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1.
Cancer Immunol Res ; 12(6): 687-703, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38592331

RESUMO

Recombinant cytokines have limited anticancer efficacy mostly due to a narrow therapeutic window and systemic adverse effects. IL18 is an inflammasome-induced proinflammatory cytokine, which enhances T- and NK-cell activity and stimulates IFNγ production. The activity of IL18 is naturally blocked by a high-affinity endogenous binding protein (IL18BP). IL18BP is induced in the tumor microenvironment (TME) in response to IFNγ upregulation in a negative feedback mechanism. In this study, we found that IL18 is upregulated in the TME compared with the periphery across multiple human tumors and most of it is bound to IL18BP. Bound IL18 levels were largely above the amount required for T-cell activation in vitro, implying that releasing IL18 in the TME could lead to potent T-cell activation. To restore the activity of endogenous IL18, we generated COM503, a high-affinity anti-IL18BP that blocks the IL18BP:IL18 interaction and displaces precomplexed IL18, thereby enhancing T- and NK-cell activation. In vivo, administration of a surrogate anti-IL18BP, either alone or in combination with anti-PD-L1, resulted in significant tumor growth inhibition and increased survival across multiple mouse tumor models. Moreover, the anti-IL18BP induced pronounced TME-localized immune modulation including an increase in polyfunctional nonexhausted T- and NK-cell numbers and activation. In contrast, no increase in inflammatory cytokines and lymphocyte numbers or activation state was observed in serum and spleen. Taken together, blocking IL18BP using an Ab is a promising approach to harness cytokine biology for the treatment of cancer.


Assuntos
Interleucina-18 , Microambiente Tumoral , Animais , Humanos , Interleucina-18/metabolismo , Camundongos , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Neoplasias/imunologia , Neoplasias/tratamento farmacológico , Ativação Linfocitária/imunologia , Ativação Linfocitária/efeitos dos fármacos , Feminino , Camundongos Endogâmicos C57BL , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
2.
J Neurosci ; 38(7): 1662-1676, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29321139

RESUMO

The embryonic formation of midbrain dopaminergic (mDA) neurons in vivo provides critical guidelines for the in vitro differentiation of mDA neurons from stem cells, which are currently being developed for Parkinson's disease cell replacement therapy. Bone morphogenetic protein (BMP)/SMAD inhibition is routinely used during early steps of stem cell differentiation protocols, including for the generation of mDA neurons. However, the function of the BMP/SMAD pathway for in vivo specification of mammalian mDA neurons is virtually unknown. Here, we report that BMP5/7-deficient mice (Bmp5-/-; Bmp7-/-) lack mDA neurons due to reduced neurogenesis in the mDA progenitor domain. As molecular mechanisms accounting for these alterations in Bmp5-/-; Bmp7-/- mutants, we have identified expression changes of the BMP/SMAD target genes MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog). Conditionally inactivating SMAD1 in neural stem cells of mice in vivo (Smad1Nes) hampered the differentiation of progenitor cells into mDA neurons by preventing cell cycle exit, especially of TH+SOX6+ (tyrosine hydroxylase, SRY-box 6) and TH+GIRK2+ (potassium voltage-gated channel subfamily-J member-6) substantia nigra neurons. BMP5/7 robustly increased the in vitro differentiation of human induced pluripotent stem cells and induced neural stem cells to mDA neurons by up to threefold. In conclusion, we have identified BMP/SMAD signaling as a novel critical pathway orchestrating essential steps of mammalian mDA neurogenesis in vivo that balances progenitor proliferation and differentiation. Moreover, we demonstrate the potential of BMPs to improve the generation of stem-cell-derived mDA neurons in vitro, highlighting the importance of sequential BMP/SMAD inhibition and activation in this process.SIGNIFICANCE STATEMENT We identify bone morphogenetic protein (BMP)/SMAD signaling as a novel essential pathway regulating the development of mammalian midbrain dopaminergic (mDA) neurons in vivo and provide insights into the molecular mechanisms of this process. BMP5/7 regulate MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog) expression to direct mDA neurogenesis. Moreover, the BMP signaling component SMAD1 controls the differentiation of mDA progenitors, particularly to substantia nigra neurons, by directing their cell cycle exit. Importantly, BMP5/7 increase robustly the differentiation of human induced pluripotent and induced neural stem cells to mDA neurons. BMP/SMAD are routinely inhibited in initial stages of stem cell differentiation protocols currently being developed for Parkinson's disease cell replacement therapies. Therefore, our findings on opposing roles of the BMP/SMAD pathway during in vitro mDA neurogenesis might improve these procedures significantly.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Neurônios Dopaminérgicos/fisiologia , Mesencéfalo/fisiologia , Células-Tronco Neurais , Neurogênese/fisiologia , Células-Tronco Pluripotentes , Transdução de Sinais/fisiologia , Proteínas Smad/fisiologia , Animais , Proteína Morfogenética Óssea 5/genética , Proteína Morfogenética Óssea 5/metabolismo , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Mesencéfalo/citologia , Camundongos , Camundongos Knockout , Proteína Smad1/genética , Proteína Smad1/metabolismo
3.
Cell Metab ; 27(2): 461-469.e6, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29233536

