Phosphoinositide-specific Phospholipase Research Articles

From Classical to Alternative Pathways of 2-Arachidonoylglycerol Synthesis: AlterAGs at the Crossroad of Endocannabinoid and Lysophospholipid Signaling.

2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid (EC), acting as a full agonist at both CB1 and CB2 cannabinoid receptors. It is synthesized on demand in postsynaptic membranes through the sequential action of phosphoinositide-specific phospholipase Cβ1 (PLCβ1) and diacylglycerol lipase α (DAGLα), contributing to retrograde signaling upon interaction with presynaptic CB1. However, 2-AG production might also involve various combinations of PLC and DAGL isoforms, as well as additional intracellular pathways implying other enzymes and substrates. Three other alternative pathways of 2-AG synthesis rest on the extracellular cleavage of 2-arachidonoyl-lysophospholipids by three different hydrolases: glycerophosphodiesterase 3 (GDE3), lipid phosphate phosphatases (LPPs), and two members of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP6-7). We propose the names of AlterAG-1, -2, and -3 for three pathways sharing an ectocellular localization, allowing them to convert extracellular lysophospholipid mediators into 2-AG, thus inducing typical signaling switches between various G-protein-coupled receptors (GPCRs). This implies the critical importance of the regioisomerism of both lysophospholipid (LPLs) and 2-AG, which is the object of deep analysis within this review. The precise functional roles of AlterAGs are still poorly understood and will require gene invalidation approaches, knowing that both 2-AG and its related lysophospholipids are involved in numerous aspects of physiology and pathology, including cancer, inflammation, immune defenses, obesity, bone development, neurodegeneration, or psychiatric disorders.

Intron retention in PI-PLC γ1 mRNA as a key mechanism affecting MMP expression in human primary fibroblast-like synovial cells.

The intron retention (IR) is a phenomenon utilized by cells to allow diverse fates at the same mRNA, leading to a different pattern of synthesis of the same protein. In this study, we analyzed the modulation of phosphoinositide-specific phospholipase C (PI-PLC) enzymes by Harpagophytum procumbensextract (HPE) in synoviocytes from joins of osteoarthritis(OA) patients. In some samples, the PI-PLC γ1 isoform mature mRNA showed the IR and, in these synoviocytes, the HPE treatment increased the phenomenon. Moreover, we highlighted that as a consequence of IR, a lower amount of PI-PLC γ1 was produced. The decrease of PI-PLC γ1 was associated with the decrease of metalloprotease-3(MMP-3),and MMP-13, and ADAMTS-5 after HPE treatment. The altered expression of MMPs is a hallmark of the onset and progression of OA, thus substances able to decrease their expression are very desirable. The interesting outcomes of this study are that 35% of analyzed synovial tissues showed the IR phenomenon in the PI-PLC γ1 mRNAand that the HPE treatment increased this phenomenon. For the first time, we found that the decrease of PI-PLC γ1 protein in synoviocytes interferes withMMP production, thus affecting the pathways involved in the MMP expression. This finding was validated by the silencing of PI-PLC γ1 in synoviocytes where the IR phenomenon was not present. Our results shed new light on the biochemical mechanisms involved in the degrading enzyme production in the joint of OA patients, suggesting a new therapeutic target and highlighting the importance of personalized medicine.

Specific Subcellular Localization of Phosphoinositide-Specific Phospholipase C enzymes in Different Human Osteosarcoma Cell Lines

The role of signal transduction in cancer progression is well established and actively studied, including in osteosarcoma. The signal transduction pathways involved in the regulation of calcium metabolism are being intensively studied, with particular regard to phosphoinositide-specific phospholipase C (PLC) signaling. This family of enzymes helps to modulate calcium metabolism and is interconnected with additional signaling molecules belonging to different pathways. The expression and subcellular localization of PLCs have been shown to differ in normal cells compared to their neoplastic counterpart in different types of cancer. We now describe the localization of the PLC enzyme family in 4 human osteosarcoma cells different in origin and malignancy (MG63, U2OS, HOS and 143B cell lines). We identified cell line-specific differences and discussed possible meaning and implications.

