RESUMO
Heterologous sensitization of adenylyl cyclase (AC) results in elevated cAMP signaling transduction that contributes to drug dependence. Inhibiting cullin3-RING ligases by blocking the neddylation of cullin3 abolishes heterologous sensitization, however, the modulating mechanism remains uncharted. Here, we report an essential role of the potassium channel tetramerization domain (KCTD) protein 2, 5, and 17, especially the dominant isoform KCTD5 in regulating heterologous sensitization of AC1 and morphine dependence via working with cullin3 and the cullin-associated and neddylation-dissociated 1 (CAND1) protein. In cellular models, we observed enhanced association of KCTD5 with Gß and cullin3, along with elevated dissociation of Gß from AC1 as well as of CAND1 from cullin3 in heterologous sensitization of AC1. Given binding of CAND1 inhibits the neddylation of cullin3, we further elucidated that the enhanced interaction of KCTD5 with both Gß and cullin3 promoted the dissociation of CAND1 from cullin3, attenuated the inhibitory effect of CAND1 on cullin3 neddylation, ultimately resulted in heterologous sensitization of AC1. The paraventricular thalamic nucleus (PVT) plays an important role in mediating morphine dependence. Through pharmacological and biochemical approaches, we then demonstrated that KCTD5/cullin3 regulates morphine dependence via modulating heterologous sensitization of AC, likely AC1 in PVT in mice. In summary, the present study revealed the underlying mechanism of heterologous sensitization of AC1 mediated by cullin3 and discovered the role of KCTD proteins in regulating morphine dependence in mice.
Assuntos
Adenilil Ciclases , Proteínas Culina , Dependência de Morfina , Animais , Adenilil Ciclases/metabolismo , Adenilil Ciclases/genética , Proteínas Culina/metabolismo , Camundongos , Dependência de Morfina/metabolismo , Células HEK293 , Humanos , Canais de Potássio/metabolismo , Canais de Potássio/genética , Camundongos Endogâmicos C57BL , Masculino , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Morfina/farmacologia , Camundongos Knockout , Transdução de Sinais , AMP Cíclico/metabolismoRESUMO
Neuroinflammation plays crucial role in the development and progression of depression. Large conductance calcium- and voltage-dependent potassium (BK) channels mediate the activation of microglia. Herein, we investigated whether BK channels could serve as a target for the treatment of inflammation-associated depression. Lipopolysaccharide (LPS, 0.83 mg/kg) was injected intraperitoneally (i.p.) to induce neuroinflammation and depressive-like behavior in 6-8 week ICR mice. Adeno-associated virus (AAV) constructs (AAV9-Iba1p-BK shRNA-EGFP (BK shRNA-AAV) or AAV9-Iba1p-NC shRNA-EGFP (NC shRNA-AAV)) were unilaterally injected intracerebroventricularly to selectively knock down BK channels in microglia. The tail suspension test (TST) and forced-swim test (FST) were used to evaluate depressive-like behavior in mice 24 h after LPS challenge. The morphology of microglia, expression of BK channels, levels of cytokines, and expression and activity of indoleamine 2,3-dioxygenase (IDO) were measured by immunohistochemistry, western blot, quantitative real time PCR, and enzyme-linked immunosorbent assay (ELISA), respectively. Either paxilline (i.p.), a specific BK channel blocker, or BK shRNA-AAV effectively inhibited the activation of microglia, reduced the production of IL-1ß in the hippocampus and suppressed the expression and activity of IDO in the hippocampus and prefrontal cortex, resulting in the amelioration of depressive-like behavior in mice. These data suggest for the first time that BK channels are involved in LPS-induced depressive-like behaviors. Thus, microglia BK channels may be a potential drug target for the depression treatment.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta , Lipopolissacarídeos , Camundongos , Animais , Lipopolissacarídeos/toxicidade , Doenças Neuroinflamatórias , Camundongos Endogâmicos ICR , Depressão/induzido quimicamente , Depressão/tratamento farmacológico , Depressão/metabolismo , RNA Interferente PequenoRESUMO
SCH23390 is a widely used D1 dopamine receptor (D1R) antagonist that also elicits some D1R-independent effects. We previously found that the benzazepine, SKF83959, an analog of SCH23390, produces positive allosteric modulation of the Sigma-1 receptor (Sig1R). SCH23390 does not bind to the orthodoxic site of Sig1R but enhances the binding of 3H (+)-pentazocine to Sig1R. In this study, we investigated whether SCH23390 functions as an allosteric modulator of Sig1R. We detected increased Sig1R dissociation from binding immunoglobulin protein (BiP) and translocation of Sig1R to the plasma membrane in response to SCH23390 in transfected HEK293T and SH-SY5Y cells, respectively. Activation of Sig1R by SCH23390 was further confirmed by inhibition of GSK3ß activity in a time- and dose-dependent manner; this effect was blocked by pretreatment with the Sig1R antagonist, BD1047, and by knockdown of Sig1R. SCH23390 also inhibited GSK3ß in wild-type mice but not in Sig1R knockout mice. Finally, we showed that SCH23390 allosterically modulated the effect of the Sig1R agonist SKF10047 on inhibition of GSK3ß. This positive allosteric effect of SCH23390 was further confirmed via promotion of neuronal protection afforded by SKF10047 in primary cortical neurons challenged with MPP+. These results provide the first evidence that SCH23390 elicits functional allosteric modulation of Sig1R. Our findings not only reveal novel pharmacological effects of SCH23390 but also indicate a potential mechanism for SCH23390-mediated D1R-independent effects. Therefore, attention should be paid to these Sig1R-mediated effects when explaining pharmacological responses to SCH23390.
