D1 Signaling Research Articles

Targeting MYO1B impairs tumorigenesis via inhibiting the SNAI2/cyclin D1 signaling in esophageal squamous cell carcinoma.

Myosin-related proteins play an important role in cancer progression. However, the clinical significance, biological functions, and mechanisms of myosin 1B (MYO1B), in esophageal squamous cell carcinoma (ESCC) remain unclear. The clinical relevance of MYO1B, SNAI2, and cyclin D1 in ESCC was determined by immunohistochemistry, Oncomine, and GEPIA databases. The oncogenic roles of MYO1B were determined by CCK8, colony formation assays, wound healing, and Transwell assay. MYO1B, SNAI2, and cyclin D1 at mRNA and protein levels in ESCC cells were detected by qPCR and Western blot analysis. In our study, we found that MYO1B expression was increased in ESCC tissue samples and correlated with tumor stage, TNM stage, and poor outcomes. Functional assays indicated that depletion of MYO1B impaired oncogenesis, and enhanced chemosensitivity in ESCC. Bioinformatic analysis and mechanistic studies illustrated that SNAI2 was a key downstream effector of MYO1B. Suppression of MYO1B downregulated expression of SNAI2, thereby inhibiting the SNAI2/cyclin D1 pathway. Furthermore, a selective inhibitor of cyclin D1 activation reversed siMYO1B cells overexpressing SNAI2-elicited aggressive phenotypes of ESCC cells. MYO1B positively correlated with SNAI2 and cyclin D1 in ESCC samples, and higher SNAI2 expression was also associated with poor prognosis in ESCC patients. Our finding demonstrated that MYO1B activates the SNAI2/cyclin D1 pathway to drive tumorigenesis and cisplatin cytotoxicity in ESCC, indicating that MYO1B is a potential therapeutic target for patients with ESCC.

The multi-kinase inhibitor afatinib serves as a novel candidate for the treatment of human uveal melanoma

PurposeUveal melanoma (UM) is the most common intraocular malignancy in adults with a poor prognosis and a high recurrence rate. Currently there is no effective treatment for UM. Multi-kinase inhibitors targeting dysregulated pro-tumorigenic signalling pathways have revolutionised anti-cancer treatment but, as yet, their efficacy in UM has not been established. Here, we identified the multi-kinase inhibitor afatinib as a highly effective agent that exerts anti-UM effects in in vitro, ex vivo and in vivo models.MethodsWe assessed the anti-cancer effects of afatinib using cell viability, cell death and cell cycle assays in in vitro and ex vivo UM models. The signaling pathways involved in the anti-UM effects of afatinib were evaluated by Western blotting. The in vivo activity of afatinib was evaluated in UM xenograft models using tumour mass measurement, PET scan, immunohistochemical staining and TUNEL assays.ResultsWe found that afatinib reduced cell viability and activated apoptosis and cell cycle arrest in multiple established UM cell lines and in patient tumour-derived primary cell lines. Afatinib impaired cell migration and enhanced reproductive death in these UM cell models. Afatinib-induced cell death was accompanied by activation of STAT1 expression and downregulation of Bcl-xL and cyclin D1 expression, which control cell survival and cell cycle progression. Afatinib attenuated HER2-AKT/ERK/PI3K signalling in UM cell lines. Consistent with these observations, we found that afatinib suppressed tumour growth in UM xenografted mice.ConclusionOur data indicate that afatinib activates UM cell death and targets the HER2-mediated cascade, which modulates STAT1-Bcl-xL/cyclin D1 signalling. Thus, targeting HER2 with agents like afatinib may be a novel therapeutic strategy to treat UM and to prevent metastasis.

Convergent dopamine and ALK4 signaling to PCBP1 controls FosB alternative splicing and cocaine behavioral sensitization.

