TM4SF1 is a molecular facilitator that distributes cargo proteins intracellularly in endothelial cells in support of blood vessel formation.

Transmembrane-4 L-six family member-1 (TM4SF1) is an atypical tetraspanin that is highly and selectively expressed in proliferating endothelial cells and plays an essential role in blood vessel development. TM4SF1 forms clusters on the cell surface called TMED (TM4SF1-enriched microdomains) and recruits other proteins that internalize along with TM4SF1 via microtubules to intracellular locations including the nucleus. We report here that tumor growth and wound healing are inhibited in Tm4sf1-heterozygous mice. Investigating the mechanisms of TM4SF1 activity, we show that 12 out of 18 signaling molecules examined are recruited to TMED on the surface of cultured human umbilical vein endothelial cells (HUVEC) and internalize along with TMED; notable among them are PLCγ and HDAC6. When TM4SF1 is knocked down in HUVEC, microtubules are heavily acetylated despite normal levels of HDAC6 protein, and, despite normal levels of VEGFR2, are unable to proliferate. Together, our studies indicate that pathological angiogenesis is inhibited when levels of TM4SF1 are reduced as in Tm4sf1-heterozygous mice; a likely mechanism is that TM4SF1 regulates the intracellular distribution of signaling molecules necessary for endothelial cell proliferation and migration.

PLC-γ-Ca2+ pathway regulates axonal TrkB endocytosis and is required for long-distance propagation of BDNF signaling.

Brain-derived neurotrophic factor (BDNF) and its tropomyosin receptor kinase B (TrkB) are important signaling proteins that regulate dendritic growth and maintenance in the central nervous system (CNS). After binding of BDNF, TrkB is endocytosed into endosomes and continues signaling within the cell soma, dendrites, and axon. In previous studies, we showed that BDNF signaling initiated in axons triggers long-distance signaling, inducing dendritic arborization in a CREB-dependent manner in cell bodies, processes that depend on axonal dynein and TrkB activities. The binding of BDNF to TrkB triggers the activation of different signaling pathways, including the ERK, PLC-γ and PI3K-mTOR pathways, to induce dendritic growth and synaptic plasticity. How TrkB downstream pathways regulate long-distance signaling is unclear. Here, we studied the role of PLC-γ-Ca2+ in BDNF-induced long-distance signaling using compartmentalized microfluidic cultures. We found that dendritic branching and CREB phosphorylation induced by axonal BDNF stimulation require the activation of PLC-γ in the axons of cortical neurons. Locally, in axons, BDNF increases PLC-γ phosphorylation and induces intracellular Ca2+ waves in a PLC-γ-dependent manner. In parallel, we observed that BDNF-containing signaling endosomes transport to the cell body was dependent on PLC-γ activity and intracellular Ca2+ stores. Furthermore, the activity of PLC-γ is required for BDNF-dependent TrkB endocytosis, suggesting a role for the TrkB/PLC-γ signaling pathway in axonal signaling endosome formation.

Paeoniflorin Alleviates Anxiety and Visceral Hypersensitivity via HPA Axis and BDNF/TrkB/PLCγ1 Pathway in Maternal Separation-induced IBS-like Rats.

BACKGROUND: Irritable Bowel Syndrome (IBS) is a prevalent gastrointestinal disorder that significantly diminishes the quality of life for affected individuals. The pathophysiology of IBS remains poorly understood, and available therapeutic options for IBS are limited. The crucial roles of brain-gut interaction, which is mediated by the Hypothalamic-Pituitary-Adrenocortical (HPA) axis and the autonomic nervous system in IBS, have attracted increasing attention. OBJECTIVE: The objective of this study was to examine the impact of paeoniflorin (PF) on anxiety and visceral hypersensitivity in maternal separation-induced IBS-like rats. METHODS: The IBS-like rat model was established through the implementation of Maternal Separation (MS) and subsequently subjected to various doses of PF administered via oral gavage for 14 days. Anxiety-like behavior was evaluated using the Open Field Test (OFT) and Elevated Plus Maze (EPM) test. The assessment of visceral sensitivity involved the utilization of the Abdominal Withdrawal Reflex (AWR) score and electromyographic (EMG) responses of the external oblique muscle in response to colorectal distention. The levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), and corticotrophin-releasing hormone (CRH) were examined by ELISA. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were employed to detect the expressions of CRH receptors 1 (CRHR1) and 2 (CRHR2). Glucocorticoid receptors (GR), mineralocorticoid receptor (MR), brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase B (TrkB), and phospholipase C γ1 (PLCγ1) were examined by Western blot. RESULTS AND DISCUSSION: The results showed that MS induced anxiety-like behavior and visceral hypersensitivity, while PF treatment attenuated these changes. Furthermore, the HPA axis hyperactivity in MS rats was attenuated by PF treatment, indicated by reduced serum ACTH, CORT, and CRH levels and recovered hippocampal CRHR1 and GR expressions. In addition, PF inhibited BDNF/TrkB signaling by downregulating the protein levels of BDNF, TrkB, and phospho-PLCγ1 in the colon. CONCLUSION: These findings suggest that PF alleviated anxiety and visceral hypersensitivity in MS-induced IBS-like rats, which may be the modulation of HPA axis activity and BDNF/TrkB/PLCγ1 signaling pathway.

