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1.
J Cancer Res Clin Oncol ; 150(7): 333, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38955827

RESUMO

OBJECTIVE: To explore the effect and mechanism of relaxin (RLX) in the growth and metastasis of livercancer after combination treatment with transarterial chemoembolization (TACE). MATERIALS AND METHODS: HCCLM3 and Huh-7 cells were adopted to evaluate the effect of tumor proliferation, migration, and invasion after RLX administration in vitro. The rabbit VX2 model was used to evaluate the biosafety, doxorubicin penetration, local tumor response, tumor metastasis, and survival benefit of RLX combined with TACE treatment. RESULTS: RLX did not affect the proliferation, migration, or invasion of HCCLM3 and Huh-7 cells, and the expression of E-cadherin and HIF-1α also remained unchanged while the MMP-9 protein was upregulated in vitro. In the rabbit VX2 model, compared to the normal saline group (NS), RLX group (RLX) and TACE mono-therapy group (TACE), the group that received TACE combined with RLX (TACE + RLX) showed an improved local tumor response and survival benefit. Furthermore, TACE combined with RLX was found to reduce tumor metastasis. This combination therapy reduced the fibrotic extracellular matrix in the tumor microenvironment, allowing for better penetration of doxorubicin, improved infiltration of CD8+ T cells and affected the secretion of cytokines. Additionally, RLX combined with TACE was able to decrease the expression of HIF-1α and PD-L1. The biosafety of TACE combined with RLX was also confirmed. CONCLUSION: RLX synergized with TACE by mitigating the fibrotic extracellular matrix and tumor hypoxic microenvironment, improving the therapeutic effect and inhibiting metastasis during the treatment of liver cancer.


Assuntos
Quimioembolização Terapêutica , Neoplasias Hepáticas , Relaxina , Animais , Quimioembolização Terapêutica/métodos , Coelhos , Relaxina/administração & dosagem , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/tratamento farmacológico , Doxorrubicina/administração & dosagem , Humanos , Terapia Combinada , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Metástase Neoplásica
2.
Dev Psychobiol ; 66(6): e22523, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38970242

RESUMO

The current literature suggests that relaxin-3/relaxin/insulin-like family peptide receptor 3 (RLN-3/RXFP-3) system is involved in the pathophysiology of affective disorders because the results of anatomical and pharmacological studies have shown that the RLN-3 signaling pathway plays a role in modulating the stress response, anxiety, arousal, depression-like behavior, and neuroendocrine homeostasis. The risk of developing mental illnesses in adulthood is increased by exposure to stress in early periods of life. The available data indicate that puberty is especially characterized by the development of the neural system and emotionality and is a "stress-sensitive" period. The presented study assessed the short-term changes in the expression of RLN-3 and RXFP-3 mRNA in the stress-dependent brain regions in male pubertal Wistar rats that had been subjected to acute stress. Three stressors were applied from 42 to 44 postnatal days (first day: a single forced swim; second day: stress on an elevated platform that was repeated three times; third day: restraint stress three times). Anxiety (open field, elevated plus maze test) and anhedonic-like behavior (sucrose preference test) were estimated during these tests. The corticosterone (CORT) levels and blood morphology were estimated. We found that the RXFP-3 mRNA expression decreased in the brainstem, whereas it increased in the hypothalamus 72 h after acute stress. These molecular changes were accompanied by the increased levels of CORT and anxiety-like behavior detected in the open field test that had been conducted earlier, that is, 24 h after the stress procedure. These findings shed new light on the neurochemical changes that are involved in the compensatory response to adverse events in pubertal male rats and support other data that suggest a regulatory interplay between the RLN-3 pathway and the hypothalamus-pituitary-adrenal axis activity in the mechanisms of anxiety-like behavior.


Assuntos
Ansiedade , Encéfalo , RNA Mensageiro , Ratos Wistar , Receptores Acoplados a Proteínas G , Estresse Psicológico , Animais , Masculino , Ratos , Estresse Psicológico/metabolismo , Estresse Psicológico/fisiopatologia , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Encéfalo/metabolismo , RNA Mensageiro/metabolismo , Comportamento Animal/fisiologia , Relaxina/metabolismo , Relaxina/genética , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genética , Maturidade Sexual/fisiologia , Proteínas do Tecido Nervoso
3.
Iowa Orthop J ; 44(1): 113-123, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38919370