ΔfosB is an alternatively spliced product of the FosB gene that is essential for dopamine-induced reward pathways and that acts as a master switch for addiction. However, the molecular mechanisms of its generation and regulation by dopamine signaling are unknown. Here, we report that dopamine D1 receptor signaling synergizes with the activin/ALK4/Smad3 pathway to potentiate the generation of ΔFosB mRNA in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) via activation of the RNA-binding protein PCBP1, a regulator of mRNA splicing. Concurrent activation of PCBP1 and Smad3 by D1 and ALK4 signaling induced their interaction, nuclear translocation, and binding to sequences in exon-4 and intron-4 of FosB mRNA. Ablation of either ALK4 or PCBP1 in MSNs impaired ΔFosB mRNA induction and nuclear translocation of ΔFosB protein in response to repeated co-stimulation of D1 and ALK4 receptors. Finally, ALK4 is required in NAc MSNs of adult mice for behavioral sensitization to cocaine. These findings uncover an unexpected mechanism for ΔFosB generation and drug-induced sensitization through convergent dopamine and ALK4 signaling.

Dopamine Negatively Regulates Insulin Secretion Through Activation of D1-D2 Receptor Heteromer.

There is increasing evidence that dopamine (DA) functions as a negative regulator of glucose-stimulated insulin secretion; however, the underlying molecular mechanism remains unknown. Using total internal reflection fluorescence microscopy, we monitored insulin granule exocytosis in primary islet cells to dissect the effect of DA. We found that D1 receptor antagonists rescued the DA-mediated inhibition of glucose-stimulated calcium (Ca2+) flux, thereby suggesting a role of D1 in the DA-mediated inhibition of insulin secretion. Overexpression of D2, but not D1, alone exerted an inhibitory and toxic effect that abolished the glucose-stimulated Ca2+ influx and insulin secretion in β-cells. Proximity ligation and Western blot assays revealed that D1 and D2 form heteromers in β-cells. Treatment with a D1-D2 heteromer agonist, SKF83959, transiently inhibited glucose-induced Ca2+ influx and insulin granule exocytosis. Coexpression of D1 and D2 enabled β-cells to bypass the toxic effect of D2 overexpression. DA transiently inhibited glucose-stimulated Ca2+ flux and insulin exocytosis by activating the D1-D2 heteromer. We conclude that D1 protects β-cells from the harmful effects of DA by modulating D2 signaling. The finding will contribute to our understanding of the DA signaling in regulating insulin secretion and improve methods for preventing and treating diabetes.

Dose-Dependent Regulation on Prefrontal Neuronal Working Memory by Dopamine D1 Agonists: Evidence of Receptor Functional Selectivity-Related Mechanisms.

Low doses of dopamine D1 agonists improve working memory-related behavior, but high doses eliminate the improvement, thus yielding an ‘inverted-U’ dose-response curve. This dose-dependency also occurs at the single neuron level in the prefrontal cortex where the cellular basis of working memory is represented. Because signaling mechanisms are unclear, we examined this process at the neuron population level. Two D1 agonists (2-methyldihydrexidine and CY208,243) having different signaling bias were tested in rats performing a spatial working memory-related T-maze task. 2-Methyldihydrexidine is slightly bias toward D1-mediated β-arrestin-related signaling as it is a full agonist at adenylate cyclase and a super-agonist at β-arrestin recruitment, whereas CY208,243 is slightly bias toward D1-mediated cAMP signaling as it has relatively high intrinsic activity at adenylate cyclase, but is a partial agonist at β-arrestin recruitment. Both compounds had the expected inverted U dose-dependency in modulating prefrontal neuronal activities, albeit with important differences. Although CY208,243 was superior in improving the strength of neuronal outcome sensitivity to the working memory-related choice behavior in the T-maze, 2-methyldihydrexidine better reduced neuron-to-neuron variation. Interestingly, at the neuron population level, both drugs affected the percentage, uniformity, and ensemble strength of neuronal sensitivity in a complicated dose-dependent fashion, but the overall effect suggested higher efficiency and potency of 2-methyldihydrexidine compared to CY208,243. The differences between 2-methyldihydrexidine and CY208,243 may be related to their specific D1 signaling. These results suggest that D1-related dose-dependent regulation of working memory can be modified differentially by functionally selective ligands, theoretically increasing the balance between desired and undesired effects.