Müller cells under hydrostatic pressure modulate retinal cell survival via TRPV1/PLCγ1 complex-mediated calcium influx in experimental glaucoma.

Glaucoma, an irreversible blinding eye disease, is currently unclear whose pathological mechanism is. This study investigated how transient receptor potential cation channel subfamily V member 1 (TRPV1), 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 (PLCγ1), and P2X purinoceptor 7 (P2X7) modulate the levels of intracellular calcium ions (Ca2+) and adenosine triphosphate (ATP) in Müller cells and retinal ganglion cells (RGCs) under conditions of elevated intraocular pressure (IOP). Müller cells were maintained at hydrostatic pressure (HP). TRPV1- and PLCG1-silenced Müller cells and P2X7-silenced RGCs were constructed by transfection with short interfering RNA (siRNAs). RGCs were cultured with the conditioned media of Müller cells under HP. A mouse model of chronic ocular hypertension (COH) was established and used to investigate the role of TRPV1 in RGCs in vivo. Müller cells and RGCs were analyzed by ATP release assays, intracellular calcium assays, CCK-8 assays, EdU (5-ethynyl-2'-deoxyuridine) staining, TUNEL staining, flow cytometry, and transmission electron microscopy. In vivo changes in inner retinal function were evaluated by hematoxylin and eosin (H&E) staining and TUNEL staining. Western blot analyses were performed to measure the levels of related proteins. Our data showed that HP increased the levels of ATP and Ca2+ influx in Müller cells, and those increases were accompanied by the upregulation of TRPV1 and p-PLCγ1 expression. Suppression of TRPV1 or PLCG1 expression in Müller cells significantly decreased the ATP levels and intracellular Ca2+ accumulation induced by HP. Knockdown of TRPV1, PLCG1, or P2X7 significantly decreased apoptosis and autophagy in RGCs cultured in the conditioned media of HP-treated Müller cells. Moreover, TRPV1 silencing decreased RGC apoptosis and autophagy in the in vivo model of COH. Collectively, inhibition of TRPV1/PLCγ1 and P2X7 expression may be a useful therapeutic strategy for managing RGC death in glaucoma.

Ginkgetin effectively mitigates collagen and AA-induced platelet activation via PLCγ2 but not cyclic nucleotide-dependent pathway in human.

Platelets assume a pivotal role in the cardiovascular diseases (CVDs). Thus, targeting platelet activation is imperative for mitigating CVDs. Ginkgetin (GK), from Ginkgo biloba L, renowned for its anticancer and neuroprotective properties, remains unexplored concerning its impact on platelet activation, particularly in humans. In this investigation, we delved into the intricate mechanisms through which GK influences human platelets. At low concentrations (0.5-1 µM), GK exhibited robust inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Intriguingly, thrombin and U46619 remained impervious to GK's influence. GK's modulatory effect extended to ATP release, P-selectin expression, intracellular calcium ([Ca2+ ]i) levels and thromboxane A2 formation. It significantly curtailed the activation of various signaling cascades, encompassing phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß and mitogen-activated protein kinases. GK's antiplatelet effect was not reversed by SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor), and GK had no effect on the phosphorylation of vasodilator-stimulated phosphoproteinSer157 or Ser239 . Moreover, neither cyclic AMP nor cyclic GMP levels were significantly increased after GK treatment. In mouse studies, GK notably extended occlusion time in mesenteric vessels, while sparing bleeding time. In conclusion, GK's profound impact on platelet activation, achieved through inhibiting PLCγ2-PKC cascade, culminates in the suppression of downstream signaling and, ultimately, the inhibition of platelet aggregation. These findings underscore the promising therapeutic potential of GK in the CVDs.