RESUMO

Background: Female athletes are at increased risk for anterior cruciate ligament (ACL) injuries. The influence of hormonal variation on female ACL injury risk remains ill-defined. Recent data suggests that the collagen-degrading menstrual hormone relaxin may cyclically impact female ACL tissue quality. This review aims to identify any correlation between menstrual relaxin peaks and rates of female ACL injury. Methods: A systematic review was performed, utilizing the MEDLINE, EMBASE, and CINAHL databases. Included studies had to directly address relaxin/female ACL interactions. The primary outcome variable was relaxin proteolysis of the ACL, at cellular, tissue, joint, and whole-organism levels. The secondary outcome variable was any discussed method of moderating relaxin levels, and the clinical results if available. Results: AllThe numerous relaxin receptors on female ACLs upregulate local collagenolysis and suppress local collagen production. Peak serum relaxin concentrations (SRC) occur during menstrual cycle days 21-24; a time phase associated with greater risk of ACL injury. Oral contraceptives (OCPs) reduce SRC, with a potential ACLprotective effect. Conclusion: A reasonable correlative and plausible causative relationship exists between peak relaxin levels and increased risk of ACL injury in females, and further investigation is warranted. Level of Evidence: III.


Assuntos
Lesões do Ligamento Cruzado Anterior , Ciclo Menstrual , Relaxina , Humanos , Relaxina/sangue , Relaxina/metabolismo , Feminino , Ciclo Menstrual/fisiologia , Traumatismos em Atletas , Atletas
4.
Peptides ; 178: 171244, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38788901

RESUMO

The neuropeptide relaxin-3 and its cognate receptor, relaxin family peptide-3 receptors (RXFP3), have been implicated in modulating learning and memory processes, but their specific roles remain unclear. This study utilized behavioral and molecular approaches to investigate the effects of putatively reversible blockade of RXFP3 in the ventral dentate gyrus (vDG) of the hippocampus on spatial and fear memory formation in rats. Male Wistar rats received bilateral vDG cannula implantation and injections of the RXFP3 antagonist, R3(BΔ23-27)R/I5 (400 ng/0.5 µL per side), or vehicle at specific time points before acquisition, consolidation, or retrieval phases of the Morris water maze and passive avoidance learning tasks. RXFP3 inhibition impaired acquisition in the passive avoidance task but not the spatial learning task. However, both memory consolidation and retrieval were disrupted in both tasks following RXFP3 antagonism. Ventral hippocampal levels of the consolidation-related kinase p70-S6 kinase (p70S6K) were reduced RXFP3 blockade. These findings highlight a key role for ventral hippocampal RXFP3 signaling in the acquisition, consolidation, and retrieval of spatial and emotional memories, extending previous work implicating this neuropeptide system in hippocampal memory processing.


Assuntos
Giro Denteado , Medo , Ratos Wistar , Receptores Acoplados a Proteínas G , Animais , Giro Denteado/metabolismo , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Masculino , Medo/fisiologia , Aprendizagem da Esquiva/fisiologia , Aprendizagem da Esquiva/efeitos dos fármacos , Memória/fisiologia , Relaxina/metabolismo , Memória Espacial/fisiologia , Memória Espacial/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Aprendizagem em Labirinto/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Receptores de Peptídeos/metabolismo
5.
Biochem Pharmacol ; 225: 116273, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38729446

RESUMO

Fibrotic changes in musculoskeletal diseases arise from the abnormal buildup of fibrotic tissue around the joints, leading to limited mobility, compromised joint function, and diminished quality of life. Relaxin (RLX) attenuates fibrosis by accelerating collagen degradation and inhibiting excessive extracellular matrix (ECM) production. Further, RLX disrupts myofibroblast activation by modulating the TGF-ß/Smads signaling pathways, which reduces connective tissue fibrosis. However, the mechanisms and effects of RLX in musculoskeletal pathologies are emerging as increasing research focuses on relaxin's impact on skin, ligaments, tendons, cartilage, joint capsules, connective tissues, and muscles. This review delineates the actions of relaxin within the musculoskeletal system and the challenges to its clinical application. Relaxin shows significant potential in both in vivo and in vitro studies for broadly managing musculoskeletal fibrosis; however, challenges such as short biological half-life and sex-specific responses may pose hurdles for clinical use.


