Fibroblast Growth Factor 23 Stimulates Cardiac Fibroblast Activity through Phospholipase C-Mediated Calcium Signaling

Fibroblast growth factor (FGF)-23 induces hypertrophy and calcium (Ca2+) dysregulation in cardiomyocytes, leading to cardiac arrhythmia and heart failure. However, knowledge regarding the effects of FGF-23 on cardiac fibrogenesis remains limited. This study investigated whether FGF-23 modulates cardiac fibroblast activity and explored its underlying mechanisms. We performed MTS analysis, 5-ethynyl-2’-deoxyuridine assay, and wound-healing assay in cultured human atrial fibroblasts without and with FGF-23 (1, 5 and 25 ng/mL for 48 h) to analyze cell proliferation and migration. We found that FGF-23 (25 ng/mL, but not 1 or 5 ng/mL) increased proliferative and migratory abilities of human atrial fibroblasts. Compared to control cells, FGF-23 (25 ng/mL)-treated fibroblasts had a significantly higher Ca2+ entry and intracellular inositol 1,4,5-trisphosphate (IP3) level (assessed by fura-2 ratiometric Ca2+ imaging and enzyme-linked immunosorbent assay). Western blot analysis showed that FGF-23 (25 ng/mL)-treated cardiac fibroblasts had higher expression levels of calcium release-activated calcium channel protein 1 (Orai1) and transient receptor potential canonical (TRPC) 1 channel, but similar expression levels of α-smooth muscle actin, collagen type IA1, collagen type Ⅲ, stromal interaction molecule 1, TRPC 3, TRPC6 and phosphorylated-calcium/calmodulin-dependent protein kinase II when compared with control fibroblasts. In the presence of ethylene glycol tetra-acetic acid (a free Ca2+ chelator, 1 mM) or U73122 (an inhibitor of phospholipase C, 1 μM), control and FGF-23-treated fibroblasts exhibited similar proliferative and migratory abilities. Moreover, polymerase chain reaction analysis revealed that atrial fibroblasts abundantly expressed FGF receptor 1 but lacked expressions of FGF receptors 2-4. FGF-23 significantly increased the phosphorylation of FGF receptor 1. Treatment with PD166866 (an antagonist of FGF receptor 1, 1 μM) attenuated the effects of FGF-23 on cardiac fibroblast activity. In conclusion, FGF-23 may activate FGF receptor 1 and subsequently phospholipase C/IP3 signaling pathway, leading to an upregulation of Orai1 and/or TRPC1-mediated Ca2+ entry and thus enhancing human atrial fibroblast activity.

Standard binding free energy and membrane desorption mechanism for a phospholipase C

Peripheral membrane proteins (PMPs) bind temporarily to cellular membranes and play important roles in signalling, lipid metabolism and membrane trafficking. Obtaining accurate membrane-PMP affinities using experimental techniques is more challenging than for protein-ligand affinities in aqueous solution. At the theoretical level, calculation of standard protein-membrane binding free energy using molecular dynamics simulations remains a daunting challenge owing to the size of the biological objects at play, the slow lipid diffusion and the large variation in configurational entropy that accompanies the binding process. To overcome these challenges, we used a computational framework relying on a series of potential-of-mean-force (PMF) calculations including a set of geometrical restraints on collective variables. This methodology allowed us to determine the standard binding free energy of a PMP to a phospholipid bilayer using an all-atom force field. Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) was chosen due to its importance as a virulence factor and owing to the host of experimental affinity data available. We computed a standard binding free energy of -8.2±1.4 kcal/mol in reasonable agreement with the reported experimental values (-6.6±0.2 kcal/mol). In light of the 2.3-μs separation PMF calculation, we investigated the mechanism whereby BtPI-PLC disengages from interactions with the lipid bilayer during separation. We describe how a short amphipathic helix engages in transitory interactions to ease the passage of its hydrophobes through the interfacial region upon desorption from the bilayer.

Characterization of the Glehnia littoralis Non-specific Phospholipase C Gene GlNPC3 and Its Involvement in the Salt Stress Response

Glehnia littoralis is a medicinal halophyte that inhabits sandy beaches and has high ecological and commercial value. However, the molecular mechanism of salt adaptation in G. littoralis remains largely unknown. Here, we cloned and identified a non-specific phospholipase C gene (GlNPC3) from G. littoralis, which conferred lipid-mediated signaling during the salt stress response. The expression of GlNPC3 was induced continuously by salt treatment. Overexpression of GlNPC3 in Arabidopsis thaliana increased salt tolerance compared to wild-type (WT) plants. GlNPC3-overexpressing plants had longer roots and higher fresh and dry masses under the salt treatment. The GlNPC3 expression pattern revealed that the gene was expressed in most G. littoralis tissues, particularly in roots. The subcellular localization of GlNPC3 was mainly at the plasma membrane, and partially at the tonoplast. GlNPC3 hydrolyzed common membrane phospholipids, such as phosphotidylserine (PS), phosphoethanolamine (PE), and phosphocholine (PC). In vitro enzymatic assay showed salt-induced total non-specific phospholipase C (NPC) activation in A. thaliana GlNPC3-overexpressing plants. Plant lipid profiling showed a significant change in the membrane-lipid composition of A. thaliana GlNPC3-overexpressing plants compared to WT after the salt treatment. Furthermore, downregulation of GlNPC3 expression by virus-induced gene silencing in G. littoralis reduced the expression levels of some stress-related genes, such as SnRK2, P5SC5, TPC1, and SOS1. Together, these results indicated that GlNPC3 and GlNPC3-mediated membrane lipid change played a positive role in the response of G. littoralis to a saline environment.