Identification and characterization of genes encoding phosphoinositide-specific phospholipase C revealed role in drought stress condition in cassava (Manihot esculenta)

Phosphoinositide-specific phospholipase C (PI-PLC) has been known as one of the key enzymes that involved in the phospholipid hydrolysis. However, the PI-PLC family in cassava has not been fully recorded. In this study, we reported a comprehensive analysis of the PI-PLC family in cassava assembly based on various bioinformatics tools. Particularly, a total of seven members of the PI-PLC family has been identified and annotated in the cassava genome. By using the full-length protein sequence of each member of the PI-PLC family in cassava, we analyzed the properties of these proteins, including the length, size, iso-electric point, instability index, aliphatic index and grand average of hydropathy. Of our interest, we investigated the expression patterns of genes encoding the PI-PLC family in various major organs/tissues in different conditions. Taken together, our study could provide a solid foundation for the PI-PLC family in cassava for further functional characterization towards the improvements of drought stress tolerance in cassava plants.

A polypeptide derived from pilose antler ameliorates CUMS-induced depression-like behavior by SENP2-PLCβ4 signaling axis

Neurogenesis is known to be closely associated with depression. We aimed to investigate whether a polypeptide monomer derived from pilose antler (polypeptide sequence LSALEGVFYP, PAP) exerts an antidepressant effect by influencing neurogenesis, and to elucidate the mechanism of its antidepressant action. Behavioral tests were performed to observe the antidepressant effect of PAP. Neurogenesis in the dentate gyrus (DG) region of hippocampus was observed by immunofluorescence. The expression of key proteins of Sentrin/SUMO-specific proteases 2 (SENP2)- Phosphoinositide-specific phospholipase C beta 4 (PLCβ4) pathway was accessed by co-immunoprecipitation (Co-IP), and the calcium homeostasis associated proteins were observed via Western blot (WB). Subsequently, temozolomide (TMZ) pharmacologically blocked neurogenesis to verify the antidepressant effect of PAP on neurogenesis. The mechanism of PAP antidepressant effect was verified by constructing a sh-SENP2 virus vector to silence SENP2 protein. Finally, corticosterone (CORT)-induced PC12 cell model was used to verify whether PAP was involved in the process of deconjugated PLCβ4 SUMOylated. The results showed that PAP improved depression-like behavior and neurogenesis induced by chronic unpredictable mild stimulation (CUMS). In addition, PAP acted on SENP2-PLCβ4 pathway to deconjugate the SUMOylation of PLCβ4 and affect calcium homeostasis. Pharmacological blockade of neurogenesis by TMZ treatment impaired the antidepressant efficacy of PAP. Knockout of SENP2 in the CUMS model attenuated the antidepressant response of PAP, and the impaired neurogenesis was not ameliorated by PAP treatment. In summary, PAP acted on the SENP2-PLCβ4 signaling pathway to inhibit the SUMOylation of PLCβ4 and maintain calcium homeostasis, thereby protecting neurogenesis and playing an antidepressant role.

Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection

Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG-CAP. Chickens immunized with rEnSAG-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.

Global analysis of putative phospholipases in Plasmodium falciparum reveals an essential role of the phosphoinositide-specific phospholipase C in parasite maturation.