Assuntos
Fibrose , Relaxina , Relaxina/uso terapêutico , Relaxina/metabolismo , Humanos , Fibrose/tratamento farmacológico , Animais , Doenças Musculoesqueléticas/tratamento farmacológico , Doenças Musculoesqueléticas/metabolismo
6.
Gen Comp Endocrinol ; 354: 114543, 2024 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-38692521

RESUMO

Relaxin3 (rln3) has been associated with various emotional and cognitive processes, including stress, anxiety, learning, memory, motivational behavior, and circadian rhythm. Notably, previous report revealed that Rln3a played an indispensable role in testicular development and male fertility in Nile tilapia (Oreochromis niloticus). However, the underlying molecular mechanisms remain largely unknown. We found that Rln3a is expressed exclusively in the diencephalon* (Di*) of the brain. Deficiency of Rln3a resulted in a significant increase in serum dopamine level and an upregulation of gene expression of gnrh1 and kisspeptin2. To further elucidate the role of Rln3a in fish fertility, we collected two different regions of Di* and hypothalamus (Hyp) tissues for subsequent RNA-seq analysis of both wild-type (rln3a+/+) and rln3a-/- male tilapia. Upon the transcriptomic data, 1136 and 755 differentially expressed genes (DEGs) were identified in the Di* and Hyp tissues, respectively. In Di*, the up-regulated genes were enriched in circadian rhythm, chemical carcinogenesis, while the down-regulated genes were enriched in type II diabetes mellitus, dopaminergic synapse, and other pathways. In Hyp, the up-regulated genes were enriched in circadian rhythm, pyrimidine metabolism, while the down-regulated genes were enriched in type I diabetes mellitus, autoimmune thyroid disease, and other pathways. Subsequently, the results of both qRT-PCR and FISH assays highlighted a pronounced up-regulation of core circadian rhythm genes, cry1b and per3, whereas genes such as clocka, clockb, and arntl exhibited down-regulation. Furthermore, the genes associated with dopamine biosynthesis were significantly increased in the Hyp. In summary, the mutation of rln3a in male tilapia resulted in notable changes in circadian rhythm and disease-linked signaling pathways in the Di* and Hyp. These changes might account for the fertility defects observed in rln3a-/- male mutants in tilapia.


Assuntos
Encéfalo , Ciclídeos , Fertilidade , Animais , Masculino , Ciclídeos/genética , Ciclídeos/metabolismo , Encéfalo/metabolismo , Fertilidade/genética , Relaxina/genética , Relaxina/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo
7.
J Proteome Res ; 23(6): 2013-2027, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38739617

RESUMO

The human relaxins belong to the Insulin/IGF/Relaxin superfamily of peptide hormones, and their physiological function is primarily associated with reproduction. In this study, we focused on a prostate tissue-specific relaxin RLN1 (REL1_HUMAN protein) and a broader tissue specificity RLN2 (REL2_HUMAN protein). Due to their structural similarity, REL1 and REL2 proteins were collectively named a 'human relaxin protein' in previous studies and were exclusively measured by immunoassays. We hypothesized that the highly selective and sensitive immunoaffinity-selected reaction monitoring (IA-SRM) assays would reveal the identity and abundance of the endogenous REL1 and REL2 in biological samples and facilitate the evaluation of these proteins for diagnostic applications. High levels of RLN1 and RLN2 transcripts were found in prostate and breast cancer cell lines by RT-PCR. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in cell lines, seminal plasma, or blood serum. The IA-SRM assay of REL2 demonstrated its undetectable levels (<9.4 pg/mL) in healthy control female and male sera and relatively high levels of REL2 in maternal sera across different gestational weeks (median 331 pg/mL; N = 120). IA-SRM assays uncovered potential cross-reactivity and nonspecific binding for relaxin immunoassays. The developed IA-SRM assays will facilitate the investigation of the physiological and pathological roles of REL1 and REL2 proteins.


Assuntos
Relaxina , Humanos , Relaxina/metabolismo , Relaxina/genética , Masculino , Feminino , Linhagem Celular Tumoral , Imunoensaio/métodos , Espectrometria de Massas/métodos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/diagnóstico , Sêmen/química , Sêmen/metabolismo
8.
Biochem Pharmacol ; 225: 116264, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38710334

