Spiraeoside inhibits mast cells activation and IgE-mediated allergic responses by suppressing phospholipase C-γ-mediated signaling.

Mast cells are responsible for IgE-mediated allergic responses through the secretion of various inflammatory cytokines and mediators. Therefore, the pharmacological regulation of mast cell activation is an important goal in the development of novel anti-allergic drugs. In this study, we found that spiraeoside (SP) inhibits mast cell activation and allergic responses in vivo. SP dose-dependently inhibited the degranulation induced by IgE-antigen (Ag) stimulation in RBL-2H3 mast cells without cytotoxic effects. At the molecular level, SP reduced the Ag-induced phosphorylation and subsequent activation of phospholipase C-γ2 (PLC-γ2). Moreover, SP inhibited the phosphorylation of spleen tyrosine kinase (Syk), linker for activation of T cells (LAT), and downstream MAPKs, such as ERK1/2, p38, and JNK, eventually attenuating expression of TNF-α and IL-4. Finally, we found that SP significantly inhibited IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Taken together, our results strongly suggest that SP suppresses IgE-mediated mast cell activation and allergic responses by inhibiting Lyn-induced PLC-γ2/MAPK signaling in mast cells.

A male mouse model for metabolic dysfunction-associated steatotic liver disease and hepatocellular carcinoma.

The lack of an appropriate preclinical model of metabolic dysfunction-associated steatotic liver disease (MASLD) that recapitulates the whole disease spectrum impedes exploration of disease pathophysiology and the development of effective treatment strategies. Here, we develop a mouse model (Streptozotocin with high-fat diet, STZ + HFD) that gradually develops fatty liver, metabolic dysfunction-associated steatohepatitis (MASH), hepatic fibrosis, and hepatocellular carcinoma (HCC) in the context of metabolic dysfunction. The hepatic transcriptomic features of STZ + HFD mice closely reflect those of patients with obesity accompanying type 2 diabetes mellitus, MASH, and MASLD-related HCC. Dietary changes and tirzepatide administration alleviate MASH, hepatic fibrosis, and hepatic tumorigenesis in STZ + HFD mice. In conclusion, a murine model recapitulating the main histopathologic, transcriptomic, and metabolic alterations observed in MASLD patients is successfully established.

PDZ domain-containing 1 (PDZK1) protein regulates phospholipase C-ß3 (PLC-ß3)-specific activation of somatostatin by forming a ternary complex with PLC-ß3 and somatostatin receptors.

Phospholipase C-ß (PLC-ß) is a key molecule in G protein-coupled receptor (GPCR)-mediated signaling. Many studies have shown that the four PLC-ß subtypes have different physiological functions despite their similar structures. Because the PLC-ß subtypes possess different PDZ-binding motifs, they have the potential to interact with different PDZ proteins. In this study, we identified PDZ domain-containing 1 (PDZK1) as a PDZ protein that specifically interacts with PLC-ß3. To elucidate the functional roles of PDZK1, we next screened for potential interacting proteins of PDZK1 and identified the somatostatin receptors (SSTRs) as another protein that interacts with PDZK1. Through these interactions, PDZK1 assembles as a ternary complex with PLC-ß3 and SSTRs. Interestingly, the expression of PDZK1 and PLC-ß3, but not PLC-ß1, markedly potentiated SST-induced PLC activation. However, disruption of the ternary complex inhibited SST-induced PLC activation, which suggests that PDZK1-mediated complex formation is required for the specific activation of PLC-ß3 by SST. Consistent with this observation, the knockdown of PDZK1 or PLC-ß3, but not that of PLC-ß1, significantly inhibited SST-induced intracellular Ca(2+) mobilization, which further attenuated subsequent ERK1/2 phosphorylation. Taken together, our results strongly suggest that the formation of a complex between SSTRs, PDZK1, and PLC-ß3 is essential for the specific activation of PLC-ß3 and the subsequent physiologic responses by SST.

Wedelolactone inhibits adipogenesis through the ERK pathway in human adipose tissue-derived mesenchymal stem cells.

Wedelolactone is an herbal medicine that is used to treat septic shock, hepatitis and venom poisoning. Although in differentiated and cancer cells, wedelolactone has been identified as anti-inflammatory, growth inhibitory, and pro-apoptotic, the effects of wedelolactone on stem cell differentiation remain largely unknown. Here, we report that wedelolactone inhibits the adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Wedelolactone reduced the formation of lipid droplets and the expression of adipogenesis-related proteins, such as CCAAT enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein aP2 (aP2). Wedelolactone mediated this process by sustaining ERK activity. In addition, inhibition of ERK activity with PD98059 resulted in reversion of the wedelolactone-mediated inhibition of adipogenic differentiation. Taken together, these results indicate that wedelolactone inhibits adipogenic differentiation through ERK pathway and suggest a novel inhibitory effect of wedelolactone on adipogenic differentiation in hAMSCs.