Neuroprotective effect of chrysophanol in Alzheimer disease via modulating the Ca2+/EGFR-PLCγ pathway.

Chrysophanol (CHR) is an anthraquinone compound found in rhubarb, and it possesses neuroprotective properties. The purpose of this study was to gain a better understanding of its role in Alzheimer's disease (AD). In vivo study, D-galactose combined with intracerebral injection of ß-protein 25-35(Aß25-35) were used to establish AD model rats. In vitro study, Aß25-35 was used to induce AD model cells. Our results indicated that CHR improves learning and memory in AD model rats and provides protection against neuronal damage in both AD model rats and cells. Additionally, we observed that CHR suppressed the protein expression of p-tau, EGFR, PLCγ, IP3R, and CAM, as well as the mRNA levels of tau, EGFR, PLCγ, IP3R, and CAM. Furthermore, we have confirmed that CHR inhibited the fluorescence expression of calcium ions (Ca2+). In conclusion, the CHR may exert neuroprotective effects in AD by reducing tau phosphorylation through the Ca2+/EGFR-PLCγ pathway.

Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models.

Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 µM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.

8-Br-cGMP suppresses tumor progression through EGFR/PLC γ1 pathway in epithelial ovarian cancer.

BACKGROUND: Cyclic guanosine monophosphate (cGMP)-dependent protein kinase I (PKG-I), a serine/threonine kinase, is important in tumor development. The present study determines that the cGMP/PKG I pathway is essential for promoting cell proliferation and survival in human ovarian cancer cells, whereas cGMP analog has been shown to lead to growth inhibition and apoptosis of various cancer cells. The role of cGMP/PKG I pathway in epithelial ovarian cancer (EOC), therefore, remains controversial. We investigated the effect of cGMP/PKG I pathway and the underlying mechanism in EOC. METHODS AND RESULTS: The results showed that exogenous 8-Bromoguanosine-3', 5'-cyclic monophosphate (8-Br-cGMP) (cGMP analog) could antagonize the effects by EGF, including suppressing proliferation, invasion and migration of EOC cells. In vivo, 8-Br-cGMP hampered the growth of the xenograft tumor. Additionally, the expressions of epidermal growth factor receptor (EGFR), matrix metallopeptidase 9 (MMP9), proliferating cell nuclear antigen and Ki67 in xenograft tumor were decreased after 8-Br-cGMP intervention. Further research demonstrated that 8-Br-cGMP decreased the phosphorylation of EGFR (Y992) and downstream proteins phospholipase Cγ1 (PLC γ1) (Y783), calmodulin kinase II (T286) and inhibited cytoplasmic Ca2+ release as well as PKC transferring to cell membrane. It's worth noting that the inhibition was 8-Br-cGMP dose-dependent and 8-Br-cGMP showed similar inhibitory effect on EOC cells compared with U-73122, a specific inhibitor of PLC γ1. CONCLUSIONS: The activation of endogenous PKG I by addition of exogenous 8-Br-cGMP could inhibit EOC development probably via EGFR/PLCγ1 signaling pathway. 8-Br-cGMP/PKG I provide a new insight and strategy for EOC treatment.

Phospholipase C Zeta in Human Spermatozoa: A Systematic Review on Current Development and Clinical Application.

During fertilization, the fusion of the spermatozoa with the oocytes causes the release of calcium from the oocyte endoplasmatic reticulum. This, in turn, triggers a series of calcium ion (Ca2+) oscillations, a process known as oocyte activation. The sperm-specific factor responsible for oocyte activation is phospholipase C zeta (PLCζ). Men undergoing intracytoplasmic sperm injection (ICSI) with their spermatozoa lacking PLCζ are incapable of generating Ca2+ oscillation, leading to fertilization failure. The immunofluorescence assay is the most used technique to assess the expression and localization of PLCζ and to diagnose patients with reduced/absent ability to activate the oocytes. In these patients, the use of assisted oocyte activation (AOA) technique can help to yield successful ICSI results and shorten the time of pregnancy. However, the production of a stable PLCζ recombinant protein represents a new powerful therapeutic approach to treating individuals with this condition. We aim to conduct a systematic review focusing on the expression, level, and localization of PLCζ, discussing the novel genetic mutation associated with its impairment. In addition, we highlight the benefits of AOA, looking at new and less invasive methods to diagnose and treat cases with PLCζ dysfunction.