Phospholipase C-related catalytically inactive protein acts as a positive regulator for insulin signalling in adipocytes

Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We previously reported that phospholipase C-related but catalytically inactive protein (PRIP) interacts with Akt, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling including insulin-stimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), Akt, and glucose uptake, were impaired in adipocytes from PRIP-knockout (KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell-surface was decreased in PRIP-KO adipocytes. Immunoprecipitation assay showed that PRIP interacted with IR. The reduced cell-surface IR in PRIP-KO adipocytes was comparable with that in WT cells when Rab5 expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which were reported to be involved in the internalisation of IR, was impaired in cells from PRIP-KO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes.

Tethered Aryl Groups Increase the Activity of Anti-Proliferative Thieno[2,3-b]Pyridines by Targeting a Lipophilic Region in the Active Site of PI-PLC

The compounds 2-amino-3-carboxamido-thieno[2,3-b]pyridines have demonstrated excellent anti-proliferative activity against human cancer cell lines, including the triple-negative breast cancer cell line MDA-MB-231. In this study, 81 novel thieno[2,3-b]pyridines were synthesised in four series to further improve their anti-proliferative activity, in particular by targeting an adjacent lipophilic pocket in the putative target enzyme phosphoinositide phospholipase C (PI-PLC). Overall, it was found that appending a propyl-aryl group at C-5 on 2-amino-3-carboxamido-thieno[2,3-b]pyridine resulted in compounds with potent biological activity, exhibiting IC50 values in the nanomolar range. The propyl linker could be an α,β-unsaturated ketone or a saturated propyl ketone, but the highest activity was obtained when allylic alcohols were the tether between thieno[2,3-b]pyridine and the appended aryl group, with compound 21r having IC50 values lower than 50 nM. Compounds with one extra carbon in the tether (i.e., a four-atom chain) were found to be considerably less active. Molecular modelling revealed this propyl tether places the newly introduced aryl ring in an untargeted lipophilic pocket within the active site of the phosphoinositide phospholipase C (PI-PLC) enzyme.

Shedding of Trypanosoma cruzi Surface Molecules That Regulate Host Cell Invasion Involves Phospholipase C and Increases Upon Sterol Depletion

Metacyclic trypomastigote (MT) forms of Trypanosoma cruzi have been shown to release into medium gp82 and gp90, the stage-specific surface molecules that regulate host cell invasion, either in vesicles or in soluble form. Here, we found that during interaction of poorly invasive G strain with the host cell, gp82 and gp90 were released in vesicle-like forms, whereas no such release by highly invasive CL strain was observed. Shedding of vesicles of varying sizes by CL and G strains was visualized by scanning electron microscopy, and the protein profile of conditioned medium (CM) of the two strains was similar, but the content of gp82 and gp90 differed, with both molecules being detected in G strain as bands of high intensity in Western blotting, whereas in CL strain, they were barely detectable. Confocal images revealed a distinct distribution of gp82 and gp90 on MT surface of CL and G strains. In cell invasion assays, addition of G strain CM resulted in decreased CL strain internalization. Depletion of gp82 in G strain CM, by treatment with specific mAb-coupled magnetic beads, increased its inhibitory effect on CL strain invasion, in contrast to CM depleted in gp90. The effect of cholesterol-depleting drug methyl-β-cyclodextrin (MβCD) on gp82 and gp90 release by MTs was also examined. G strain MTs, untreated or treated with MβCD, were incubated in serum-containing medium or in nutrient-depleted PBS++, and the CM generated under these conditions was analyzed by Western blotting. In PBS++, gp82 and gp90 were released at lower levels by untreated MTs, as compared with MβCD-treated parasites. CM from untreated and MβCD-treated G strain, generated in PBS++, inhibited CL strain internalization. Treatment of CL strain MTs with MβCD resulted in increased gp82 and gp90 shedding and in decreased host cell invasion. The involvement of phospholipase C (PLC) on gp82 and gp90 shedding was also investigated. The CM from G strain MTs pretreated with specific PLC inhibitor contained lower levels of gp82 and gp90, as compared with untreated parasites. Our results contribute to shed light on the mechanism by which T. cruzi releases surface molecules implicated in host cell invasion.