RESUMO

Ghrelin, an appetite-stimulatory hormone secreted by the stomach, was discovered as a ligand for the growth hormone secretagogue receptor (GHSR). Through GHSR, ghrelin stimulates growth hormone (GH) secretion, a function that evolved to protect against starvation-induced hypoglycemia. Though the biology mediated by ghrelin has been described in great detail, regulation of ghrelin action is poorly understood. Here, we report the discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous antagonist of GHSR. LEAP2 is produced in the liver and small intestine, and its secretion is suppressed by fasting. LEAP2 fully inhibits GHSR activation by ghrelin and blocks the major effects of ghrelin in vivo, including food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. In contrast, neutralizing antibodies that block endogenous LEAP2 function enhance ghrelin action in vivo. Our findings reveal a mechanism for fine-tuning ghrelin action in response to changing environmental conditions.


Assuntos
Hepcidinas/metabolismo , Receptores de Grelina/antagonistas & inibidores , Animais , Cirurgia Bariátrica , Restrição Calórica , Ingestão de Alimentos , Jejum , Feminino , Grelina/antagonistas & inibidores , Grelina/metabolismo , Hormônio do Crescimento/metabolismo , Humanos , Intestino Delgado/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Ligação Proteica , Ratos , Receptores de Grelina/metabolismo
4.
J Biol Chem ; 292(5): 1925-1933, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-27994053

RESUMO

Apelin-36 was discovered as the endogenous ligand for the previously orphan receptor APJ. Apelin-36 has been linked to two major types of biological activities: cardiovascular (stimulation of cardiac contractility and suppression of blood pressure) and metabolic (improving glucose homeostasis and lowering body weight). It has been assumed that both of these activities are modulated through APJ. Here, we demonstrate that the metabolic activity of apelin-36 can be separated from canonical APJ activation. We developed a series of apelin-36 variants in which evolutionarily conserved residues were mutated, and evaluated their ability to modulate glucose homeostasis and body weight in chronic mouse models. We found that apelin-36(L28A) retains full metabolic activity, but is 100-fold impaired in its ability to activate APJ. In contrast to its full metabolic activity, apelin-36(L28A) lost the ability to suppress blood pressure in spontaneously hypertensive rats (SHR). We took advantage of these findings to develop a longer-acting variant of apelin-36 that could modulate glucose homeostasis without impacting blood pressure (or activating APJ). Apelin-36-[L28C(30kDa-PEG)] is 10,000-fold less potent than apelin-36 at activating the APJ receptor but retains its ability to significantly lower blood glucose and improve glucose tolerance in diet-induced obese mice. Apelin-36-[L28C(30kDa-PEG)] provides a starting point for the development of diabetes therapeutics that are devoid of the blood pressure effects associated with canonical APJ activation.