Abstract 115: Combined treatment of letrozole or olaparib with aspirin inhibits cell proliferation and induces apoptosis through upregulation of wild p53 and modulation of aurora kinases in human breast cancer cell lines

Abstract Although, there are several breakthroughs in the treatment of breast cancer on the past few decades, high incidence of relapse and progression after conventional therapies is deeply concerning, indicating a great need for developing new therapeutics for breast cancer. In the present study, the anti-cancer effects of combinational therapy of Letrozole (Aromatase inhibitor) or Olaparib (PARP inhibitor) with Aspirin, non-steroidal anti-inflammatory drugs (NSAIDs), were examined on well differentiated ER-positive and poorly differentiated triple negative breast cancer cell lines, T-47D and MDA-MB-231, respectively. In addition, the effect of combined treatment was also examined by detecting cell survival, cell cycle analysis, and alterations in signaling pathway that play a role in breast cancer progression such as β -catenin, STAT3, cyclin D1, Bax, c-PARP, wild and Mutant p53. In addition the effect of combined treatments were examined on gene expression levels of Cytochromes; CYP1A1, CYP1B1, Aurora kinases; Aurora-A and Aurora-B were determined by RT-PCR.Our data showed that combined treatments of 50 nmol Letrozole or 250 nmol Olaparib with 5 mmol, 2.5mmol Aspirin for 24 and 48 h, respectively, promoted apoptosis induction through enhanced histone release and caspase-3 activation compared to control and cells treated with each drug alone. Furthermore, apoptotic induction was associated with upregulation of Bax and downregulation of Bcl-2, cyclin D1, β-catenin, and STAT3 signaling pathways in both tested cell lines compared to control, suggesting that Aspirin may enhance the sensitivity of tested cells to Letrozole or Olaparib in both (ER) positive and triple negative breast cancer cell lines. In addition, cell cycle analysis showed a cell arrest at G0/G1 phase in both cell lines. Interestingly, in T-47D cells, combined treatment of Letrozole with Aspirin significantly upregulated wild p53 at protein level and downregulated CYP1A1, CYP1B1, Aurora-A, and Aurora-B expression at gene level compared to control, suggesting that combined treatment may inhibit breast cancer cell proliferation via down-regulation of Aurora-A and Aurora-B. In MDA-MB-231 cells, combined treatment of Olaparib with Aspirin showed a significant downregulation of Mutant p53 at protein level and CYP1A1, CYP1B1, Aurora-B and upregulation of Aurora-A at gene level compared to control, suggesting that there is a crosstalk between Olaparib/Aspirin and P53 In conclusion, combined treatment of Letrozole or Olaparib with Aspirin might be a promising approach to inhibit both ER (+) and triple negative breast cancer cell lines survival and proliferation. Citation Format: Marwa Elsayed ElKamel, Salah Sheweita, Ahmed Sultan. Combined treatment of letrozole or olaparib with aspirin inhibits cell proliferation and induces apoptosis through upregulation of wild p53 and modulation of aurora kinases in human breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 115.

A double-blind, randomized, placebo-controlled proof of concept study of the efficacy and safety of Lu AF11167 for persistent negative symptoms in people with schizophrenia

Lu AF11167 is a selective, high-affinity inhibitor of PDE10A that modulates dopamine D1 and D2 receptor-mediated intraneuronal signalling without binding to these receptors. This randomized, double-blind, parallel-group, placebo-controlled study (NCT03793712) with open-label extension (NCT03929497) evaluated the efficacy of two fixed-flexible doses (1-2mg/day and 3-4mg/day) of Lu AF11167 in stable, non-acute patients with schizophrenia and persistent prominent negative symptoms. The studies were discontinued following a futility analysis of the double-blind study, and we report data collected up to study termination. Of the 210 patients screened, 162 were randomized, 111 completed the double-blind study and 96 entered the open-label study before early termination. The withdrawal rate due to impending relapse was low and comparable across treatment groups (n = 2-4 per group in the double-blind study and n = 1 in the open-label extension). Double-blind treatment with Lu AF11167 3-4mg was not superior to placebo in the reduction of Brief Negative Symptom Scale (BNSS) total scores from Baseline to Week 12 (primary endpoint); adjusted mean changes were -6.8 with placebo, -5.7 with Lu AF11167 1-2 mg group and -6.0 with Lu AF11167 3-4mg. Treatment with Lu AF11167 1-2mg also failed to separate from placebo on the primary endpoint. Neither dose group showed significant improvements versus placebo on any of the secondary efficacy measures exploring effect of treatment on overall symptomology, negative symptoms, positive symptoms, or functioning. Administration of Lu AF11167 was safe and well tolerated and adverse events were not a major reason for withdrawal from the study.