For its replication within red blood cells, the malaria parasite depends on a highly active and regulated lipid metabolism. Enzymes involved in lipid metabolic processes such as phospholipases are, therefore, potential drug targets. Here, using reverse genetics approaches, we show that only 1 out of the 19 putative phospholipases expressed in asexual blood stages of Plasmodium falciparum is essential for proliferation in vitro, pointing toward a high level of redundancy among members of this enzyme family. Using conditional mislocalization and gene disruption techniques, we show that this essential phosphoinositide-specific phospholipase C (PI-PLC, PF3D7_1013500) has a previously unrecognized essential role during intracellular parasite maturation, long before its previously perceived role in parasite egress and invasion. Subsequent lipidomic analysis suggests that PI-PLC mediates cleavage of phosphatidylinositol bisphosphate (PIP2) in schizont-stage parasites, underlining its critical role in regulating phosphoinositide levels in the parasite. IMPORTANCE The clinical symptoms of malaria arise due to repeated rounds of replication of Plasmodium parasites within red blood cells (RBCs). Central to this is an intense period of membrane biogenesis. Generation of membranes not only requires de novo synthesis and acquisition but also the degradation of phospholipids, a function that is performed by phospholipases. In this study, we investigate the essentiality of the 19 putative phospholipase enzymes that the human malaria parasite Plasmodium falciparum expresses during its replication within RBCs. We not only show that a high level of functional redundancy exists among these enzymes but, at the same time, also identify an essential role for the phosphoinositide-specific phospholipase C in parasite development and cleavage of the phospholipid phosphatidylinositol bisphosphate.

Phospholipase Family Enzymes in Lung Cancer: Looking for Novel Therapeutic Approaches.

Lung cancer (LC) is the second most common neoplasm in men and the third most common in women. In the last decade, LC therapies have undergone significant improvements with the advent of immunotherapy. However, the effectiveness of the available treatments remains insufficient due to the presence of therapy-resistant cancer cells. For decades, chemotherapy and radiotherapy have dominated the treatment strategy for LC; however, relapses occur rapidly and result in poor survival. Malignant lung tumors are classified as either small- or non-small-cell lung carcinoma (SCLC and NSCLC). Despite improvements in the treatment of LC in recent decades, the benefits of surgery, radiotherapy, and chemotherapy are limited, although they have improved the prognosis of LC despite the persistent low survival rate due to distant metastasis in the late stage. The identification of novel prognostic molecular markers is crucial to understand the underlying mechanisms of LC initiation and progression. The potential role of phosphatidylinositol in tumor growth and the metastatic process has recently been suggested by some researchers. Phosphatidylinositols are lipid molecules and key players in the inositol signaling pathway that have a pivotal role in cell cycle regulation, proliferation, differentiation, membrane trafficking, and gene expression. In this review, we discuss the current understanding of phosphoinositide-specific phospholipase enzymes and their emerging roles in LC.

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6–19 rams at two very distant points in the mating season (July as Early Breeding Season –EBS– and November as Late Breeding Season –LBS–). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.

Endoplasmic reticulum localization of phosphoinositide specific phospholipase C enzymes in U73122 cultured human osteoblasts

&lt;abstract&gt; &lt;p&gt;Different signal transduction pathways contribute to the differentiation and metabolic activities of osteoblasts, with special regard to the calcium-related pathway of phosphoinositide specific phospholipase C (PLC) enzyme family. PLC enzymes were demonstrated to be involved in the differentiation of osteoblasts and differently localize in the nucleus, cytoplasm or both depending on the isoform. The amino-steroid molecule U-73122 inhibits the enzymes belonging to the PLC family. In addition to the temporary block of the enzymatic activity, U-73122 promotes off-target effects, including modulation of the expression of selected &lt;italic&gt;PLC&lt;/italic&gt; genes and different localization of PLC enzymes, depending on the cell line, in different cell lines.&lt;/p&gt; &lt;p&gt;In order to evaluate possible off-target effects of the molecule in human osteoblasts, we investigated the expression of &lt;italic&gt;PLC&lt;/italic&gt; genes and the localization of PLC enzymes in cultured human osteoblasts (hOBs) in the presence of low dose U-73122.&lt;/p&gt; &lt;p&gt;Our results confirm that all &lt;italic&gt;PLC&lt;/italic&gt; genes are transcribed in hOBs, that probably splicing variants of selected &lt;italic&gt;PLC&lt;/italic&gt; genes are expressed and that all PLC enzymes are present in hOBs, except for PLC δ3 in quiescent hOBs at seeding. Our results confirm literature data excluding toxicity of U-73122 on cell survival. Our results indicate that U-73122 did not significantly affect the transcription of &lt;italic&gt;PLC&lt;/italic&gt; genes. It acts upon the localization of PLC enzymes, as PLC enzymes are detected in cell protrusions or pseudopodia-like structures, at the nuclear or the plasma membrane, in membrane ruffles and/or in the endoplasmic reticulum.&lt;/p&gt; &lt;/abstract&gt;