Forward-Projection Architecture for Fast Iterative Image Reconstruction in X-ray CT.

Iterative image reconstruction can dramatically improve the image quality in X-ray computed tomography (CT), but the computation involves iterative steps of 3D forward- and back-projection, which impedes routine clinical use. To accelerate forward-projection, we analyze the CT geometry to identify the intrinsic parallelism and data access sequence for a highly parallel hardware architecture. To improve the efficiency of this architecture, we propose a water-filling buffer to remove pipeline stalls, and an out-of-order sectored processing to reduce the off-chip memory access by up to three orders of magnitude. We make a floating-point to fixed-point conversion based on numerical simulations and demonstrate comparable image quality at a much lower implementation cost. As a proof of concept, a 5-stage fully pipelined, 55-way parallel separable-footprint forward-projector is prototyped on a Xilinx Virtex-5 FPGA for a throughput of 925.8 million voxel projections/s at 200 MHz clock frequency, 4.6 times higher than an optimized 16-threaded program running on an 8-core 2.8-GHz CPU. A similar architecture can be applied to back-projection for a complete iterative image reconstruction system. The proposed algorithm and architecture can also be applied to hardware platforms such as graphics processing unit and digital signal processor to achieve significant accelerations.

A novel glucagon analog with an extended half-life, HM15136, normalizes glucose levels in rodent models of congenital hyperinsulinism.

Congenital hyperinsulinism (CHI) is a rare genetic condition characterized by uncontrolled insulin secretion, resulting in hypoglycemia. Although glucagon has lately been regarded as a therapeutic option for CHI, its use is severely hampered by its poor solubility and stability at physiological pH, as well as its short duration of action. To address these constraints, we developed HM15136, a novel long-acting glucagon analog composed of a glucagon analog conjugated to the Fc fragment of human immunoglobulin G4 via a polyethylene glycol linker. In this study, we established that HM15136 was more soluble than natural glucagon (≥ 150 mg/mL vs 0.03 mg/mL). Next, we confirmed that HM15136 activated glucagon receptor in vitro and induced glycogenolysis and gluconeogenesis in rat primary hepatocytes. Pharmacokinetics (PK)/Pharmacodynamics (PD) analysis of HM15136 shows that HM15136 has a markedly longer half-life (36 h vs. < 5 min) and increased bioavailability (90%) compared to native glucagon in mice. Further, HM15136 could effectively reverse acute hypoglycemia induced by insulin challenge, and multiple doses of HM15136 could sustain increased blood glucose levels in CHI rats. In conclusion, our findings indicate that HM15136 promotes sustained elevation of blood glucose, demonstrating the potential for development as a once-weekly therapy for CHI.

Wnt5a stimulates chemotactic migration and chemokine production in human neutrophils.

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (ß-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.

Phospholipase C-γ1 involved in brain disorders.

Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) is an important signaling regulator involved in various cellular processes. In brain, PLC-γ1 is highly expressed and participates in neuronal cell functions mediated by neurotrophins. Consistent with essential roles of PLC-γ1, it is involved in development of brain and synaptic transmission. Significantly, abnormal expression and activation of PLC-γ1 appears in various brain disorders such as epilepsy, depression, Huntington's disease and Alzheimer's disease. Thus, PLC-γ1 has been implicated in brain functions as well as related brain disorders. In this review, we discuss the roles of PLC-γ1 in neuronal functions and its pathological relevance to diverse brain diseases.

Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling.

Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca(2+) signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca(2+) source is enough for PLC-η1 activation. Furthermore, the IP(3) receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca(2+) mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca(2+) mobilization from the ER, and therefore amplifies GPCR-mediated signaling.

Subtype-specific roles of phospholipase C-ß via differential interactions with PDZ domain proteins.