MRCKα is a novel regulator of prolactin-induced lactogenesis in bovine mammary epithelial cells.

Myotonic dystrophy-related Cdc42-binding kinase alpha (MRCKα) is an integral component of signaling pathways controlling vital cellular processes, including cytoskeletal reorganization, cell proliferation and cell survival. In this study, we investigated the physiological role of MRCKα in milk protein and fat production in dairy cows, which requires a dynamic and strict organization of the cytoskeletal network in bovine mammary epithelial cells (BMEC). Within a selection of 9 Holstein cows, we found that both mRNA and protein expression of MRCKα in the mammary gland were upregulated during lactation and correlated positively (r > 0.89) with the mRNA and protein levels of β-casein. Similar positive correlations (r > 0.79) were found in a primary culture of BMEC stimulated with prolactin for 24 h. In these cells, silencing of MRCKα decreased basal β-casein, sterol-regulatory element binding protein (SREBP)-1 and cyclin D1 protein level, phosphorylation of mTOR, triglyceride secretion, cell number and viability—while overexpression of MRCKα displayed the reversed effect. Notably, silencing of MRCKα completely prevented the stimulatory action of prolactin on the same parameters. These data demonstrate that MRCKα is a critical mediator of prolactin-induced lactogenesis via stimulation of the mTOR/SREBP1/cyclin D1 signaling pathway.

TUBB4A interacts with MYH9 to protect the nucleus during cell migration and promotes prostate cancer via GSK3β/β-catenin signalling

Human tubulin beta class IVa (TUBB4A) is a member of the β-tubulin family. In most normal tissues, expression of TUBB4A is little to none, but it is highly expressed in human prostate cancer. Here we show that high expression levels of TUBB4A are associated with aggressive prostate cancers and poor patient survival, especially for African-American men. Additionally, in prostate cancer cells, TUBB4A knockout (KO) reduces cell growth and migration but induces DNA damage through increased γH2AX and 53BP1. Furthermore, during constricted cell migration, TUBB4A interacts with MYH9 to protect the nucleus, but either TUBB4A KO or MYH9 knockdown leads to severe DNA damage and reduces the NF-κB signaling response. Also, TUBB4A KO retards tumor growth and metastasis. Functional analysis reveals that TUBB4A/GSK3β binds to the N-terminal of MYH9, and that TUBB4A KO reduces MYH9-mediated GSK3β ubiquitination and degradation, leading to decreased activation of β-catenin signaling and its relevant epithelial-mesenchymal transition. Likewise, prostate-specific deletion of Tubb4a reduces spontaneous tumor growth and metastasis via inhibition of NF-κB, cyclin D1, and c-MYC signaling activation. Our results suggest an oncogenic role of TUBB4A and provide a potentially actionable therapeutic target for prostate cancers with TUBB4A overexpression.

Prognostic Value and Potential Mechanism of MTFR2 in Lung Adenocarcinoma.

Mitochondrial fission regulator 2 (MTFR2) belongs to the MTFR1 family, which plays a crucial role in regulating oxidative phosphorylation. Recent studies indicate that it also participates in cancer carcinogenesis and development; however, the clinical significance of MTFR2 in lung adenocarcinoma has not been fully confirmed. Our current study investigated the relationships between clinical characteristics and MTFR2 expression based on The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GSE31210) dataset, and clinical histopathological sample cohort. In addition, Kaplan–Meier and Cox regression analyses were additionally performed to evaluate the association between MTFR2 expression and patient survival. Gene set enrichment analysis (GESA) was conducted to spot possible pathways associated with MTFR2. Moreover, a single-sample GESA (ssGESA) was performed to evaluate the association between MTFR2 expression and immune cell infiltration. Cell colony formation assay, CCK-8 assay, cell cycle assay, and transwell assay were performed to verify the cell proliferation, migration, and invasion abilities after interfering with MTFR2 in lung cancer cells. Western blot assay was applied to identify the underlying protein levels. The results indicated that the elevated MTFR2 expression in lung adenocarcinoma samples correlated with T stage (P < 0.001), N stage (P = 0.005), M stage (P = 0.015), pathological stage (P = 0.002), and TP53 status (P < 0.001). Patients with a higher MTFR2 expression correlated with poorer overall survival (P < 0.01) and progression-free survival (P = 0.002). Knockdown of MTFR2 inhibited cell proliferation, migration, and invasion via AKT-cyclin D1 signaling and EMT pathways. Moreover, MTFR2 expression significantly positively correlated with Th2 cells (P < 0.001). Taken together, MTFR2 could serve as a novel prognostic indicator and therapeutic target for lung adenocarcinoma.