The hypermorphic PLCγ2 S707Y variant dysregulates microglial cell function - Insight into PLCγ2 activation in brain health and disease, and opportunities for therapeutic modulation.

Phospholipase C-gamma 2 (PLCγ2) is highly expressed in hematopoietic and immune cells, where it is a key signalling node enabling diverse cellular functions. Within the periphery, gain-of-function (GOF) PLCγ2 variants, such as the strongly hypermorphic S707Y, cause severe immune dysregulation. The milder hypermorphic mutation PLCγ2 P522R increases longevity and confers protection in central nervous system (CNS) neurodegenerative disorders, implicating PLCγ2 as a novel therapeutic target for treating these CNS indications. Currently, nothing is known about what consequences strong PLCγ2 GOF has on CNS functionality, and more precisely on the specific biological functions of microglia. Using the PLCγ2 S707Y variant as a model of chronic activation we investigated the functional consequences of strong PLCγ2 GOF on human microglia. PLCγ2 S707Y expressing human inducible pluripotent stem cells (hiPSC)-derived microglia exhibited hypermorphic enzymatic activity under both basal and stimulated conditions, compared to PLCγ2 wild type. Despite the increase in PLCγ2 enzymatic activity, the PLCγ2 S707Y hiPSC-derived microglia display diminished functionality for key microglial processes including phagocytosis and cytokine secretion upon inflammatory challenge. RNA sequencing revealed a downregulation of genes related to innate immunity and response, providing molecular support for the phenotype observed. Our data suggests that chronic activation of PLCγ2 elicits a detrimental phenotype that is contributing to unfavourable CNS functions, and informs on the therapeutic window for targeting PLCγ2 in the CNS. Drug candidates targeting PLCγ2 will need to precisely mimic the effects of the PLCγ2 P522R variant on microglial function, but not those of the PLCγ2 S707Y variant.

Sperm-Induced Ca2+ Release in Mammalian Eggs: The Roles of PLCζ, InsP3, and ATP.

Mammalian egg activation at fertilization is triggered by a long-lasting series of increases in cytosolic Ca2+ concentration. These Ca2+ oscillations are due to the production of InsP3 within the egg and the subsequent release of Ca2+ from the endoplasmic reticulum into the cytosol. The generation of InsP3 is initiated by the diffusion of sperm-specific phospholipase Czeta1 (PLCζ) into the egg after gamete fusion. PLCζ enables a positive feedback loop of InsP3 production and Ca2+ release which then stimulates further InsP3 production. Most cytosolic Ca2+ increases in eggs at fertilization involve a fast Ca2+ wave; however, due to the limited diffusion of InsP3, this means that InsP3 must be generated from an intracellular source rather than at the plasma membrane. All mammalian eggs studied generated Ca2+ oscillations in response to PLCζ, but the sensitivity of eggs to PLCζ and to some other stimuli varies between species. This is illustrated by the finding that incubation in Sr2+ medium stimulates Ca2+ oscillations in mouse and rat eggs but not eggs from other mammalian species. This difference appears to be due to the sensitivity of the type 1 InsP3 receptor (IP3R1). I suggest that ATP production from mitochondria modulates the sensitivity of the IP3R1 in a manner that could account for the differential sensitivity of eggs to stimuli that generate Ca2+ oscillations.

Novel ACTL7A variants in males lead to fertilization failure and male infertility.

