ingestão de Energia , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hormônios Hipotalâmicos/metabolismo , Hipotálamo/metabolismo , Hormônios Peptídicos/metabolismo , Perciformes/fisiologia , Animais , Aquicultura , China , Ingestão de Energia/efeitos dos fármacos , Feminino , Proteínas de Peixes/administração & dosagem , Proteínas de Peixes/genética , Proteínas de Peixes/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônio do Crescimento/agonistas , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Hormônios Hipotalâmicos/administração & dosagem , Hormônios Hipotalâmicos/genética , Hormônios Hipotalâmicos/farmacologia , Hipotálamo/efeitos dos fármacos , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Especificidade de Órgãos , Hormônios Peptídicos/administração & dosagem , Hormônios Peptídicos/genética , Hormônios Peptídicos/farmacologia , Perciformes/crescimento & desenvolvimento , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Isoformas de Proteínas/administração & dosagem , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Distribuição Aleatória , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/agonistas , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Receptores da Somatotropina/agonistas , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Técnicas de Cultura de Tecidos/veterinária , Ganho de Peso
14.
Pol J Pathol ; 69(1): 48-52, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29895126

RESUMO

Encouraged by our previous finding of growth hormone-releasing hormone receptor (GHRH-R) expression in metaplastic and neoplastic apocrine breast epithelium, we examined a small series of skin adnexal tumours with various degrees of apocrine (oxyphilic) differentiation, as well as normal axillary and anogenital apocrine sweat glands, for the expression of GHRH-R. Sections of formalin-fixed paraffin-embedded tissue blocks were immunostained for gross cystic disease fluid protein-15 (GCDFP-15) and androgen receptor (AR), to prove apocrine differentiation and correlate it with areas of GHRH-R expression. All but one of 19 tumours with apocrine epithelium and all five benign apocrine glands stained with both anti-GHRH-R antibodies used, each labelling a different domain of the receptor. Non-apocrine areas of the tumours and four eccrine tumours without oxyphilic features did not stain, but most sebaceous glands and some eccrine glands were labelled. Our data suggest that anti-GHRH-R antibodies highlight apocrine differentiation at extramammary sites also. Although GHRH-R seems to have a sensitivity comparable to classic apocrine markers such as AR and GCDFP-15, it seems to be inferior in specificity. The GHRH-R labelling of apocrine glands and neoplastic epithelium might also interfere with the emerging anti-GHRH targeted treatment of some malignancies acting on these receptors.


Assuntos
Glândulas Apócrinas/química , Biomarcadores Tumorais/análise , Células Epiteliais/química , Neoplasias de Anexos e de Apêndices Cutâneos/química , Receptores de Neuropeptídeos/análise , Receptores de Hormônios Reguladores de Hormônio Hipofisário/análise , Neoplasias das Glândulas Sudoríparas/química , Glândulas Apócrinas/patologia , Biópsia , Proteínas de Transporte/análise , Diagnóstico Diferencial , Células Epiteliais/patologia , Glicoproteínas/análise , Humanos , Imuno-Histoquímica , Proteínas de Membrana Transportadoras , Neoplasias de Anexos e de Apêndices Cutâneos/patologia , Valor Preditivo dos Testes , Receptores Androgênicos/análise , Neoplasias das Glândulas Sudoríparas/patologia
15.
Prostate ; 78(12): 915-926, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29748961

RESUMO

BACKGROUND: Therapeutic strategies should be designed to transform aggressive prostate cancer phenotypes to a chronic situation. To evaluate the effects of the new growth hormone-releasing hormone receptor (GHRH-R) antagonists: MIA-602, MIA-606, and MIA-690 on processes associated with cancer progression as cell proliferation, adhesion, migration, and angiogenesis. METHODS: We used three human prostate cell lines (RWPE-1, LNCaP, and PC3). We analyzed several molecules such as E-cadherin, ß-catenin, Bcl2, Bax, p53, MMP2, MMP9, PCNA, and VEGF and signaling mechanisms that are involved on effects exerted by GHRH-R antagonists. RESULTS: GHRH-R antagonists decreased cell viability and provoked a reduction in proliferation in LNCaP and PC3 cells. Moreover, GHRH-R antagonists caused a time-dependent increase of cell adhesion in all three cell lines and retarded the wound closure with the highest value with MIA-690 in PC3 cells. GHRH-R antagonists also provoked a large number of cells in SubG0 phase revealing an increase in apoptotic cells in PC3 cell line. CONCLUSIONS: Taken all together, GHRH-R antagonists of the MIAMI series appear to be inhibitors of tumor progression in prostate cancer and should be considered for use in future therapeutic strategies on this malignancy.