Cordycepin Suppresses The Malignant Phenotypes of Colon Cancer Cells through The GSK3ß/ß-catenin/cyclin D1 Signaling Pathway.

Objective Cordycepin, also known as 3ˊ-deoxyadenosine, is the main bioactive ingredient of Cordyceps militaris and possesses various pharmacological effects. This study was performed to investigate the role of cordycepin in regulating the biological behaviors of colon cancer cells and the potential mechanism behind it. Materials and Methods In this experimental study, after treatment of colon cancer cells with different concentrations of cordycepin, inhibition of proliferation was detected by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Colon cancer cell migration and invasion abilities were analyzed by wound healing and Transwell assays. Flow cytometry was performed to detect cell apoptosis. A lung metastasis model in nude mice was utilized to examine the effect of cordycepin on the metastasis of colon cancer cells in vivo. Western blot was used to quantify GSK3β, β-catenin and cyclin D1 expression levels. Results Cordycepin inhibited colon cancer cell proliferation, migration and invasion, induced apoptosis in vitro, and inhibited lung metastasis of colon cancer cells in vivo. GSK-3β inhibitor (CHIR99021) treatment abolished the effects of cordycepin on cell viability, migration, invasion and apoptosis. Additionally, cordycepin promoted the expressions of GSK3β, and inhibited β-catenin and cyclin D1 in colon cancer cells, while co-treatment with CHIR99021 reversed the above effects. ConclusionCordycepin suppresses the malignant phenotypes of colon cancer through the GSK3β/β-catenin/cyclin D1 signaling pathway.

Evening Primrose Oil Enhances Tamoxifen's Anticancer Activity against Breast Cancer Cells by Inducing Apoptosis, Inhibiting Angiogenesis, and Arresting the Cell Cycle.

Background: Despite advancements in cancer treatment, breast cancer (BC) is still one of the leading causes of death among women. The majority of anti-breast-cancer medications induce serious side effects and multidrug resistance. Although several natural compounds, such as evening primrose oil (EPO), have been shown to have anticancer properties when used alone, their combination with the anticancer medicine tamoxifen (TAM) has yet to be investigated. The present study aimed to investigate the anticancer efficacy of EPO, alone or in combination with TAM, in the BC cell lines MCF-7 and MDA-MB-231, as well as to elucidate the mechanism of action. Methods: The MTT assay was used to investigate the cytotoxic effect of EPO on the two cell lines, and we discovered an acceptable IC50 that was comparable to TAM. The ELISA, qRT-PCR, flow cytometry and colorimetric techniques were used. Results: The combination of EPO and TAM suppressed the VEGF level, VEGF gene expression and Cyclin D1 signaling pathways, arrested the cell cycle, and induced the apoptotic signaling pathways by increasing the Bax/Bcl-2 ratio and caspase 3 activity; this revealed significant anti-tumor activity. Conclusions: The most significant finding of this study was the confirmation of the anticancer activity of the natural product EPO, which potentiated the activity of the anticancer drug TAM against MCF-7 and MDA-MB-231 BC cell lines through the induction of apoptosis, inhibiting angiogenesis and halting cell proliferation.

Heterogeneous nuclear ribonucleoprotein K promotes the progression of lung cancer by inhibiting the p53-dependent signaling pathway.

BackgroundHeterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid‐binding protein. Reportedly, hnRNPK is overexpressed in many human tumors, and such overexpression is associated with poor prognosis, implicating the role of hnRNPK as an oncogene during tumorigenesis. In this study, hnRNPK expression in lung cancer tissues was investigated.MethodsBriefly, hnRNPK was knocked down in lung cancer cell lines, and effects of knockdown on the cell proliferation, migration, and cell cycle were assessed using a cell counting kit‐8 (CCK‐8) assay, colony formation assay, transwell assay and flow cytometry. The effects of hnRNPK knockdown on the p53‐dependent signaling pathway were examined using western blotting. Finally, the effect of hnRNPK knockdown on tumor growth was verified in vivo using a lung cancer xenograft mouse model.ResultshnRNPK knockdown inhibited the cell proliferation, migration and cell cycle. In addition to phenotypic changes, hnRNPK knockdown upregulated expressions of pCHK1, pCHK2, and p53，p21，cyclin D1, thereby mediating the DNA damage response (DDR). The regulatory function of hnRNPK during p53/p21/cyclin D1 signaling in hnRNPK‐knockdown A549 cells was confirmed by suppressed the protein expression of associated signaling pathways, which inhibited DDR.ConclusionhnRNPK plays a crucial role in the progression of lung cancer, ultimately affecting survival rate. Inhibition of progression of lung cancer cells induced by hnRNPK‐knockdown is dependent on activation of p53 by the p53/p21/cyclin D1 pathway.

A multifunctional composite hydrogel as an intrinsic and extrinsic coregulator for enhanced therapeutic efficacy for psoriasis

BackgroundPsoriasis is a chronic relapsing immunological skin disease characterized by multiple cross-talk inflammatory circuits which are relevantly associated with abnormal cross-reactivity between immune cells and keratinocytes (KCs). It may be inadequate to eradicate complicated pathogenesis only via single-mode therapy. To provide optimal combinatory therapeutics, a nanocomposite-based hydrogel was constructed by loading methotrexate (MTX) into ZnO/Ag to realize combined multiple target therapy of psoriasis.ResultsIn this composite hydrogel, ZnO hybrid mesoporous microspheres were utilized both as drug carriers and reactive oxygen species (ROS)-scavenging nanoparticles. A proper amount of Ag nanoparticle-anchored ZnO nanoparticles (ZnO/Ag) was functionalized with inherent immunoregulatory property. The experiments showed that ZnO/Ag nanoparticles could exhibit a self-therapeutic effect that was attributed to reducing innate cytokine profiles by inactivating p65 in proinflammatory macrophages and abrogating secretion of adaptive cytokines in KCs by downregulating ROS-mediated STAT3-cyclin D1 signaling. A preferable antipsoriatic efficacy was achieved via topical administration of this hydrogel on the imiquimod (IMQ)-induced psoriasis mice model, demonstrating the superior transdermal delivery and combined enhancement of therapeutic efficacy caused by intrinsic nanoparticles and extrinsic MTX.ConclusionThis composite hydrogel could serve as a multifunctional, nonirritating, noninvasive and effective transcutaneous nanoagent against psoriasis.Graphical

Dopamine D2 receptors in the nucleus accumbens modulate erectile function in a rat model of nonorganic erectile dysfunction.

BackgroundThe central molecular mechanisms of nonorganic erectile dysfunction remains unknown.ObjectiveThis study aimed to investigate the association of dopaminergic neurons projecting to the nucleus accumbens of male rats with nonorganic erectile dysfunction.Materials/methodsNonorganic erectile dysfunction was induced by chronic mild stress. The sucrose consumption test, sexual behavior test, and apomorphine test were carried out to select depression‐like rats with erectile dysfunction. These rats were considered as nonorganic erectile dysfunction model rats. Dopamine D1/D2 receptor agonist/antagonist was infused into the nucleus accumbens to observe the effect on sexual behavior. Dopaminergic projections to the nucleus accumbens were labeled with both the retrograde tracer FluoroGold injected into the nucleus accumbens and tyrosine hydroxylase. The expression level of tyrosine hydroxylase in dopaminergic neurons projecting to the nucleus accumbens in the ventral tegmental area was measured. The expression levels of dopamine D1/D2 receptors and tyrosine hydroxylase in the nucleus accumbens were also measured.ResultsNonorganic erectile dysfunction was proved by the sucrose consumption test, sexual behavior test, and apomorphine test in model rats. After central infusion of the dopamine D2 receptor agonist into the nucleus accumbens, the recovery of erectile function, sexual arousal, and motivation were indicated by increased intromission ratio and decreased mount latency. Decreased expression levels of dopamine D2 receptors and tyrosine hydroxylase in the nucleus accumbens and decreased expression level of tyrosine hydroxylase in the dopaminergic neurons projecting to the nucleus accumbens were observed in model rats.DiscussionThese results suggest the impairment of dopaminergic neurons projecting to the nucleus accumbens and dopamine D2 signaling in the nucleus accumbens, causing the suppression of erectile function, sexual arousal, and motivation.ConclusionThese results suggest that the impaired dopamine D2 receptor pathway in the nucleus accumbens may be one of the main pathway involved in the development of nonorganic erectile dysfunction in the present model.