RESUMO

The retrosplenial cortex (RSC) plays a central role in processing contextual fear conditioning. In addition to corticocortical and thalamocortical projections, the RSC receives subcortical inputs, including a substantial projection from the nucleus incertus in the pontine tegmentum. This GABAergic projection contains the neuropeptide, relaxin-3 (RLN3), which inhibits target neurons via its Gi/o-protein-coupled receptor, RXFP3. To assess this peptidergic system role in contextual fear conditioning, we bilaterally injected the RSC of adult rats with an adeno-associated-virus (AAV), expressing the chimeric RXFP3 agonist R3/I5 or a control AAV, and subjected them to contextual fear conditioning. The R3/I5 injected rats did not display any major differences to control-injected and naïve rats but displayed a significantly delayed extinction. Subsequently, we employed acute bilateral injections of the specific RXFP3 agonist peptide, RXFP3-Analogue 2 (A2), into RSC. While the administration of A2 before each extinction trial had no impact on the extinction process, treatment with A2 before each acquisition trial resulted in delayed extinction. In related anatomical studies, we detected an enrichment of RLN3-immunoreactive nerve fibers in deep layers of the RSC, and a higher level of co-localization of RXFP3 mRNA with vesicular GABA transporter (vGAT) mRNA than with vesicular glutamate transporter-1 (vGLUT1) mRNA across the RSC, consistent with an effect of RLN3/RXFP3 signalling on the intrinsic, inhibitory circuits within the RSC. These findings suggest that contextual conditioning processes in the RSC involve, in part, RLN3 afferent modulation of local inhibitory neurons that provides a stronger memory acquisition which, in turn, retards the extinction process.


Assuntos
Extinção Psicológica , Medo , Receptores Acoplados a Proteínas G , Animais , Masculino , Medo/fisiologia , Medo/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Ratos , Extinção Psicológica/fisiologia , Extinção Psicológica/efeitos dos fármacos , Relaxina/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/efeitos dos fármacos , Giro do Cíngulo/metabolismo , Giro do Cíngulo/efeitos dos fármacos , Giro do Cíngulo/fisiologia , Receptores de Peptídeos
9.
Biochem Pharmacol ; 225: 116305, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38768763

RESUMO

Heart failure (HF) prevalence is rising due to reduced early mortality and demographic change. Relaxin (RLN) mediates protective effects in the cardiovascular system through Relaxin-receptor 1 (RXFP1). Cardiac overexpression of RXFP1 with additional RLN supplementation attenuated HF in the pressure-overload transverse aortic constriction (TAC) model. Here, we hypothesized that robust transgenic RXFP1 overexpression in cardiomyocytes (CM) protects from TAC-induced HF even in the absence of RLN. Hence, transgenic mice with a CM-specific overexpression of human RXFP1 (hRXFP1tg) were generated. Receptor functionality was demonstrated by in vivo hemodynamics, where the administration of RLN induced positive inotropy strictly in hRXFP1tg. An increase in phospholamban-phosphorylation at serine 16 was identified as a molecular correlate. hRXFP1tg were protected from TAC without additional RLN administration, presenting not only less decline in systolic left ventricular (LV) function but also abrogated LV dilation and pulmonary congestion compared to WT mice. Molecularly, transgenic hearts exhibited not only a significantly attenuated fetal and fibrotic gene activation but also demonstrated less fibrotic tissue and CM hypertrophy in histological sections. These protective effects were evident in both sexes. Similar cardioprotective effects of hRXFP1tg were detectable in a RLN-knockout model, suggesting an alternative mechanism of receptor activation through intrinsic activity, alternative endogenous ligands or crosstalk with other receptors. In summary, CM-specific RXFP1 overexpression provides protection against TAC even in the absence of endogenous RLN. This suggests RXFP1 overexpression as a potential therapeutic approach for HF, offering baseline protection with optional RLN supplementation for specific activation.


Assuntos
Miócitos Cardíacos , Receptores Acoplados a Proteínas G , Receptores de Peptídeos , Relaxina , Animais , Humanos , Masculino , Camundongos , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/prevenção & controle , Insuficiência Cardíaca/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Relaxina/genética , Relaxina/metabolismo
10.
Biochem Pharmacol ; 225: 116323, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38815632

RESUMO

Relaxin's role in differentiated thyroid cancer (DTC) has been suggested but its characterization in a large clinical sample remains limited. We performed immunohistochemistry for relaxin-2 (RLN2), CD68 (total macrophages), CD163 (M2 macrophages) on tissue microarrays from 181 subjects with non-distant metastatic DTC, and 185 subjects with benign thyroid tissue. Mean pixels/area for each marker was compared between tumor and adjacent tissue via paired-t test and between DTC and benign subjects via t-test assuming unequal variances. RNA qPCR was performed for expression of RLN2, RLN1, and RXFP1 in cell lines. Amongst 181 cases, the mean age was 46 years, 75 % were females. Tumoral tissue amongst the DTC cases demonstrated higher mean expression of RLN2 (53.04 vs. 9.79; p < 0.0001) compared to tumor-adjacent tissue. DTC tissue also demonstrated higher mean expression of CD68 (14.46 vs. 4.79; p < 0.0001), and CD163 (23.13 vs. -0.73; p < 0.0001) than benign thyroid. These markers did not differ between tumor-adjacent and benign thyroid tissue groups; and amongst cases, did not differ by demographic or clinicopathologic features. RLN1 and RXFP1 expression was detected in a minority of the cell lines, while RLN2 was expressed by 6/7 cell lines. In conclusion, widespread RLN2 expression in DTC tissue and most cell lines demonstrates that RLN2 acts in a paracrine manner, and that RLN1 and RXFP1 are probably not involved in thyroid cancer cell signaling. RLN2 is a biomarker for thyroid carcinogenesis, being associated with but not secreted by immunosuppressive macrophages. These findings will guide further investigations for therapeutic avenues against thyroid cancer.