The phosphoinositide-specific phospholipase C1 modulates flowering time and grain size in rice

Preferential expression of OsPLC1 is detected at the heading stage of rice, OsPLC1 overexpression results in early flowering, increased-grain size and yield; however, opposing phenotypes produced in the osplc1 mutants. Abstract: The importance of phospholipase C (PLC) in plant development has been demonstrated in several studies. OsPLC1, a member of PI-PLC in rice, although its role in the response to salt stress of rice seedlings has been reported, its functions in the growth and development of rice is elusive. Here, we report that OsPLC1 expression could be detectable in various tissues throughout the developmental stages of rice, and the highest expression level of OsPLC1 was detected at the heading stage. OsPLC1 overexpression (OE) produced rice plants with early flowering, whereas OsPLC1 loss-of-function led to delay in flowering. The expression levels of subset genes, which are involved in the control of flowering time in rice, were altered in the plants of OE and osplc1. In addition, the enlargement of grain size was observed in OE plants, however, the reduction of grain size was noticed in osplc1 mutants. The increase in the grain size and the grain yield of OE lines were associated with the improvement of cell length and expression levels of a set of genes related to cell expansion, contrarily, the decrease in osplc1 mutant grain size and yield were linked to declined cell length and expression levels of related genes. No significant differences, in terms of the grain quality of mature seeds, were found in OE and osplc1 mutants, with compared to those in Nipponbare (Nip). In summary, our study suggests that OsPLC1 could modulate rice flowering time and grain size.

Role of phospholipase C in catecholamine-induced increase in myocardial protein synthesis

The activation of the α1-adrenoceptor-(α1-AR) by norepinephrine results in the G-protein (Gqα) mediated increase in the phosphoinositide-specific phospholipase C (PLC) activity. The byproducts of PLC hydrolytic activity, namely, 1,2-diacylglycerol and inositol-1,4,5-trisphosphate, are important downstream signal transducers for increased protein synthesis in the cardiomyocyte and the subsequent hypertrophic response. In this article, evidence was outlined to demonstrate the role of cardiomyocyte PLC isozymes in the catecholamine-induced increase in protein synthesis by using a blocker of α1-AR and an inhibitor of PLC. The discussion was focused on the α1-AR-Gqα-PLC-mediated hypertrophic signalling pathway from the viewpoint that it may compliment the other β1-AR-Gs protein-adenylyl cyclase signal transduction mechanisms in the early stages of cardiac hypertrophy development, but may become more relevant at the late stage of cardiac hypertrophy. From the information provided here, it is suggested that some specific PLC isozymes may potentially serve as important targets for the attenuation of cardiac hypertrophy in the vulnerable patient population at-risk for heart failure.

32Pi Labeled Transgenic Wheat Shows the Accumulation of Phosphatidylinositol 4,5-bisphosphate and Phosphatidic Acid Under Heat and Osmotic Stress.