Dopamine signaling modulates microglial NLRP3 inflammasome activation: implications for Parkinson\u2019s disease

BackgroundParkinson’s disease (PD) is characterized by the loss of nigral dopaminergic neurons leading to impaired striatal dopamine signaling, α-synuclein- (α-syn-) rich inclusions, and neuroinflammation. Degenerating neurons are surrounded by activated microglia with increased secretion of interleukin-1β (IL-1β), driven largely by the NLRP3 inflammasome. A critical role for microglial NLRP3 inflammasome activation in the progression of both dopaminergic neurodegeneration and α-syn pathology has been demonstrated in parkinsonism mouse models. Fibrillar α-syn activates this inflammasome in mouse and human macrophages, and we have shown previously that the same holds true for primary human microglia. Dopamine blocks microglial NLRP3 inflammasome activation in the MPTP model, but its effects in this framework, highly relevant to PD, remain unexplored in primary human microglia and in other in vivo parkinsonism models.MethodsBiochemical techniques including quantification of IL-1β secretion and confocal microscopy were employed to gain insight into dopamine signaling-mediated inhibition of the NLRP3 inflammasome mechanism in primary human microglia and the SYN120 transgenic mouse model. Dopamine and related metabolites were applied to human microglia together with various inflammasome activating stimuli. The involvement of the receptors through which these catecholamines were predicted to act were assessed with agonists in both species.ResultsWe show in primary human microglia that dopamine, l-DOPA, and high extracellular K+, but not norepinephrine and epinephrine, block canonical, non-canonical, and α-syn-mediated NLRP3 inflammasome-driven IL-1β secretion. This suggests that dopamine acts as an inflammasome inhibitor in human microglia. Accordingly, we provide evidence that dopamine exerts its inhibitory effect through dopamine receptor D1 and D2 (DRD1 and DRD2) signaling. We also show that aged mice transgenic for human C-terminally truncated (1–120) α-syn (SYN120 tg mice) display increased NLRP3 inflammasome activation in comparison to WT mice that is diminished upon DRD1 agonism.ConclusionsDopamine inhibits canonical, non-canonical, and α-syn-mediated activation of the NLRP3 inflammasome in primary human microglia, as does high extracellular K+. We suggest that dopamine serves as an endogenous repressor of the K+ efflux-dependent microglial NLRP3 inflammasome activation that contributes to dopaminergic neurodegeneration in PD, and that this reciprocation may account for the specific vulnerability of these neurons to disease pathology.

An oncogenic splice variant of PDGFR\u03b1 in adult glioblastoma as a therapeutic target for selective CDK4/6 inhibitors

Understanding human genome alterations is necessary to optimize genome-based cancer therapeutics. However, some newly discovered mutations remain as variants of unknown significance (VUS). Here, the mutation c.1403A > G in exon 10 of the platelet-derived growth factor receptor-alpha (PDGFRA) gene, a VUS found in adult glioblastoma multiforme (GBM), was introduced in human embryonal kidney 293 T (HEK293T) cells using genome editing to investigate its potential oncogenic functions. Genome editing was performed using CRISPR/Cas9; the proliferation, drug sensitivity, and carcinogenic potential of genome-edited cells were investigated. We also investigated the mechanism underlying the observed phenotypes. Three GBM patients carrying the c.1403A > G mutation were studied to validate the in vitro results. The c.1403A > G mutation led to a splice variant (p.K455_N468delinsN) because of the generation of a 3’-acceptor splice site in exon 10. PDGFRA-mutated HEK293T cells exhibited a higher proliferative activity via PDGFRα and the cyclin-dependent kinase (CDK)4/CDK6-cyclin D1 signaling pathway in a ligand-independent manner. They showed higher sensitivity to multi-kinase, receptor tyrosine kinase, and CDK4/CDK6 inhibitors. Of the three GBM patients studied, two harbored the p.K455_N468delinsN splice variant. The splicing mutation c.1403A > G in PDGFRA is oncogenic in nature. Kinase inhibitors targeting PDGFRα and CDK4/CDK6 signaling should be evaluated for treating GBM patients harboring this mutation.