Assuntos
Neoplasias da Próstata/tratamento farmacológico , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Hormônios Reguladores de Hormônio Hipofisário/antagonistas & inibidores , Sermorelina/análogos & derivados , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Masculino , Células PC-3 , Neoplasias da Próstata/patologia , Fase de Repouso do Ciclo Celular , Sermorelina/farmacologia , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacos , beta Catenina/análise
16.
Circ Res ; 122(10): 1395-1408, 2018 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-29618597

RESUMO

RATIONALE: Vascular calcification (VC) is a marker of the severity of atherosclerotic disease. Hormones play important roles in regulating calcification; estrogen and parathyroid hormones exert opposing effects, the former alleviating VC and the latter exacerbating it. To date no treatment strategies have been developed to regulate clinical VC. OBJECTIVE: The objective of this study was to investigate the effect of growth hormone-releasing hormone (GHRH) and its agonist (GHRH-A) on the blocking of VC in a mouse model. METHODS AND RESULTS: Young adult osteoprotegerin-deficient mice were given daily subcutaneous injections of GHRH-A (MR409) for 4 weeks. Significant reductions in calcification of the aortas of MR409-treated mice were paralleled by markedly lower alkaline phosphatase activity and a dramatic reduction in the expression of transcription factors, including the osteogenic marker gene Runx2 and its downstream factors, osteonectin and osteocalcin. The mechanism of action of GHRH-A was dissected in smooth muscle cells isolated from human and mouse aortas. Calcification of smooth muscle cells induced by osteogenic medium was inhibited in the presence of GHRH or MR409, as evidenced by reduced alkaline phosphatase activity and Runx2 expression. Inhibition of calcification by MR409 was partially reversed by MIA602, a GHRH antagonist, or a GHRH receptor-selective small interfering RNA. Treatment with MR409 induced elevated cytosolic cAMP and its target, protein kinase A which in turn blocked nicotinamide adenine dinucleotide phosphate oxidase activity and reduced production of reactive oxygen species, thus blocking the phosphorylation of nuclear factor κB (p65), a key intermediate in the ligand of receptor activator for nuclear factor-κ B-Runx2/alkaline phosphatase osteogenesis program. A protein kinase A-selective small interfering RNA or the chemical inhibitor H89 abolished these beneficial effects of MR409. CONCLUSIONS: GHRH-A controls osteogenesis in smooth muscle cells by targeting cross talk between protein kinase A and nuclear factor κB (p65) and through the suppression of reactive oxygen species production that induces the Runx2 gene and alkaline phosphatase. Inflammation-mediated osteogenesis is thereby blocked. GHRH-A may represent a new pharmacological strategy to regulate VC.


Assuntos
Fragmentos de Peptídeos/uso terapêutico , Calcificação Vascular/prevenção & controle , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Aorta/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Meios de Cultura/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hormônio Liberador de Hormônio do Crescimento , Transplante de Coração , Humanos , Isoquinolinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese , Osteoprotegerina/deficiência , Fragmentos de Peptídeos/farmacologia , RNA Interferente Pequeno/genética , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/antagonistas & inibidores , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Sulfonamidas/farmacologia , Fator de Transcrição RelA/metabolismo , Calcificação Vascular/fisiopatologia
17.
Br J Haematol ; 181(4): 476-485, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29663325

RESUMO

Growth hormone-releasing hormone (GHRH) is secreted by the hypothalamus and acts on the pituitary gland to stimulate the release of growth hormone (GH). GHRH can also be produced by human cancers, in which it functions as an autocrine/paracrine growth factor. We have previously shown that synthetic antagonistic analogues of GHRH are able to successfully suppress the growth of 60 different human cancer cell lines representing over 20 cancers. Nevertheless, the expression of GHRH and its receptors in leukaemias has never been examined. Our study demonstrates the presence of GHRH receptor (GHRH-R) on 3 of 4 human acute myeloid leukaemia (AML) cell lines-K-562, THP-1, and KG-1a-and significant inhibition of proliferation of these three cell lines in vitro following incubation with the GHRH antagonist MIA-602. We further show that this inhibition of proliferation is associated with the upregulation of pro-apoptotic genes and inhibition of Akt signalling in leukaemic cells. Treatment with MIA-602 of mice bearing xenografts of these human AML cell lines drastically reduced tumour growth. The expression of GHRH-R was further confirmed in 9 of 9 samples from patients with AML. These findings offer a new therapeutic approach to this malignancy and suggest a possible role of GHRH-R signalling in the pathology of AML.