The ensuing heat stress drastically affects wheat plant growth and development, consequently compromising its grain yield. There are many thermoregulatory processes/mechanisms mediated by ion channels, lipids, and lipid-modifying enzymes that occur in the plasma membrane and the chloroplast. With the onset of abiotic or biotic stresses, phosphoinositide-specific phospholipase C (PI-PLC), as a signaling enzyme, hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which is further phosphorylated into phosphatidic acid (PA) as a secondary messenger and is involved in multiple processes. In the current study, a phospholipase C (PLC) signaling pathway was investigated in spring wheat (Triticum aestivum L.) and evaluated its four AtPLC5 overexpressed (OE)/transgenic lines under heat and osmotic stresses through 32Pi radioactive labeling. Naturally, the wheat harbors only a small amount of PIP2. However, with the sudden increase in temperature (40°C), PIP2 levels start to rise within 7.5 min in a time-dependent manner in wild-type (Wt) wheat. While the Phosphatidic acid (PA) level also elevated up to 1.6-fold upon exposing wild-type wheat to heat stress (40°C). However, at the anthesis stage, a significant increase of ∼4.5-folds in PIP2 level was observed within 30 min at 40°C in AtPLC5 over-expressed wheat lines. Significant differences in PIP2 level were observed in Wt and AtPLC5-OE lines when treated with 1200 mM sorbitol solution. It is assumed that the phenomenon might be a result of the activation of PLC/DGK pathways. Together, these results indicate that heat stress and osmotic stress activate several lipid responses in wild-type and transgenic wheat and can explain heat and osmotic stress tolerance in the wheat plant.

Expression and Localization of Phosphoinositide-Specific Phospholipases C in Cultured, Differentiating and Stimulated Human Osteoblasts

Casoni SD, Romanelli A, Checchi M, Truocchio S, Ferretti M, Palumbo C, et al. Expression and Localization of Phosphoinositide-Specific Phospholipases C in Cultured, Differentiating and Stimulated Human Osteoblasts. J Cell Signal. 2022;3(1):44-61.

High Resolution Proteomic Analysis of Subcellular Fractionated Boar Spermatozoa Provides Comprehensive Insights Into Perinuclear Theca-Residing Proteins.

The perinuclear theca (PT) is a highly condensed, largely insoluble protein structure that surrounds the nucleus of eutherian spermatozoa. Recent reports have indicated that the PT unexpectedly houses several somatic proteins, such as core histones, which may be important post-fertilization during re-modelling of the male pronucleus, yet little is known regarding the overall proteomic composition of the PT. Here, we report the first in depth, label-free proteomic characterization of the PT of boar spermatozoa following the implementation of a long-established subcellular fractionation protocol designed to increase the detection of low abundance proteins. A total of 1,802 proteins were identified, a result that represents unparalleled depth of coverage for the boar sperm proteome and exceeds the entire annotated proteome of the Sus scrofa species so far. In the PT structure itself, we identified 813 proteins and confirmed the presence of previously characterized PT proteins including the core histones H2A, H2B, H3 and H4, as well as Ras-related protein Rab-2A (RAB2A) and Rab-2B (RAB2B) amongst other RAB proteins. In addition to these previously characterized PT proteins, our data revealed that the PT is replete in proteins critical for sperm-egg fusion and egg activation, including: Izumo family members 1–4 (IZUMO1-4) and phosphoinositide specific phospholipase ζ (PLCZ1). Through Ingenuity Pathway Analysis, we found surprising enrichment of endoplasmic reticulum (ER) proteins and the ER-stress response in the PT. This is particularly intriguing as it is currently held that the ER structure is lost during testicular sperm maturation. Using the String and Cytoscape tools to visualize protein-protein interactions revealed an intricate network of PT protein complexes, including numerous proteasome subunits. Collectively, these data suggest that the PT may be a unique site of cellular homeostasis that houses an abundance of protein degradation machinery. This fits with previous observations that the PT structure dissociates first within the oocyte post-fertilization. It remains to be explored whether proteasome subunits within the PT actively assist in the protein degradation of paternal cell structures post-fertilization and how aberrations in PT protein content may delay embryonic development.

Role of PLCγ1 in the modulation of cell migration and cell invasion in glioblastoma.