Since we first identified the PLC-ß isozyme, enormous studies have been conducted to investigate the functional roles of this protein (Min et al., 1993; Suh et al.,1988). It is now well-known that the four PLC-ß subtypes are major effector molecules in GPCR-mediated signaling, especially for intracellular Ca2+ signaling. Nonetheless, it is still poorly understood why multiple PLC-ß subtype exist. Most cells express multiple subtypes of PLC-ß in different combinations, and each subtype is involved in somewhat different signaling pathways. Therefore, studying the differential roles of each PLC-ß subtype is a very interesting issue. In this regard, we focus here on PDZ domain proteins which are novel PLC-ß interacting proteins. As scaffolders, PDZ domain proteins recruit various target proteins ranging from membrane receptors to cytoskeletal proteins to assemble highly organized signaling complexes; this can give rise to efficiency and diversity in cellular signaling. Because PLC-ß subtypes have different PDZ-binding motifs, it is possible that they are engaged with different PDZ domain proteins, and in turn participate in distinct physiological responses. To date, several PDZ domain proteins, such as the NHERF family, Shank2, and Par-3, have been reported to selectively interact with certain PLC-ß subtypes and GPCRs. Systematic predictions of potential binding partners also suggests differential binding properties between PLC-ß subtypes. Furthermore, we elucidated parallel signaling processes for multiple PLC-ß subtypes, which still perform distinct functions resulting from differential interactions with PDZ domain proteins within a single cell. Therefore, these results highlight the novel function of PDZ domain proteins as intermediaries in subtype-specific role of PLC-ß in GPCR-mediated signaling. Future studies will focus on the physiological meanings of this signaling complex formation by different PDZ domain proteins and PLC-ß subtypes. It has been observed for a long time that the expression of certain PLC-ß subtype fluctuates during diverse physiological conditions. For example, the expression of PLC-ß1 is selectively increased during myoblast and adipocyte differentiation (Faenza et al., 2004; O'Carroll et al., 2009). Likewise, PLC-ß2 is highly up-regulated during breast cancer progression and plays a critical role in cell migration and mitosis (Bertagnolo et al., 2007). Although PLC-ß3 is selectively down-regulated in neuroendocrine tumors, the expression of PLC-ß1 is increased in small cell lung carcinoma (Stalberg et al., 2003; Strassheim et al., 2000). In our hypothetical model, it is most likely that up- and down regulation of certain PLC-ß subtypes are due to their selective coupling with specific GPCR-mediated signaling, implicated in these pathophysiologic conditions. Therefore, better understanding of selective coupling between PLC-ß subtypes, PDZ domain proteins, and GPCRs will shed light on new prognosis and therapy of diverse diseases, and provide potential targets for drug development.

Ochratoxin A inhibits adipogenesis through the extracellular signal-related kinases-peroxisome proliferator-activated receptor-γ pathway in human adipose tissue-derived mesenchymal stem cells.

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with nephrotoxic, carcinogenic, and apoptotic potential. Although the toxic effects of OTA in various cell types are well characterized, it is not known whether OTA has an effect on stem cell differentiation. In this study, we demonstrate that OTA inhibits adipogenesis in human adipose tissue-derived mesenchymal stem cells, as indicated by decreased accumulation of intracellular lipid droplets. Further, OTA significantly reduces expression of adipocyte-specific markers, including peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (aP2). At the molecular level, OTA phosphorylates PPAR-γ2 through extracellular signal-related kinase activation and inhibits PPAR-γ activity. We also found that treatment with the mitogen-activated protein kinase kinase inhibitor, PD98059, significantly blocked the OTA-induced inhibition of adipogenesis. These results indicate that OTA suppresses adipogenesis in an extracellular signal-related kinase-dependent manner. Taken together, our results suggest a novel effect of OTA on adipocyte differentiation in human adipose tissue-derived mesenchymal stem cells and the possibility that OTA might affect the differentiation of other types of stem cells.

A double point mutation in PCL-gamma1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation.

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.

Subtype-specific role of phospholipase C-beta in bradykinin and LPA signaling through differential binding of different PDZ scaffold proteins.

Among phospholipase C (PLC) isozymes (beta, gamma, delta, epsilon, zeta and eta), PLC-beta plays a key role in G-protein coupled receptor (GPCR)-mediated signaling. PLC-beta subtypes are often overlapped in their distribution, but have unique knock-out phenotypes in organism, suggesting that each subtype may have the different role even within the same type of cells. In this study, we examined the possibility of the differential coupling of each PLC-beta subtype to GPCRs, and explored the molecular mechanism underlying the specificity. Firstly, we found that PLC-beta1 and PLC-beta 3 are activated by bradykinin (BK) or lysophosphatidic acid (LPA), respectively. BK-triggered phosphoinositides hydrolysis and subsequent Ca(2+) mobilization were abolished specifically by PLC-beta1 silencing, whereas LPA-triggered events were by PLC-beta 3 silencing. Secondly, we showed the evidence that PDZ scaffold proteins is a key mediator for the selective coupling between PLC-beta subtype and GPCR. We found PAR-3 mediates physical interaction between PLC-beta1 and BK receptor, while NHERF2 does between PLC-beta 3 and LPA(2) receptor. Consistently, the silencing of PAR-3 or NHERF2 blunted PLC signaling induced by BK or LPA respectively. Taken together, these data suggest that each subtype of PLC-beta is selectively coupled to GPCR via PDZ scaffold proteins in given cell types and plays differential role in the signaling of various GPCRs.