Assuntos
Apoptose/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Leucemia Mieloide Aguda/tratamento farmacológico , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Hormônios Reguladores de Hormônio Hipofisário/antagonistas & inibidores , Sermorelina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Animais , Feminino , Humanos , Células K562 , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sermorelina/farmacologia , Células THP-1 , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Clin Res Pediatr Endocrinol ; 10(3): 284-288, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-29537382

RESUMO

Congenital isolated growth hormone deficiency (IGHD) type 1b is an autosomal recessive genetic condition caused by mutations of growth hormone (GH)-1 or the growth hormone releasing hormone receptor (GHRH-R) genes. Affected subjects present with symptoms of growth hormone deficiency (GHD) with low but detectable levels of growth hormone (GH), short stature and responsiveness to GH therapy. We describe a 13-month old girl with severe growth failure who showed a low GH response to two GH provocation tests and a modest increase of insulin-like growth factor-1 (IGF-1) to an IGF-1 generation test. Whole exome sequencing revealed a novel homozygous variant of the GHRH-R gene (c.97C>T), leading to a premature stop codon. Administration of recombinant human GH improved linear growth. This is the first report of a c.97C>T mutation of the GHRH-R gene.


Assuntos
Nanismo/genética , Doenças da Hipófise/genética , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Códon sem Sentido , Feminino , Humanos , Lactente
19.
Growth Horm IGF Res ; 41: 71-74, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29571594

RESUMO

Growth hormone (GH) and the insulin-like growth factor I (IGF-I) have cell proliferative and differentiation properties. Whether these hormones have a role in mutagenesis is unknown. Nevertheless, severe IGF-I deficiency seems to confer protection against the development of neoplasms. Here, we report five cases of adult patients with severe and congenital isolated GH deficiency (IGHD) due to the c.57+1G>A mutation in the GHRH receptor gene, who developed tumors. Four GH-naïve subjects presented skin tumors: a 42-year-old man with a fibroepithelial polyp, a 53-year-old woman and two men (59 and 56 years old) with epidermoid skin cancers. One of these died from it after three surgeries and radiotherapy. The fifth patient was a 25-year-old woman, who had intermittently received GH replacement therapy (GHRT) from age 11 to 18, who developed an ependymoma extending from the fourth ventricle to the end of the thoracic spine. She underwent three surgical procedures, without obvious evidence of tumor recurrence during the six years follow up. These observations suggest that severe IGHD does not protect completely from development of tumors.


Assuntos
Biomarcadores/metabolismo , Nanismo Hipofisário/complicações , Hormônio do Crescimento Humano/deficiência , Mutação , Neoplasias/epidemiologia , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Prognóstico , Índice de Gravidade de Doença
20.
Eur J Pharm Sci ; 117: 341-350, 2018 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-29526766

RESUMO

Extra-hypothalamic growth hormone-releasing hormone (GHRH) plays an important role in infertility. The female infertility models were formed by intraperitoneally injecting cyclophosphamide in 5-week-old Chinese hamster once in a week for 5 weeks. All the models mated with healthy male hamster in the ratio of 1:1 in the experimental 6-8th week and the couples were separated to breed in the 9-10th week. 20 mg/kg of cyclophosphamide induced temporary interference of reproduction and did not cause significant difference in the weight of body, bilateral ovaries, or liver. By intramuscularly injecting twice in a week during the experimental 4-10th week, 2, 4, 8 mg/kg of Grin induced 30, 42.9, 60% of total pregnancy rates in a dose-dependent manner whereas 200 U/kg of hMG induced 50% of total pregnancy rates. The single cyclophosphamide dose caused strongly eosinophilic ovarian cells, scattered early follicles, many atretic follicles, and no corpora luteum was observed. The hMG group individually presents many follicles at all levels, especially secondary ones in the ovarian cortex and medulla. Much of loose connective tissue, vacuoles, and sparse interstitial cells distribute in the medulla. Grin induced many follicles at all dose levels and corpora lutea in the cortex, and the compactly aligned interstitial cells occurred in the whole ovarian tissue. The less TUNEL staining and higher expression of ki67 showed the proliferation and protection effect of Grin on ovarian cells. Grin obviously promotes fertility by up-regulating ovarian GHRH receptor and strengthening the development and maturation of follicles without triggering central and ovarian GH secretion.


Assuntos
Fármacos para a Fertilidade Feminina/administração & dosagem , Fertilidade/efeitos dos fármacos , Hormônio Liberador de Hormônio do Crescimento/administração & dosagem , Infertilidade Feminina/tratamento farmacológico , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Receptores de Neuropeptídeos/agonistas , Receptores de Hormônios Reguladores de Hormônio Hipofisário/agonistas , Animais , Cricetulus , Ciclofosfamida , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Infertilidade Feminina/induzido quimicamente , Infertilidade Feminina/metabolismo , Infertilidade Feminina/fisiopatologia , Injeções Intramusculares , Masculino , Folículo Ovariano/metabolismo , Folículo Ovariano/fisiopatologia , Ovário/metabolismo , Ovário/fisiopatologia , Gravidez , Taxa de Gravidez , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Regulação para Cima
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