Elevated extracellular vesicular Nm23-H1 subdues the pro-migratory potential of breast cancer cell-derived extracellular vesicles.

Metastasis is a key determinant in cancer mortality which is often associated with decreased levels of Nm23-H1, a well-established metastasis suppressor. Despite lacking a secretion signal peptide, Nm23-H1 has been reported to be present in the extracellular space and enclosed within extracellular vesicles (EVs). While the presence of Nm23-H1 proteins in EVs released by cancer cells has been observed through proteomics profiling, the role of vesicular Nm23-H1 remains unclear. Here, we investigated the function of vesicular Nm23-H1 using MDA-MB-231 (highly metastatic, low Nm23-H1) and MCF-7 (low/non-metastatic, high Nm23-H1) breast cancer cell models. Our findings confirm that Nm23-H1 is indeed encapsulated within EVs, and its levels can be manipulated through overexpression and knockdown approaches. Functional assays revealed that EVs derived from MDA-MB-231 cells that contained high levels of Nm23-H1 exhibit impaired pro-migratory properties, suggesting that vesicular Nm23-H1 may act as a metastasis suppressor. Furthermore, EVs with increased levels of Nm23-H1 altered the transcript levels of multiple cancer-related genes in recipient cells and stimulated type I interferon signaling through STAT1 phosphorylation. These results suggest the existence of an unconventional signaling pathway mediated by the uptake of EVs enriched with Nm23-H1, which may contribute to the anti-metastatic effect of Nm23-H1 in the tumor microenvironment. Additionally, our study demonstrates that elevated Nm23-H1 levels can impact the abundance of various other proteins encapsulated within breast cancer cell-derived EVs, such as SUSD2 (Sushi Domain Containing 2) which can also modulate metastasis.

Inhibition of adenylyl cyclase by GTPase-deficient Gαi is mechanistically different from that mediated by receptor-activated Gαi.

Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. Numerous disorders are associated with GPCRs that utilize Gi proteins to inhibit adenylyl cyclase (AC) as well as regulate other effectors. Several early studies have successfully defined the AC-interacting domains of several members of Gαi by measuring the loss of activity upon homologous replacements of putative regions of constitutive active Gαi mutants. However, whether such findings can indeed be translated into the context of a receptor-activated Gαi have not been rigorously verified. To address this issue, an array of known and new chimeric mutations was introduced into GTPase-deficient Q204L (QL) and R178C (RC) mutants of Gαi1, followed by examinations on their ability to inhibit AC. Surprisingly, most chimeras failed to abolish the constitutive activity brought on by the QL mutation, while some were able to eliminate the inhibitory activity of RC mutants. Receptor-mediated inhibition of AC was similarly observed in the same chimeric constructs harbouring the pertussis toxin (PTX)-resistant C351I mutation. Moreover, RC-bearing loss-of-function chimeras appeared to be hyper-deactivated by endogenous RGS protein. Molecular docking revealed a potential interaction between AC and the α3/ß5 loop of Gαi1. Subsequent cAMP assays support a cooperative action of the α3/ß5 loop, the α4 helix, and the α4/ß6 loop in mediating AC inhibition by Gαi1-i3. Our results unveiled a notable functional divergence between constitutively active mutants and receptor-activated Gαi1 to inhibit AC, and identified a previously unknown AC-interacting domain of Gαi subunits. These results collectively provide valuable insights on the mechanism of AC inhibition in the cellular environment.

Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of Gs Protein to the Melatonin MT1 Receptor.