Generation of Hydrogen Peroxide and Downstream Protein Kinase D1 Signaling Is a Common Feature of Inducers of Pancreatic Acinar-to-Ductal Metaplasia.

Pancreatic acinar-to-ductal metaplasia (ADM) is a reversible process that occurs after pancreatic injury, but becomes permanent and leads to pancreatic lesions in the presence of an oncogenic mutation in KRAS,. While inflammatory macrophage-secreted chemokines, growth factors that activate epidermal growth factor receptor (EGFR) and oncogenic KRAS have been implicated in the induction of ADM, it is currently unclear whether a common underlying signaling mechanism exists that drives this process. In this study, we show that different inducers of ADM increase levels of hydrogen peroxide, most likely generated at the mitochondria, and upregulate the expression of Protein Kinase D1 (PKD1), a kinase that can be activated by hydrogen peroxide. PKD1 expression in acinar cells affects their survival and mediates ADM, which is in part due to the PKD1 target NF-κB. Overall, our data implicate ROS-PKD1 signaling as a common feature of different inducers of pancreatic ADM.

Suppression of CDCA3 inhibits prostate cancer progression via NF‑κB/cyclin D1 signaling inactivation and p21 accumulation.

Dysregulation of the cell cycle contributes to tumor progression. Cell division cycle-associated 3 (CDCA3) is a known trigger of mitotic entry and has been demonstrated to be constitutively upregulated in tumors. It is therefore associated with carcinogenic properties reported in various cancers. However, the role of CDCA3 in prostate cancer is unclear. In the present study, western blotting and analysis of gene expression profiling datasets determined that CDCA3 expression was upregulated in prostate cancer and was associated with a poor prognosis. CDCA3 knockdown in DU145 and PC-3 cells led to decreased cell proliferation and increased apoptosis, with increased protein expression levels of cleaved-caspase3. Further experiments demonstrated that downregulated CDCA3 expression levels induced G0/G1 phase arrest, which was attributed to increased p21 protein expression levels and decreased cyclin D1 expression levels via the regulation of NF-κB signaling proteins (NFκB-p105/p50, IKKα/β, and pho-NFκB-p65). In conclusion, these results indicated that CDCA3 may serve a crucial role in prostate cancer and consequently, CDCA3 knockdown may be used as a potential therapeutic target.

Dopamine receptor D1 signaling stimulates lipolysis and browning of white adipocytes

Adipocytes express several kinds of catecholamine receptors, including adrenergic receptors, and dopamine receptors. Signaling pathways mediated by catecholamine receptors, such as β3-adrenergic receptor pathway, can induce body energy expenditure via activating thermogenesis of adipose tissue. However, the roles of adipose dopamine receptors on adipocytes are still unclear. Here, we investigate the role of dopamine receptor D1 (DRD1) on adipocytes. To this end, we use DRD1 agonist Fenoldopam and antagonist SCH23390 to stimulate and inhibit DRD1 signaling, respectively. We found that, compared with control group mice, Fenoldopam-treated and SCH23390-treated high-fat-diet (HFD)-fed mice showed smaller and bigger white adipose tissue/adipocyte sizes, respectively. Meanwhile, activating of DRD1 signaling enhanced intracellular levels of cAMP, phosphorylation levels of protein kinase A substrates, and hormone-sensitive lipase, a key enzyme for lipolysis in mature 3T3-L1 adipocytes and white adipose tissue of HFD-fed mice. As a result, the levels of free fatty acid or glycerol were increased, indicating stimulation of lipolysis by DRD1 activation. Moreover, activating DRD1 can induce the browning of adipocytes, as indicated by enhanced phosphorylation of P38 MAP kinase, increased expression of beige cell markers (PGC-1α, UCP-1, and CD81), mitochondrion content, and expression of β-oxidation related genes. All of these effects were reduced after treating with SCH23390 both in vitro and in HFD-fed mice. Collectively, our study indicated that DRD1 signaling stimulates lipolysis and browning of white adipocytes in vitro and in vivo. Understanding the functions of DRD1 on human adipocytes and adipose tissues will help us to design novel strategies to treat obesity.