Assuntos
Benzazepinas , Receptores de Dopamina D1 , Receptores sigma , Receptor Sigma-1 , Receptores sigma/metabolismo , Receptores sigma/antagonistas & inibidores , Humanos , Animais , Benzazepinas/farmacologia , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Células HEK293 , Camundongos , Antagonistas de Dopamina/farmacologia , Masculino , Camundongos Endogâmicos C57BLRESUMO
The blood-brain barrier (BBB)/blood-tumor barrier (BTB) impedes brain entry of most brain-targeted drugs, whether they are water-soluble or hydrophobic. Endothelial WNT signaling and neoplastic pericytes maintain BTB low permeability by regulating tight junctions. Here, we proposed nitazoxanide (NTZ) and ibrutinib (IBR) co-loaded ICAM-1-targeting nanoparticles (NI@I-NPs) to disrupt the BTB in a time-dependent, reversible, and size-selective manner by targeting specific ICAM-1, inactivating WNT signaling and depleting pericytes in tumor-associated blood vessels in breast cancer brain metastases. At the optimal NTZ/IBR mass ratio (1:2), BTB opening reached the optimum effect at 48-72 h without any sign of intracranial edema and cognitive impairment. The combination of NI@I-NPs and chemotherapeutic drugs (doxorubicin and etoposide) extended the median survival of mice with breast cancer brain metastases. Targeting BTB endothelial WNT signaling and tumor pericytes via NI@I-NPs could open the BTB to improve chemotherapeutic efficiency against brain metastases.
Assuntos
Barreira Hematoencefálica , Neoplasias Encefálicas , Nanopartículas , Pericitos , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Pericitos/metabolismo , Pericitos/efeitos dos fármacos , Feminino , Humanos , Nanopartículas/administração & dosagem , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Tiazóis/administração & dosagem , Tiazóis/farmacologia , Linhagem Celular Tumoral , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Camundongos Endogâmicos BALB C , Via de Sinalização Wnt/efeitos dos fármacos , Camundongos , Sistemas de Liberação de Medicamentos , Adenina/análogos & derivadosRESUMO
PTENα/ß, two variants of PTEN, play a key role in promoting tumor growth by interacting with WDR5 through their N-terminal extensions (NTEs). This interaction facilitates the recruitment of the SET1/MLL methyltransferase complex, resulting in histone H3K4 trimethylation and upregulation of oncogenes such as NOTCH3, which in turn promotes tumor growth. However, the molecular mechanism underlying this interaction has remained elusive. In this study, we determined the first crystal structure of PTENα-NTE in complex with WDR5, which reveals that PTENα utilizes a unique binding motif of a sequence SSSRRSS found in the NTE domain of PTENα/ß to specifically bind to the WIN site of WDR5. Disruption of this interaction significantly impedes cell proliferation and tumor growth, highlighting the potential of the WIN site inhibitors of WDR5 as a way of therapeutic intervention of the PTENα/ß associated cancers. These findings not only shed light on the important role of the PTENα/ß-WDR5 interaction in carcinogenesis, but also present a promising avenue for developing cancer treatments that target this pathway.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , PTEN Fosfo-Hidrolase , Animais , Humanos , Camundongos , Motivos de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos Nus , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/químicaRESUMO
Lipogenesis is often highly upregulated in breast cancer brain metastases to adapt to intracranial low lipid microenvironments. Lipase inhibitors hold therapeutic potential but their intra-tumoral distribution is often blocked by the bloodâtumor barrier (BTB). BTB activates its Wnt signaling to maintain barrier properties, e.g., Mfsd2a-mediated BTB low transcytosis. Here, we reported VCAM-1-targeting nano-wogonin (W@V-NPs) as an adjuvant of nano-orlistat (O@V-NPs) to intensify drug delivery and inhibit lipogenesis of brain metastases. W@V-NPs were proven to be able to inactivate BTB Wnt signaling, downregulate BTB Mfsd2a, accelerate BTB vesicular transport, and enhance tumor accumulation of O@V-NPs. With the ability to specifically kill cancer cells in a lipid-deprived environment with IC50 at 48 ng/mL, W@V-NPs plus O@V-NPs inhibited the progression of brain metastases with prolonged survival of model mice. The combination did not induce brain edema, cognitive impairment, and systemic toxicity in healthy mice. Targeting Wnt signaling could safely modulate the BTB to improve drug delivery and metabolic therapy against brain metastases.