Melatonin is a neuroendocrine hormone that regulates the circadian rhythm and many other physiological processes. Its functions are primarily exerted through two subtypes of human melatonin receptors, termed melatonin type-1 (MT1) and type-2 (MT2) receptors. Both MT1 and MT2 receptors are generally classified as Gi-coupled receptors owing to their well-recognized ability to inhibit cAMP accumulation in cells. However, it remains an enigma as to why melatonin stimulates cAMP production in a number of cell types that express the MT1 receptor. To address if MT1 can dually couple to Gs and Gi proteins, we employed a highly sensitive luminescent biosensor (GloSensorTM) to monitor the real-time changes in the intracellular cAMP level in intact live HEK293 cells that express MT1 and/or MT2. Our results demonstrate that the activation of MT1, but not MT2, leads to a robust enhancement on the forskolin-stimulated cAMP formation. In contrast, the activation of either MT1 or MT2 inhibited cAMP synthesis driven by the activation of the Gs-coupled ß2-adrenergic receptor, which is consistent with a typical Gi-mediated response. The co-expression of MT1 with Gs enabled melatonin itself to stimulate cAMP production, indicating a productive coupling between MT1 and Gs. The possible existence of a MT1-Gs complex was supported through molecular modeling as the predicted complex exhibited structural and thermodynamic characteristics that are comparable to that of MT1-Gi. Taken together, our data reveal that MT1, but not MT2, can dually couple to Gs and Gi proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP levels in cells that express different complements of MT1, MT2, and G proteins.

Disulfidptosis-related PABPC3 promotes tumor progression and inhibits immune activity in osteosarcoma.

BACKGROUND: Osteosarcoma is a very aggressive bone tumor mainly affecting teens and young adults. Disulfidptosis is a metabolic-related form of regulated cell death. However, the interconnection between disulfidptosis and osteosarcoma has not been explored. METHODS: In the present study, disulfidptosis-related clusters were identified in osteosarcoma using the nonnegative matrix factorization clustering method. PABPC3 was identified as a hazardous gene in osteosarcoma using machine learning algorithms, CoxBoost, and Random Survival Forest. The prognostic value, pathway annotation, immune characteristics, and drug prediction of PABPC3 were systematically explored. MTT (i.e., 3-(4, 5-dimethyl thiazol-2-yl)-2,5-diphenytetrazolium bromide), EdU (ie. 5-ethyny-2'-deoxvuridine), and Transwell assays were used for in vitro validation of PABPC3. RESULTS: The disulfidptosis-related clusters could distinguish survival outcomes of osteosarcoma patients. PABPC3 could predict survival outcomes, immune activity, and drug response in osteosarcoma patients. Besides, PABPC3 was proven to facilitate the proliferation and migration of osteosarcoma. CONCLUSIONS: The present study is expected to establish the bridge between disulfidptosis and osteosarcoma. PABPC3 is expected to be further explored as a therapeutic target in osteosarcoma.

Multi-target regulatory mechanism of Yang Xin Tang - a traditional Chinese medicine against dementia.

BACKGROUND: Yang Xin Tang (YXT) is a traditional Chinese herbal preparation which has been reported to improve cognitive function and memory in patients with dementia. As the underlying mechanism of action of YXT has not been elucidated, we examined the effects of YXT and its major herbal components in regulating gene transcription and molecular targets related to Alzheimer's disease (AD). METHODS: Aqueous and ethanol extracts of YXT and selected herbal components were prepared and validated by standard methods. A series of biochemical and cellular assays were employed to assess the ability of the herbal extracts to inhibit acetylcholinesterase, reduce ß-amyloid aggregation, stimulate the differentiation of neural progenitor cells, suppress cyclooxygenase, and protect neurons against ß-amyloid or N-methyl-D-aspartate-induced cytotoxicity. The effects of YXT on multiple molecular targets were further corroborated by a panel of nine reporter gene assays. RESULTS: Extracts of YXT and two of its constituent herbs, Poria cocos and Poria Sclerotium pararadicis, significantly inhibited ß-amyloid aggregation and ß-amyloid-induced cytotoxicity. A protective effect of the YXT extract was similarly observed against N-methyl-D-aspartate-induced cytotoxicity in primary neurons, and this activity was shared by extracts of Radix Astragali and Rhizoma Chuanxiong. Although the YXT extract was ineffective, extracts of Poria cocos, Poria Sclerotium pararadicis and Radix Polygalae inhibited acetylcholine esterase, with the latter also capable of upregulating choline acetyltransferase. YXT and its components significantly inhibited the activities of the pro-inflammatory cyclooxygenases. Additionally, extracts of YXT and several of its constituent herbs significantly stimulated the phosphorylation of extracellular signal-regulated kinases and cAMP-responsive element binding protein, two molecular targets involved in learning and memory, as well as in the regulation of neurogenesis. CONCLUSIONS: Several constituents of YXT possess multiple regulatory effects on known therapeutic targets of AD that range from ß-amyloid to acetylcholinesterase. The demonstrated neuroprotective and neurogenic actions of YXT lend credence to its use as an alternative medicine for treating AD.