Phosphoinositide-specific phospholipases C (PLCs) are a class of enzymes involved in several cell activities, such as cell cycle regulation, proliferation, differentiation and cytoskeletal dynamics. Among these enzymes, PLCγ1 is one of the most expressed PLCs in the brain, contributing to a complex network in the developing nervous system. Several studies have shown that PLCγ1 signaling imbalance is linked to several brain disorders, including glioblastoma, the most aggressive brain tumor in adults. Indeed, it has been demonstrated a link between PLCγ1 inhibition and the arrest of glioma cell motility of fetal rat brain aggregates and the impairment of cell invasion abilities following its down-regulation. This study aims to determine the pathological influence of PLCγ1 in glioblastoma, through a translational study which combines in silico data, data from glioblastoma patients' samples and data on engineered cell lines. We found out that PLCγ1 gene expression correlates with the pathological grade of gliomas, and it is higher in fifty patients' glioblastoma tissue samples compared to twenty healthy controls. Moreover, it was demonstrated that PLCγ1 silencing in U87-MG leads to a reduction in cell migration and invasion abilities. The opposite trend was observed following PLCγ1 overexpression, suggesting an interesting possible involvement of PLCγ1 in gliomas' aggressiveness.

Suppressing phosphoinositide-specific phospholipases Cγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing autophagy, thereby ameliorating articular cartilage degeneration.

Phosphoinositide-specific phospholipases C-γ1 (PLC-γ1) signaling has been shown to modulate osteoarthritis (OA) chondrocyte metabolism. However, the role of PLC-γ1 in OA osteoblasts remains unclear. Herein, whether and how PLC-γ1 was involved in mineralization in OA subchondral bone osteoblasts were investigated. Primary non-OA and OA osteoblasts of human and rat isolated from the subchondral bone or the calvaria were cultured in vitro, as well as mouse pre-osteoblastic cell line MC3T3-E1 cells. Rat knee OA model was induced by anterior cruciate ligament transection (ACLT), in which bone canal was carried out from the surface of lateral epicondyle of femur using micro-electric drill. Morphological characteristics of subchondral bone structure and articular cartilage were assessed using CT, micro-CT, and Safranin O/Fast green staining, respectively. Mineralization was measured by alizarin red staining. The expression and production of genes involved in osteoblastic phenotype and mineralization were evaluated by qPCR, western blotting, and immunohistochemistry assays, respectively. The inhibitions were performed using inhibitors and ShRNAs. The decreased relative bone density and thickness in the early stage of OA and the increased one in the late stage of OA were observed in subchondral bone of ACLT-rat model. Decreased ALP and OCN levels and absorbance values of ARS content were observed in in vitro osteoblasts isolated from 2 w post-ACLT rat model, as well as IL-1β-treated (for maintaining and mimicking inflammatory status) human OA and rat osteoblasts. Decreased Atg7 level and LC3BII/I ratio in combination with an increase in the P62 level, was concomitant with decreased ALP and OCN mRNA levels and absorbance values of ARS content in OA or IL-1β-treated osteoblasts. Specific inhibition of PLC-γ1 by ShRNAs or inhibitor (U73122) elevated ALP and OCN mRNA levels and absorbance values of ARS content accompanied with increased Atg7 level and LC3BII/I ratio in combination with a decrease in the P62 level in OA osteoblasts. Furthermore, the promoting effect of PLC-γ1 inhibition on ALP and OCN mRNA levels and absorbance values of ARS content was reversed by endoplasmic reticulum (ER) stress activator HA15, as well as autophagic inhibitors CQ and 3MA. Injection with PLC-γ1 inhibitor U73122 from the surface of lateral epicondyle of femur reduced aberrant subchondral bone formation and attenuated articular cartilage degeneration in ACLT-rat. Aberrant changes of OA subchondral bone structure were concomitant with altered osteoblastic phenotype and mineralization. Impaired autophagy contributed to decreased osteoblastic mineralization in the early stage of OA. PLC-γ1 inhibition promoted osteoblastic mineralization through increasing autophagy in OA osteoblasts, which was partially attributed to suppression of ER stress. Targeting PLC-γ1 in subchondral bone osteoblasts could be more efficacious for OA therapy through treating the bone and cartilage at the same time. In summary, we hypothesize that suppressing PLCγ1 promotes mineralization of osteoarthritic subchondral bone osteoblasts via increasing autophagy, thereby ameliorating articular cartilage degeneration.