Assuntos
Adipocinas/farmacologia , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apelina , Receptores de Apelina , Pressão Sanguínea/efeitos dos fármacos , Camundongos , Ratos , Ratos Endogâmicos SHR
5.
PLoS One ; 10(10): e0139697, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26444681

RESUMO

Studying the development of mesodiencephalic dopaminergic (mdDA) neurons provides an important basis for better understanding dopamine-associated brain functions and disorders and is critical for establishing cell replacement therapy for Parkinson's disease. The transcription factors Otx2 and Lmx1b play a key role in the development of mdDA neurons. However, little is known about the genes downstream of Otx2 and Lmx1b in the pathways controlling the formation of mdDA neurons in vivo. Here we report on our investigation of Lmx1b as downstream target of Otx2 in the formation of mdDA neurons. Mouse mutants expressing Otx2 under the control of the En1 promoter (En1+/Otx2) showed increased Otx2 expression in the mid-hindbrain region, resulting in upregulation of Lmx1b and expansion of mdDA neurons there. In contrast, Lmx1b-/- mice showed decreased expression of Otx2 and impairments in several aspects of mdDA neuronal formation. To study the functional interaction between Otx2 and Lmx1b, we generated compound mutants in which Otx2 expression was restored in mice lacking Lmx1b (En1+/Otx2;Lmx1b-/-). In these animals Otx2 was not sufficient to rescue any of the aberrations in the formation of mdDA neurons caused by the loss of Lmx1b, but rescued the loss of ocular motor neurons. Gene expression studies in Lmx1b-/- embryos indicated that in these mutants Wnt1, En1 and Fgf8 expression are induced but subsequently lost in the mdDA precursor domain and the mid-hindbrain organizer in a specific, spatio-temporal manner. In summary, we demonstrate that Otx2 critically depends on Lmx1b for the formation of mdDA neurons, but not for the generation of ocular motor neurons. Moreover, our data suggest that Lmx1b precisely maintains the expression pattern of Wnt1, Fgf8 and En1, which are essential for mid-hindbrain organizer function and the formation of mdDA neurons.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Proteínas com Homeodomínio LIM/genética , Mesencéfalo/fisiologia , Fatores de Transcrição Otx/genética , Fatores de Transcrição/genética , Animais , Padronização Corporal/genética , Diferenciação Celular/genética , Dopamina/genética , Embrião de Mamíferos/fisiologia , Feminino , Fator 8 de Crescimento de Fibroblasto/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Rombencéfalo/fisiologia , Proteína Wnt1/genética
6.
Mol Cell Neurosci ; 45(1): 1-11, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20493948

RESUMO

The locus coeruleus (LC) which is the major noradrenergic nucleus in the brain develops under the influence of Bmps secreted by the roof plate and Fgf8 emitted from the mid-hindbrain organizer. We studied the development of the LC in different Bmp mouse mutants and report the absence of this nucleus in Bmp5(-/-);Bmp7(-/-) double knockouts. Notably, genes marking organizers and neuronal populations adjacent to the LC precursor field are unperturbed in Bmp5(-/-);Bmp7(-/-) animals. In addition, we found that in En1(+/Otx2) mutants in which the caudal Otx2 expression domain and thereby the mid-hindbrain organizer are shifted caudally, LC neurons are concomitantly reduced along with Bmp5/7. Complementing these results, Otx1(-/-);Otx2(+/-) mutants, in which the mid-hinbrain organizer is shifted rostrally, show a rostrally extended Bmp5 expression area and an increase in LC neurons. Taken together, our data indicate that LC development requires either Bmp5 or Bmp7, and one is able to compensate for the loss of the other. In addition, we conclude that the position of the mid-hindbrain organizer determines the size of the LC and propose that Bmp5/7 play an important role in mediating this organizer function.


Assuntos
Proteína Morfogenética Óssea 5/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Locus Cerúleo/citologia , Locus Cerúleo/embriologia , Mesencéfalo/fisiologia , Norepinefrina/metabolismo , Rombencéfalo/fisiologia , Animais , Apoptose , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Locus Cerúleo/metabolismo , Mesencéfalo/citologia , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/fisiologia , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Rombencéfalo/citologia , Células-Tronco/citologia , Células-Tronco/fisiologia
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