Modeling the Heterodimer Interfaces of Melatonin Receptors.

Melatonin receptors are Class A G protein-coupled receptors (GPCRs) that regulate a plethora of physiological activities in response to the rhythmic secretion of melatonin from the pineal gland. Melatonin is a key regulator in the control of circadian rhythm and has multiple functional roles in retinal physiology, memory, immunomodulation and tumorigenesis. The two subtypes of human melatonin receptors, termed MT1 and MT2, utilize overlapping signaling pathways although biased signaling properties have been reported in some cellular systems. With the emerging concept of GPCR dimerization, melatonin receptor heterodimers have been proposed to participate in system-biased signaling. Here, we used computational approaches to map the dimerization interfaces of known heterodimers of melatonin receptors, including MT1/MT2, MT1/GPR50, MT2/GPR50, and MT2/5-HT2C . By homology modeling and membrane protein docking analyses, we have identified putative preferred interface interactions within the different pairs of melatonin receptor dimers and provided plausible structural explanations for some of the unique pharmacological features of specific heterodimers previously reported. A thorough understanding of the molecular basis of melatonin receptor heterodimers may enable the development of new therapeutic approaches against aliments involving these heterodimeric receptors.

Re-examining the 'Dissociation Model' of G protein activation from the perspective of Gßγ signaling.

G protein-coupled receptors (GPCRs) represent a major group of drug targets with tremendous pharmacological value. Signals arising from GPCRs are primarily transduced via two functional components of their corresponding G proteins, the Gα subunit and the Gßγ dimer that dissociate from each other upon activation of the heterotrimer (Gαßγ). The Gßγ dimer has become an increasingly popular subject in GPCR signaling, owing to its numerous effectors and notable roles in signal integration. Because Gßγ dimers participate in a wide range of intracellular processes that regulate cellular physiology, they are often implicated in the pathology of various diseases. Yet, one caveat to the current 'Dissociation Model' on GPCR signaling is that unequivocal Gßγ signals are biasedly detected with Gi/o -coupled receptors, while Gßγ signals from Gs - or Gq -coupled receptors seem to play an auxiliary role. In this review, we revisit the evidence for or against the 'Dissociation Model' and discuss in detail several hypotheses that may explain such disparity and provide alternative interpretations to accommodate the 'biased Gßγ signals' observed in different biological systems. The issue of whether unique combinations of Gßγ dimer can confer signaling specificity is also discussed in the context of physiological relevance.

Age Associated Decrease of MT-1 Melatonin Receptor in Human Dermal Skin Fibroblasts Impairs Protection Against UV-Induced DNA Damage.

The human body follows a physiological rhythm in response to the day/night cycle which is synchronized with the circadian rhythm through internal clocks. Most cells in the human body, including skin cells, express autonomous clocks and the genes responsible for running those clocks. Melatonin, a ubiquitous small molecular weight hormone, is critical in regulating the sleep cycle and other functions in the body. Melatonin is present in the skin and, in this study, we showed that it has the ability to dose-dependently stimulate PER1 clock gene expression in normal human dermal fibroblasts and normal human epidermal keratinocytes. Then we further evaluated the role of MT-1 melatonin receptor in mediating melatonin actions on human skin using fibroblasts derived from young and old subjects. Using immunocytochemistry, Western blotting and RT-PCR, we confirmed the expression of MT-1 receptor in human skin fibroblasts and demonstrated a dramatic age-dependent decrease in its level in mature fibroblasts. We used siRNA technology to transiently knockdown MT-1 receptor in fibroblasts. In these MT-1 knockdown cells, UV-dependent oxidative stress (H2O2 production) was enhanced and DNA damage was also increased, suggesting a critical role of MT-1 receptor in protecting skin cells from UV-induced DNA damage. These studies demonstrate that the melatonin pathway plays a pivotal role in skin aging and damage. Moreover, its correlation with skin circadian rhythm may offer new approaches for decelerating skin aging by modulating the expression of melatonin receptors in human skin.