Assuntos
Biomarcadores Tumorais , Relaxina , Neoplasias da Glândula Tireoide , Humanos , Relaxina/metabolismo , Relaxina/genética , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/diagnóstico , Feminino , Pessoa de Meia-Idade , Masculino , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Adulto , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Linhagem Celular Tumoral , Antígenos CD/genética , Antígenos CD/metabolismo , Idoso , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética
11.
Int J Biol Macromol ; 270(Pt 1): 132165, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38729472

RESUMO

Relaxin 3 is a neuropeptide that plays a crucial role in reproductive functions of mammals. Previous studies have confirmed that rln3a plays an important role in the male reproduction of tilapia. To further understand the significance of its paralogous gene rln3b in male fertility, we generated a homozygous mutant line of rln3b in Nile tilapia. Our findings indicated that rln3b mutation delayed spermatogenesis and led to abnormal testes structure. Knocking out rln3b gene resulted in a decrease in sperm count, sperm motility and male fish fertility. TUNEL detection revealed a small amount of apoptosis in the testes of rln3b-/- male fish at 390 days after hatching (dah). RT-qPCR analysis demonstrated that mutation of rln3b gene caused a significant downregulation of steroid synthesis-related genes such as cyp17a1, cyp11b2, germ cell marker gene, Vasa, and gonadal somatic cell marker genes of amh and amhr2. Furthermore, we found a significant down-regulation of hypothalamic-pituitary-gonadal (HPG) axis-related genes, while a significantly up-regulation of the dopamine synthetase gene in the rln3b-/- male fish. Taken together, our data strongly suggested that Rln3b played a crucial role in the fertility of XY tilapia by regulating HPG axis genes.


Assuntos
Hipogonadismo , Espermatogênese , Testículo , Tilápia , Animais , Masculino , Tilápia/genética , Hipogonadismo/genética , Espermatogênese/genética , Testículo/metabolismo , Relaxina/genética , Relaxina/metabolismo , Fertilidade/genética , Motilidade dos Espermatozoides/genética , Mutação , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo
12.
Elife ; 122024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38819436

RESUMO

The nucleus incertus (NI), a conserved hindbrain structure implicated in the stress response, arousal, and memory, is a major site for production of the neuropeptide relaxin-3. On the basis of goosecoid homeobox 2 (gsc2) expression, we identified a neuronal cluster that lies adjacent to relaxin 3a (rln3a) neurons in the zebrafish analogue of the NI. To delineate the characteristics of the gsc2 and rln3a NI neurons, we used CRISPR/Cas9 targeted integration to drive gene expression specifically in each neuronal group, and found that they differ in their efferent and afferent connectivity, spontaneous activity, and functional properties. gsc2 and rln3a NI neurons have widely divergent projection patterns and innervate distinct subregions of the midbrain interpeduncular nucleus (IPN). Whereas gsc2 neurons are activated more robustly by electric shock, rln3a neurons exhibit spontaneous fluctuations in calcium signaling and regulate locomotor activity. Our findings define heterogeneous neurons in the NI and provide new tools to probe its diverse functions.


Assuntos
Neurônios , Peixe-Zebra , Animais , Neurônios/fisiologia , Neurônios/metabolismo , Relaxina/metabolismo , Relaxina/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Sistemas CRISPR-Cas , Rombencéfalo/fisiologia , Rombencéfalo/metabolismo
13.
Biochem Pharmacol ; 224: 116238, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38677442