BACKGROUND: Total fertilization failure occurs in 1%-3% of all intracytoplasmic sperm injection cycles. Genetic defects are found to be crucial causes responsible for total fertilization failure after intracytoplasmic sperm injection. However, the reported genes only elucidate a small proportion of total fertilization failure cases, and more genetic defects are required to be explored. OBJECTIVE: To investigate the genetic causes of male-related fertilization failure and explore the potential underlying mechanism. MATERIAL AND METHODS: Whole-exome sequencing was performed on male patients suffering from fertilization failure, and Sanger sequencing was used to confirm the detected mutations. The effects of genetic mutations on protein function were analyzed using bioinformatic tools and immunofluorescence assays. RESULTS: Two males with ACTL7A mutations were enrolled. One carried two compound heterozygous mutations (c.1118G>A:p.R373H; c.1204G>A:p.G402S), the other had a homozygous mutation (c.1117C>T:p.R373C) and was from a consanguineous family with a recessive inheritance pattern. All the variants were located in the actin domain and were predicted to be pathogenic, affecting the number of hydrogen bonds or the arrangement of nearby protein structures. Furthermore, the protein expression of actin-like protein 7A was absent in affected spermatozoa by using immunofluorescence staining and western blotting, confirming the pathogenicity of the variants. In addition, the phospholipase C zeta 1 was barely absent, and acrosome peanut agglutinin signals were attenuated and unevenly distributed, indicating acrosome dysfunction. In addition, intracytoplasmic sperm injection with artificial oocyte activation treatment could increase the fertilization rate in oocytes injected with affected spermatozoa. DISCUSSION: Our study identified three ACTL7A pathogenic missense mutations in two males with fertilization failure. It expands the mutational and phenotypic spectrum of ACTL7A gene and provides information on the pathogenesis and therapeutic strategies of fertilization defects induced by ACTL7A pathogenic variants. CONCLUSION: ACTL7A variants affected the expression and localization of actin-like protein 7A in the affected spermatozoa and subsequently decreased the expression of phospholipase C zeta 1, which caused fertilization failure and male infertility.

Biomolecular Condensation of SH2 Domain-Containing Proteins on Membranes.

The plasma membrane serves as an effective platform for signal transduction of membrane receptor pathways. Activation of the T-cell receptor (TCR) triggers the formation of membrane-associated condensates that are formed through liquid-liquid phase separation. These condensates are assembled by multivalent interactions between the tyrosine-phosphorylated receptor/adaptor and the SH2 domain-containing protein at membrane-proximal milieu. Here, we describe a biochemical reconstitution system that has been implemented to decipher the mechanisms of phospholipase PLCγ1-mediated LAT condensate formation. To characterize the interaction between specific phosphotyrosine-SH2 pair, we developed a total internal reflection fluorescence (TIRF) microscopy-based system to quantify the binding preference of each SH2 domain to specific tyrosine in the context of membranes. An assay to determine the condensate-mediated protection of phosphotyrosines from being dephosphorylated by phosphatase is also elaborated. These assays could be applied to study other transmembrane receptor pathway as well as condensates formed on endomembrane systems including the endoplasmic reticulum, mitochondrion, and Golgi apparatus.

Vascular endothelial growth factor B regulates insulin secretion in ß cells of type 2 diabetes mellitus mice via PLCγ and the IP3R­evoked Ca2+/CaMK2 signaling pathway.

Vascular endothelial growth factor B (VEGFB) plays a crucial role in glucolipid metabolism and is highly associated with type 2 diabetes mellitus (T2DM). The role of VEGFB in the insulin secretion of ß cells remains unverified. Thus, the present study aimed to discuss the effect of VEGFB on regulating insulin secretion in T2DM development, and its underlying mechanism. A high­fat diet and streptozocin (STZ) were used for inducing T2DM in mice model, and VEGFB gene in islet cells of T2DM mice was knocked out by CRISPR Cas9 and overexpressed by adeno­Associated Virus (AAV) injection. The effect of VEGFB and its underlying mechanism was assessed by light microscopy, electron microscopy and fluorescence confocal microscopy, enzyme­linked immunosorbent assay, mass spectrometer and western blot analysis. The decrement of insulin secretion in islet ß cell of T2DM mice were aggravated and blood glucose remained at a high level after VEGFB knockout (KO). However, glucose tolerance and insulin sensitivity of T2DM mice were improved after the AAV­VEGFB186 injection. VEGFB KO or overexpression can inhibit or activate PLCγ/IP3R in a VEGFR1­dependent manner. Then, the change of PLCγ/IP3R caused by VEGFB/VEGFR1 will alter the expression of key factors on the Ca2+/CaMK2 signaling pathway such as PPP3CA. Moreover, VEGFB can cause altered insulin secretion by changing the calcium concentration in ß cells of T2DM mice. These findings indicated that VEGFB activated the Ca2+/CaMK2 pathway via VEGFR1­PLCγ and IP3R pathway to regulate insulin secretion, which provides new insight into the regulatory mechanism of abnormal insulin secretion in T2DM.