GPCRs in Autocrine and Paracrine Regulations.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of integral membrane protein receptors. As signal detectors, the several 100 known GPCRs are responsible for sensing the plethora of endogenous ligands that are critical for the functioning of our endocrine system. Although GPCRs are typically considered as detectors for first messengers in classical signal transduction pathways, they seldom operate in isolation in complex biological systems. Intercellular communication between identical or different cell types is often mediated by autocrine or paracrine signals that are generated upon activation of specific GPCRs. In the context of energy homeostasis, the distinct complement of GPCRs in each cell type bridges the autocrine and paracrine communication within an organ, and the various downstream signaling mechanisms regulated by GPCRs can be integrated in a cell to produce an ultimate output. GPCRs thus act as gatekeepers that coordinate and fine-tune a response. By examining the role of GPCRs in activating and receiving autocrine and paracrine signals, one may have a better understanding of endocrine diseases that are associated with GPCR mutations, thereby providing new insights for treatment regimes.

Promoting axon regeneration in the adult CNS by modulation of the melanopsin/GPCR signaling.

Cell-type-specific G protein-coupled receptor (GPCR) signaling regulates distinct neuronal responses to various stimuli and is essential for axon guidance and targeting during development. However, its function in axonal regeneration in the mature CNS remains elusive. We found that subtypes of intrinsically photosensitive retinal ganglion cells (ipRGCs) in mice maintained high mammalian target of rapamycin (mTOR) levels after axotomy and that the light-sensitive GPCR melanopsin mediated this sustained expression. Melanopsin overexpression in the RGCs stimulated axonal regeneration after optic nerve crush by up-regulating mTOR complex 1 (mTORC1). The extent of the regeneration was comparable to that observed after phosphatase and tensin homolog (Pten) knockdown. Both the axon regeneration and mTOR activity that were enhanced by melanopsin required light stimulation and Gq/11 signaling. Specifically, activating Gq in RGCs elevated mTOR activation and promoted axonal regeneration. Melanopsin overexpression in RGCs enhanced the amplitude and duration of their light response, and silencing them with Kir2.1 significantly suppressed the increased mTOR signaling and axon regeneration that were induced by melanopsin. Thus, our results provide a strategy to promote axon regeneration after CNS injury by modulating neuronal activity through GPCR signaling.

Injured adult retinal axons with Pten and Socs3 co-deletion reform active synapses with suprachiasmatic neurons.

Despite advances in promoting axonal regeneration after adult central nervous system injury, elicitation of a large number of lesion-passing axons reform active synaptic connections with natural target neurons remains limited. By deleting both Pten and Socs3 in retinal ganglion cells, we report that optic nerve axons after prechiasm lesion robustly reinnervate the hypothalamus, form new synapses with neurons in the suprachiasmatic nucleus (SCN), and re-integrate with the existing circuitry. Photic or electric stimulation of the retinal axons induces neuronal response in SCN. However both the innervation pattern and evoked responses are not completely restored by the regenerating axons, suggesting that combining with other strategies is necessary to overcome the defective rewiring. Our results support that boosting the intrinsic growth capacity in injured neurons promotes axonal reinnervation and rewiring.

Cycloastragenol is a potent telomerase activator in neuronal cells: implications for depression management.

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression-like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression.

A molecular and chemical perspective in defining melatonin receptor subtype selectivity.