RESUMO

INSL5 and relaxin-3 are relaxin family peptides with important roles in gut and brain function, respectively. They mediate their actions through the class A GPCRs RXFP4 and RXFP3. RXFP4 has been proposed to be a therapeutic target for colon motility disorders whereas RXFP3 targeting could be effective for neurological conditions such as anxiety. Validation of these targets has been limited by the lack of specific ligands and the availability of robust ligand-binding assays for their development. In this study, we have utilized NanoBiT complementation to develop a SmBiT-conjugated tracer for use with LgBiT-fused RXFP3 and RXFP4. The low affinity between LgBiT:SmBiT should result in a low non-specific luminescence signal and enable the quantification of binding without the tedious separation of non-bound ligands. We used solid-phase peptide synthesis to produce a SmBiT-labelled RXFP3/4 agonist, R3/I5, where SmBiT was conjugated to the B-chain N-terminus via a PEG12 linker. Both SmBiT-R3/I5 and R3/I5 were synthesized and purified in high purity and yield. Stable HEK293T cell lines expressing LgBiT-RXFP3 and LgBiT-RXFP4 were produced and demonstrated normal signaling in response to the synthetic R3/I5 peptide. Binding was first characterized in whole-cell binding kinetic assays validating that the SmBiT-R3/I5 bound to both cell lines with nanomolar affinity with minimal non-specific binding without bound and free SmBiT-R3/I5 separation. We then optimized membrane binding assays, demonstrating easy and robust analysis of both saturation and competition binding from frozen membranes. These assays therefore provide an appropriate rigorous binding assay for the high-throughput analysis of RXFP3 and RXFP4 ligands.


Assuntos
Proteínas , Receptores Acoplados a Proteínas G , Receptores de Peptídeos , Relaxina , Relaxina/metabolismo , Relaxina/química , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Ligantes , Células HEK293 , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genética , Proteínas/metabolismo , Proteínas/química , Insulina/metabolismo , Ligação Proteica/fisiologia , Peptídeos/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos
14.
Expert Opin Ther Pat ; 34(1-2): 71-81, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38573177

RESUMO

INTRODUCTION: The neuropeptide relaxin-3/RXFP3 system belongs to the relaxin/insulin superfamily and is involved in many important physiological processes, such as stress responses, appetite control, and motivation for reward. Although relaxin-3 is the endogenous agonist for RXFP3, it can also bind to and activate RXFP1 and RXFP4. Consequently, research has been focused on the development of RXFP3-specific peptides and small-molecule ligands to validate the relaxin-3/RXFP3 system as a novel drug target. AREAS COVERED: This review provides an overview of patents on the relaxin-3/RXFP3 system covering ligand development and pharmacological studies since 2003. Related patents and literature reports were obtained from established sources including SciFinder, Google Patents, and Espacenet for patents and SciFinder, PubMed, and Google Scholar for literature reports. EXPERT OPINION: There has been an increasing amount of patent activities around relaxin-3/RXFP3, highlighting the importance of this novel neuropeptide system for drug discovery. The development of relaxin-3 derived peptides and small-molecule modulators, as well as behavioral studies in rodents, have shown that the relaxin-3/RXFP3 system is a promising drug target for treating various metabolic and neuropsychiatric diseases including obesity, anxiety, and alcohol addiction.


Assuntos
Neuropeptídeos , Relaxina , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Relaxina/metabolismo , Patentes como Assunto , Insulina/metabolismo , Receptores de Peptídeos/agonistas , Receptores de Peptídeos/metabolismo
15.
Reprod Biol ; 24(2): 100864, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38640630

RESUMO

Deregulation of the relaxin family peptide system (RFPS) appears to increase the risk of range of cancers, including epithelial ovarian cancers (EOC). The present study examines the effect of relaxin family peptide receptor 1 (RXFP1) level on the biological properties of human epithelial ovarian adenocarcinoma cells (OVCAR4 and SKOV3). RXFP1 was downregulated (RXFP1↓) in the cells using the RXFP1 sgRNA CRISPR All-in-One Lentivirus set (pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro), and upregulated (RXFP1↑) using the RXFP1 CRISPRa sgRNA Lentivector (pLenti-U6-sgRNA-PGK-Neo) kit, which activates the RXFP1 gene when paired with dCas9-SAM. The changes taking place during adhesion to extracellular matrix (ECM) proteins were assessed in multi-well plates coated with collagen, fibronectin, laminin and gelatin. Cellular viability was monitored based on mitochondrial metabolic activity (MTT Assay, Alamar Blue Assay) and adenosine triphosphate production (ATP Assay). The rate of cell proliferation was determined based on the percentage of Ki67 immunoreactive cells and the numbers of cells in particular cell-cycle phases. The mesenchymal-like (Boyden Chamber Assay) and amoeboid-like movements (Wound Healing Assay) of ovarian cancer cells were also analyzed after transfection. RXFP1 downregulation decreased the adhesion properties of ovarian cancer cells and increased the tendency for apoptosis under stressful conditions. In contrast, RXFP1 upregulation had pro-proliferative, pro-survival and promigratory effects. Our findings confirm that the relaxin-2/RXFP1 signaling pathway plays a role in the promotion of growth and progression of ovarian cancer.