Myeloid-specific knockout of SHP2 regulates PI3K/PLCγ signaling pathway to protect against early myocardial infarction injury.

OBJECTIVES: To study the effects of myeloid-specific knockout of SHP2 on early myocardial infarction and explore its molecular mechanism. METHODS: The model of myocardial infarction was established by using SHP2 in myeloid-specific knockout mice, and the effect of SHP2MAC-KO on myocardial function was detected by echocardiography. The effects of SHP2 on myocardial infarct size in myeloid-specific knockout mice was examined by TTC assay and Masson staining. Then, the detection of apoptosis was performed using TUNEL staining and inflammatory cell infiltration was observed using immunohistochemical staining. Moreover, macrophages in mouse hearts were selected by Flow Cytometry and treated with PI3K inhibitors respectively. Western blotting was then used to detect protein expression of p-SHP2 and PI3K/PLCγ signaling pathway. The phagocytic ability of cells was detected by endocytosis test, and the expression of inflammatory cytokines was detected by ELISA. RESULTS: Specific knockout of SHP2 in mice with myocardial infarction can improve the cardiac function, decrease infarct size, and reduce apoptosis as well as inflammatory cell infiltration. It also can mediate the PI3K/PLCγ signaling pathway in macrophages, which in turn enhances the endocytosis of macrophages and reduces the expression of inflammatory cytokines in macrophages. CONCLUSIONS: Myeloid-specific knockout of SHP2 regulates PI3K/PLCγ signaling pathway to protect against early myocardial infarction injury.

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIß activation in gastrointestinal stromal tumor cells.

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase gene, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes Golgi retention of KIT. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT cannot trigger downstream activation. In the Golgi/trans-Golgi network (TGN), KIT activates the PKD2-phosphatidylinositol 4-kinase IIIß (PKD2-PI4KIIIß) pathway through phospholipase Cγ2 (PLCγ2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in the release of KIT from the Golgi/TGN. Our findings show the molecular mechanisms underlying KIT mislocalization and provide evidence for a strategy for inhibition of oncogenic signaling.

Protaetia brevitarsis Extract Attenuates RANKL-Induced Osteoclastogenesis by Inhibiting the JNK/NF-κB/PLCγ2 Signaling Pathway.

Protaetia brevitarsis (PB)-derived bioactive substances have been used as food and medicine in many Asian countries because of their antioxidant, antidiabetic, anti-cancer, and hepatoprotective properties. However, the effect of PB extracts (PBE) on osteoclast differentiation is unclear. In this study, we investigated the effect of PBE on RANKL-induced osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs). To investigate the cytotoxicity of PBE, the viability of BMMs was confirmed via MTT assay. Tartrate-resistant acid phosphatase (TRAP) staining and pit assays were performed to confirm the inhibitory effect of PBE on osteoclast differentiation and bone resorption. The expression levels of osteoclast differentiation-related genes and proteins were evaluated using quantitative real-time PCR and Western blotting. PBE attenuated osteoclastogenesis in BMMs in TRAP and pit assays without cytotoxicity. The expression levels of osteoclast marker genes and proteins induced by RANKL were decreased after PBE treatment. PBE suppressed osteoclastogenesis by inhibiting the RANKL-induced activated JNK/NF-κB/PLCγ2 signaling pathway and the expression of NFATc1 and c-Fos. Collectively, these results suggest that PBE could be a potential therapeutic strategy or functional product for osteoclast-related bone disease.

Co-Stimulation of AGEs and LPS Induces Inflammatory Mediators through PLCγ1/JNK/NF-κB Pathway in MC3T3-E1 Cells.