Melatonin is primarily synthesized and secreted by the pineal gland during darkness in a normal diurnal cycle. In addition to its intrinsic antioxidant property, the neurohormone has renowned regulatory roles in the control of circadian rhythm and exerts its physiological actions primarily by interacting with the G protein-coupled MT1 and MT2 transmembrane receptors. The two melatonin receptor subtypes display identical ligand binding characteristics and mediate a myriad of signaling pathways, including adenylyl cyclase inhibition, phospholipase C stimulation and the regulation of other effector molecules. Both MT1 and MT2 receptors are widely expressed in the central nervous system as well as many peripheral tissues, but each receptor subtype can be linked to specific functional responses at the target tissue. Given the broad therapeutic implications of melatonin receptors in chronobiology, immunomodulation, endocrine regulation, reproductive functions and cancer development, drug discovery and development programs have been directed at identifying chemical molecules that bind to the two melatonin receptor subtypes. However, all of the melatoninergics in the market act on both subtypes of melatonin receptors without significant selectivity. To facilitate the design and development of novel therapeutic agents, it is necessary to understand the intrinsic differences between MT1 and MT2 that determine ligand binding, functional efficacy, and signaling specificity. This review summarizes our current knowledge in differentiating MT1 and MT2 receptors and their signaling capacities. The use of homology modeling in the mapping of the ligand-binding pocket will be described. Identification of conserved and distinct residues will be tremendously useful in the design of highly selective ligands.

Cyclic AMP signalling through PKA but not Epac is essential for neurturin-induced biphasic ERK1/2 activation and neurite outgrowths through GFRα2 isoforms.

Cyclic AMP (cAMP) and neurotrophic factors are known to interact closely to promote neurite outgrowth and neuronal regeneration. Glial cell line-derived neurotrophic factor (GDNF) and its family member neurturin (NTN) transduce signal through a multi-component receptor complex consisting of GDNF family receptor alpha 2 (GFRα2) and Ret receptor tyrosine kinase. Neurons from GFRα2-deficient mice do not promote axonal initiation when stimulated by NTN, consistent with the role of GFRα2 in neuronal outgrowth. Multiple alternatively spliced isoforms of GFRα2 are known to be expressed in the nervous system. GFRα2a and GFRα2c but not GFRα2b promoted neurite outgrowth. It is currently unknown if cAMP signalling is differentially regulated by these isoforms. In this study, NTN activation of GFRα2a and GFRα2c but not GFRα2b induced biphasic ERK1/2 activation and phosphorylation of the major cAMP target CREB. Interestingly, inhibition of cAMP signalling significantly impaired GFRα2a and GFRα2c-mediated neurite outgrowth while cAMP agonists cooperated with GFRα2b to induce neurite outgrowth. Importantly, the specific cAMP effector PKA but not Epac was essential for NTN-induced neurite outgrowth, through transcription and translation-dependent activation of late phase ERK1/2. Taken together, these results not only demonstrated the essential role of cAMP-PKA signalling in NTN-induced biphasic ERK1/2 activation and neurite outgrowth, but also suggested cAMP-PKA signalling as a hitherto unrecognized underlying mechanism contributing to the differential neuritogenic activities of GFRα2 isoforms.

Glial cells isolated from dorsal root ganglia express prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors.

Isolated cells from adult rat dorsal root ganglia (DRG) are frequently used as a model system to study responses of primary sensory neurons to nociceptor sensitizing agents such as prostaglandin E(2) and prostacyclin, which are presumed to act only on the neurons in typical mixed cell cultures. In the present study, we evaluated the expression of prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors in cultures of mixed DRG cells and in purified DRG glia. We show here that EP(4) and IP receptor agonists stimulated adenylyl cyclase activity in both mixed DRG cells and in purified DRG glia, and that these responses were specifically inhibited by EP(4) and IP receptor antagonists, respectively. The presence of EP(4) and IP receptors in DRG glia was further confirmed by the expression of EP(4) and IP receptor immunoreactivity and mRNA. With the increasing awareness of neuron-glial interactions within intact DRG and the use of isolated DRG cells in the study of mechanisms underlying nociception, it will be essential to consider the role played by EP(4) and IP receptor-expressing glial cells when evaluating prostanoid-induced sensitization of DRG neurons.