Involvement of phospholipase C in the norepinephrine-induced hypertrophic response in Cardiomyocytes

Norepinephrine (NE) is known to mediate cardiomyocyte hypertrophy through the G protein coupled a1 -adrenoceptor (a1 -AR) and the activation of the phosphoinositide-specific phospholipase C (PLC). Since the by-products of PLC activity are important downstream signal transducers for cardiac hypertrophy, the role of and the regulatory mechanisms involved in the activation of PLC isozymes in cardiac hypertrophy are highlighted in this review. The discussion is focused to underscore PLC in different experimental models of cardiac hypertrophy, as well as in isolated adult and neonatal cardiomyocytes treated with NE. Particular emphasis is laid concerning the a1 -AR-PLC-mediated hypertrophic signalling pathway. From the information provided, it is evident that the specific activation of PLC isozymes is a primary signalling event in the a1 -AR mediated response to NE as well as initiation and progression of cardiac hypertrophy. Furthermore, the possibility of PLC involvement in the perpetuation of cardiac hypertrophy is also described. It is suggested that specific PLC isozymes may serve as viable targets for the prevention of cardiac hypertrophy in patient population at-risk for the development of heart failure.

Prediction of genetic alteration of phospholipase C isozymes in brain disorders: Studies with deep learning.

Genetic mutations leading to the development of various diseases, such as cancer, diabetes, and neurodegenerative disorders, can be attributed to multiple mechanisms and exposure to diverse environments. These disorders further increase gene mutation rates and affect the activity of translated proteins, both phenomena associated with cellular responses. Therefore, maintaining the integrity of genetic and epigenetic information is critical for disease suppression and prevention. With the advent of genome sequencing technologies, large-scale genomic data-based machine learning tools, including deep learning, have been used to predict and identify somatic inactivation or negative dominant expression of target genes in various diseases. Although deep learning studies have recently been highlighted for their ability to distinguish between the genetic information of diseases, conventional wisdom is also necessary to explain the correlation between genotype and phenotype. Herein, we summarize the current understanding of phosphoinositide-specific phospholipase C isozymes (PLCs) and an overview of their associations with genetic variation, as well as their emerging roles in several diseases. We also predicted and discussed new findings of cryptic PLC splice variants by deep learning and the clinical implications of the PLC genetic variations predicted using these tools.

The rice phosphoinositide-specific phospholipase C3 is involved in responses to osmotic stresses via modulating ROS homeostasis

Four members of phosphoinositide-specific phospholipase C (PI-PLC) are predicted in rice genome. Although the involvement of OsPLC1 and OsPLC4 in the responses of rice to salt and drought stresses has been documented, the role of OsPLC3 in which, yet, is elusive. Here, we report that OsPLC3 was ubiquitously expressed in various tissues during the development of rice. The expression of YFP-tagged OsPLC3 was observed at the plasma membrane (PM), cytoplasm and nucleus of rice protoplasts, onion epidermal cells and tobacco leaves. The catalytic activity of OsPLC3 was measured using the thin-layer chromatography (TLC) method. The inhibition of OsPLC3 expression was detected in the treatments of NaCl and mannitol. Overexpression (OE) of OsPLC3 produced plants showing more sensitive to osmotic stresses when they were compared to the wild-type (HJ) and osplc3 mutants, the phenomena such as decreased plant fresh weight and increased water loss rate (WLR) were observed. Under the treatment of NaCl or mannitol, expressions of a subset osmotic stress-related genes were altered, in both OE and osplc3 mutant lines. In addition, the expressions and the enzyme activities of reactive oxygen species (ROS) scavengers were significantly decreased in OE lines, leading to over-accumulation of ROS together with less osmotic adjustment substances including proline, soluble sugars and soluble proteins in OE plants which caused the growth inhibition. Thus, our results suggested that, via modulating ROS homeostasis, OsPLC3 is involved in responses to the osmotic stress in rice.