Advanced glycation end-products (AGEs) are increased under hyperglycemia in vivo and are associated with the onset of diabetes. According to previous studies, AGEs exacerbate inflammatory diseases. However, the mechanism by which AGEs aggravate osteoblast inflammation remains unknown. Therefore, the aim of this study was to determine the effects of AGEs on the production of inflammatory mediators in MC3T3-E1 cells and the underlying molecular mechanisms. Co-stimulation with AGEs and lipopolysaccharide (LPS) was found to increase the mRNA and protein levels of cyclooxygenase 2 (COX2), interleukin-1α (IL-1α), S100 calcium-binding protein A9 (S100A9), and the production of prostaglandin E2 (PGE2) compared to no stimulation (untreated control) or individual stimulation with LPS or AGEs. In contrast, the phospholipase C (PLC) inhibitor, U73122, inhibited these stimulatory effects. Co-stimulation with AGEs and LPS also increased the nuclear translocation of nuclear factor-kappa B (NF-κB) compared to no stimulation (untreated control) or individual stimulation with LPS or AGE. However, this increase was inhibited by U73122. Co-stimulation with AGEs and LPS-induced phosphorylated phospholipase Cγ1 (p-PLCγ1) and phosphorylated c-Jun N-terminal kinase (p-JNK) expression compared to no stimulation or individual stimulation with LPS or AGEs. U73122 inhibited the effects induced by co-stimulation. siPLCγ1 did not increase the expression of p-JNK and the translocation of NF-κB. Overall, co-stimulation with AGEs and LPS may promote inflammation mediators in MC3T3-E1 cells by activating the nuclear translocation of NF-κB via PLCγ1-JNK activation.

Citropten Ameliorates Osteoclastogenesis Related to MAPK and PLCγ/Ca2+ Signaling Pathways through the Regulation of Amyloid Beta.

Osteoporosis and Alzheimer's disease are typical types of dementia in seniors, which share common risk factors. Previous studies have shown that citizens with osteoporosis are more likely than healthy individuals to be at risk of Alzheimer's disease. Citropten, found in Citrus aurantifolia, has been reported to have several pharmacological activities; however, its antiosteoclastogenic activity remains unknown. Here, receptor activator nuclear factor κB ligand (RANKL)-induced osteoclast differentiation, formation, and function in the presence of amyloid beta (Aß) were attenuated by citropten in the RAW 264.7 cell line. The expression of osteoclast specific genes and proteins indicated that citropten pretreatment lowers the MAPK and PLCγ/Ca2+ signaling pathways. Molecular docking simulations revealed that citropten interacts with the active sites of proteins in the calcium signaling pathway, which have negative binding affinities. These findings indicate that, through Aß regulation, the RANKL-induced osteoclast can be suppressed by citropten, suggesting that citropten is a potential candidate for treating osteoclastogenesis-related diseases.

A VEGFB-Based Peptidomimetic Inhibits VEGFR2-Mediated PI3K/Akt/mTOR and PLCγ/ERK Signaling and Elicits Apoptotic, Antiangiogenic, and Antitumor Activities.

Vascular endothelial growth factor receptor 2 (VEGFR2) mediates VEGFA signaling mainly through the PI3K/AKT/mTOR and PLCγ/ERK1/2 pathways. Here we unveil a peptidomimetic (VGB3) based on the interaction between VEGFB and VEGFR1 that unexpectedly binds and neutralizes VEGFR2. Investigation of the cyclic and linear structures of VGB3 (named C-VGB3 and L-VGB3, respectively) using receptor binding and cell proliferation assays, molecular docking, and evaluation of antiangiogenic and antitumor activities in the 4T1 mouse mammary carcinoma tumor (MCT) model showed that loop formation is essential for peptide functionality. C-VGB3 inhibited proliferation and tubulogenesis of human umbilical vein endothelial cells (HUVECs), accounting for the abrogation of VEGFR2, p-VEGFR2 and, subsequently, PI3K/AKT/mTOR and PLCγ/ERK1/2 pathways. In 4T1 MCT cells, C-VGB3 inhibited cell proliferation, VEGFR2 expression and phosphorylation, the PI3K/AKT/mTOR pathway, FAK/Paxillin, and the epithelial-to-mesenchymal transition cascade. The apoptotic effects of C-VGB3 on HUVE and 4T1 MCT cells were inferred from annexin-PI and TUNEL staining and activation of P53, caspase-3, caspase-7, and PARP1, which mechanistically occurred through the intrinsic pathway mediated by Bcl2 family members, cytochrome c, Apaf-1 and caspase-9, and extrinsic pathway via death receptors and caspase-8. These data indicate that binding regions shared by VEGF family members may be important in developing novel pan-VEGFR inhibitors that are highly relevant in the pathogenesis of angiogenesis-related diseases.