Involvement of calcium mobilization from caffeine-sensitive stores in mechanically induced cell cycle arrest in the dinoflagellate Crypthecodinium cohnii.

Mechanical loads can profoundly alter cell growth and cell proliferation. The dinoflagellates are especially sensitive to mechanical stimulation. Many species will be arrested in cell cycle in response to turbulence or shear stress. We demonstrate here that mechanical shaking and caffeine, the ryanodine-receptor agonist, induced an elevation of cytosolic calcium in the dinoflagellate Crypthecodinium cohnii. Dantrolene, a ryanodine-receptor antagonist, dose-dependently inhibited both shaking-induced and caffeine-induced calcium release. Similar to the effect of mechanical shaking, caffeine alone dose-dependently and reversibly induced cell cycle arrest in dinoflagellates. Prolonged shaking substantially abolished the magnitude of caffeine-induced calcium release and vice-versa, suggesting that both agents released calcium from similar stores through ryanodine receptors. Fluorescence-conjugated ryanodine gave positive labeling, which could be blocked by ryanodine, in the cortice of C. cohnii cells. In addition, caffeine or shaking mobilized intracellular chlortetracycline (CTC)-positive membrane-bound calcium, which could be similarly depleted by t-BuBHQ, a SERCA pump inhibitor. Prior treatment with shaking or caffeine also inhibited the ability of the other agent in mobilizing CTC-positive calcium. CTC-positive microsomal fractions could also be induced to release calcium by caffeine and cADPR, the ryanodinee receptor modulator. t-BuBHQ, but not calcium ionophores, induced cell cycle arrest, and the calcium chelator BAPTA-AM was unable to rescue caffeine-induced cell cycle arrest. These data culminate to suggest that mobilization or depletion of caffeine-sensitive calcium stores, but not calcium elevation per se, is involved in the induction of cell cycle arrest by mechanical stimulation. The present study establishes the role of caffeine-sensitive calcium stores in the regulation of cell cycle progression.

Immuno-regulatory effects of CKBM on the activities of mitogen-activated protein kinases and the release of cytokines in THP-1 monocytic cells.

CKBM is an herbal formula composed of five Chinese medicinal herbs (Panax ginseng, Schisandra chinensis, Fructus crataegi, Ziziphus jujube and Glycine Max) supplemented with processed Saccharomyces cerevisiae. Previous studies have demonstrated that CKBM is capable of triggering the release of IL-6 and TNFalpha from human peripheral blood mononuclear cells, and its anti-tumorigenic activity has been demonstrated in nude mice with gastric cancer. In this report, we utilized the THP-1 monocytic cell line as a cellular model to investigate how CKBM regulates the intracellular signaling of monocytes and the subsequent release of the produced cytokines. In terms of mitogen-activated protein kinase (MAPK) cascades, CKBM (20%) had no significant effect on ERK, but was linked to an inhibitory effect on JNK and a stimulatory effect on p38 MAPK. The differential responsiveness of JNK and p38 was dependent on the duration of treatment, as well as on the dosage of CKBM. Treatment of CKBM alone induced the release of IL-10 and IFNgamma, but not IL-1beta, IL-4, IL-6 and TNFbeta, while increase of intracellular Ca2+ concentration by A23187 triggered the release of IL-10 only. Interestingly, A23187 synergized with the activities of CKBM-treated THP-1 cells in terms of IL-1beta and IFNgamma production, while the IL-10 production showed no synergistic relationship between CKBM and A23187. This A23187-induced synergism was associated with a dose-dependent character towards CKBM administration. In view of the intracellular Ca2+ elevation during monocyte activation, our results suggest that CKBM can serve as a promoting agent for modulating the functions of monocytes.