Assuntos
Proliferação de Células , Neoplasias Ovarianas , Receptores Acoplados a Proteínas G , Humanos , Feminino , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Neoplasias Ovarianas/metabolismo , Linhagem Celular Tumoral , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genética , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/genética , Regulação Neoplásica da Expressão Gênica , Movimento Celular , Relaxina/metabolismo , Adesão Celular
16.
J Med Chem ; 67(6): 4442-4462, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38502780

RESUMO

Relaxin H2 is a clinically relevant peptide agonist for relaxin family peptide receptor 1 (RXFP1), but a combination of this hormone's short plasma half-life and the need for injectable delivery limits its therapeutic potential. We sought to overcome these limitations through the development of a potent small molecule (SM) RXFP1 agonist. Although two large SM HTS campaigns failed in identifying suitable hit series, we uncovered novel chemical space starting from the only known SM RXFP1 agonist series, represented by ML290. Following a design-make-test-analyze strategy based on improving early dose to man ranking, we discovered compound 42 (AZ7976), a highly selective RXFP1 agonist with sub-nanomolar potency. We used AZ7976, its 10 000-fold less potent enantiomer 43 and recombinant relaxin H2 to evaluate in vivo pharmacology and demonstrate that AZ7976-mediated heart rate increase in rats was a result of RXFP1 agonism. As a result, AZ7976 was selected as lead for continued optimization.


Assuntos
Relaxina , Humanos , Masculino , Ratos , Animais , Relaxina/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas
17.
J Med Chem ; 67(6): 4419-4441, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38502782

RESUMO

Optimization of the highly potent and selective, yet metabolically unstable and poorly soluble hRXFP1 agonist AZ7976 led to the identification of the clinical candidate, AZD5462. Assessment of RXFP1-dependent cell signaling demonstrated that AZD5462 activates a highly similar panel of downstream pathways as relaxin H2 but does not modulate relaxin H2-mediated cAMP second messenger responsiveness. The therapeutic potential of AZD5462 was assessed in a translatable cynomolgus monkey heart failure model. Following 8 weeks of treatment with AZD5462, robust improvements in functional cardiac parameters including LVEF were observed at weeks 9, 13, and 17 without changes in heart rate or mean arterial blood pressure. AZD5462 was well tolerated in both rat and cynomolgus monkey and has successfully completed phase I studies in healthy volunteers. In summary, AZD5462 is a small molecule pharmacological mimetic of relaxin H2 signaling at RXFP1 and holds promise as a potential therapeutic approach to treat heart failure patients.


Assuntos
Insuficiência Cardíaca , Relaxina , Humanos , Ratos , Animais , Relaxina/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Macaca fascicularis/metabolismo , Receptores de Peptídeos/metabolismo , Insuficiência Cardíaca/tratamento farmacológico
18.
Biochem Pharmacol ; 223: 116130, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38490518

RESUMO

Organ scarring, referred to as fibrosis, results from a failed wound-healing response to chronic tissue injury and is characterised by the aberrant accumulation of various extracellular matrix (ECM) components. Once established, fibrosis is recognised as a hallmark of stiffened and dysfunctional tissues, hence, various fibrosis-related diseases collectively contribute to high morbidity and mortality in developed countries. Despite this, these diseases are ineffectively treated by currently-available medications. The pro-fibrotic cytokine, transforming growth factor (TGF)-ß1, has emerged as the master regulator of fibrosis progression, owing to its ability to promote various factors and processes that facilitate rapid ECM synthesis and deposition, whilst negating ECM degradation. TGF-ß1 signal transduction is tightly controlled by canonical (Smad-dependent) and non-canonical (MAP kinase- and Rho-associated protein kinase-dependent) intracellular protein activity, whereas its pro-fibrotic actions can also be facilitated by the Wnt/ß-catenin pathway. This review outlines the pathological sequence of events and contributing roles of TGF-ß1 in the progression of fibrosis, and how the Wnt/ß-catenin pathway contributes to tissue repair in acute disease settings, but to fibrosis and related tissue dysfunction in synergy with TGF-ß1 in chronic diseases. It also outlines the anti-fibrotic and related signal transduction mechanisms of the hormone, relaxin, that are mediated via its negative modulation of TGF-ß1 and Wnt/ß-catenin signaling, but through the promotion of Wnt/ß-catenin activity in acute disease settings. Collectively, this highlights that the crosstalk between TGF-ß1 signal transduction and the Wnt/ß-catenin cascade may provide a therapeutic target that can be exploited to broadly treat and reverse established fibrosis.


Assuntos
Relaxina , Humanos , Relaxina/uso terapêutico , beta Catenina/metabolismo , Doença Aguda , Via de Sinalização Wnt , Fator de Crescimento Transformador beta1 , Fibrose
19.
Biochem Pharmacol ; 223: 116136, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38494063

RESUMO

Relaxin-2 (RLX), a critical hormone in pregnancy, has been investigated as a therapy for heart failure. In most studies, the peptide was delivered continuously, subcutaneously for 2 weeks in animals or intravenously for 2-days in human subjects, for stable circulating [RLX]. However, pulsatile hormone levels may better uncover the normal physiology. This premise was tested by subcutaneously injecting Sprague Dawley rats (250 g, N = 2 males, 2 females/group) with human RLX (0, 30, 100, or 500 µg/kg), every 12 h for 1 day, then measuring changes in Nav1.5, connexin43, and ß-catenin, 24 h later. Pulsatile RLX was measured by taking serial blood draws, post-injection. After an injection, RLX reached a peak in âˆ¼ 60 min, fell to 50 % in 5-6 h; injections of 0, 30, 100 or 500 µg/kg yielded peak levels of 0, 11.26 ± 3.52, 58.33 ± 16.10, and 209.42 ± 29.04 ng/ml and residual levels after 24-hrs of 0, 4.9, 45.1 and 156 pg/ml, respectively. The 30 µg/kg injections had no effect and 100 µg/kg injections increased Nav1.5 (25 %), Cx43 (30 %) and ß-catenin (90 %). The 500 µg/kg injections also increased Nav1.5 and Cx43 but were less effective at upregulating ß-catenin (up by 25 % vs. 90 %). Periodic injections of 100 µg/kg were highly effective at increasing the expression of Nav1.5 and Cx43 which are key determinants of conduction velocity in the heart and the suppression of arrhythmias. Periodic RLX is effective at eliciting changes in cardiac protein expression and may be a better strategy for its longer-term delivery in the clinical setting.


Assuntos
Relaxina , Gravidez , Ratos , Masculino , Animais , Feminino , Humanos , Relaxina/metabolismo , beta Catenina , Conexina 43/genética , Ratos Sprague-Dawley , Arritmias Cardíacas
20.
Biochem Pharmacol ; 223: 116157, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38518995

RESUMO

Recombinant human relaxin-2 (serelaxin) has been widely proven as a novel drug with myriad effects at different cardiovascular levels, which support its potential therapeutic efficacy in several cardiovascular diseases (CVD). Considering these effects, together with the influence of relaxin-2 on adipocyte physiology and adipokine secretion, and the connection between visceral adipose tissue (VAT) dysfunction and the development of CVD, we could hypothesize that relaxin-2 may regulate VAT metabolism. Our objective was to evaluate the impact of a 2-week serelaxin treatment on the proteome and lipidome of VAT from Sprague-Dawley rats. We found that serelaxin increased 1 polyunsaturated fatty acid and 6 lysophosphatidylcholines and decreased 4 triglycerides in VAT employing ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) based platforms, and that regulates 47 phosphoproteins using SWATH/MS analysis. Through RT-PCR, we found that serelaxin treatment also caused an effect on VAT lipolysis through an increase in the mRNA expression of hormone-sensitive lipase (HSL) and a decrease in the expression of adipose triglyceride lipase (ATGL), together with a reduction in the VAT expression of the fatty acid transporter cluster of differentiation 36 (Cd36). Serelaxin also caused an anti-inflammatory effect in VAT by the decrease in the mRNA expression of tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß), chemerin, and its receptor. In conclusion, our results highlight the regulatory role of serelaxin in the VAT proteome and lipidome, lipolytic function, and inflammatory profile, suggesting the implication of several mechanisms supporting the potential benefit of serelaxin for the prevention of obesity and metabolic disorders.


Assuntos
Doenças Cardiovasculares , Relaxina , Humanos , Ratos , Animais , Metabolismo dos Lipídeos , Proteoma , Gordura Intra-Abdominal/metabolismo , Lipidômica , Relaxina/farmacologia , Relaxina/metabolismo , Ratos Sprague-Dawley , Vasodilatadores/farmacologia , Doenças Cardiovasculares/metabolismo , RNA Mensageiro/genética , Tecido Adiposo/metabolismo , Proteínas Recombinantes/metabolismo
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