Regulation of myofibroblast activities: Calcium pulls some strings behind the scene

Myofibroblast-induced remodeling of collagenous extracellular matrix is a key component of our body's strategy to rapidly and efficiently repair damaged tissues; thus myofibroblast activity is considered crucial in assuring the mechanical integrity of vital organs and tissues after injury. Typical examples of beneficial myofibroblast activities are scarring after myocardial infarct and repair of damaged connective tissues including dermis, tendon, bone, and cartilage. However, deregulation of myofibroblast contraction causes the tissue deformities that characterize hypertrophic scars as well as organ fibrosis that ultimately leads to heart, lung, liver and kidney failure. The phenotypic features of the myofibroblast, within a spectrum going from the fibroblast to the smooth muscle cell, raise the question as to whether it regulates contraction in a fibroblast- or muscle-like fashion. In this review, we attempt to elucidate this point with a particular focus on the role of calcium signaling. We suggest that calcium plays a central role in myofibroblast biological activity not only in regulating contraction but also in mediating intracellular and extracellular mechanical signals, structurally organizing the contractile actin-myosin cytoskeleton, and establishing lines of intercellular communication.     

Generation of cells that ignore the effects of PIP3 on cytoskeleton

Comment on: Tang M, et al. Mol Biol Cell 2011; 22:437-47.     

Tyrosine-protein kinase inhibition in human erythrocytes by polyphosphoinositides (PIP and PIP2).

In human erythrocytes Ser/Thr- and Tyr-phosphorylations of cytoplasmic domain of band 3 are catalyzed by casein kinase I and Tyr-protein kinase respectively, both distributed between cytosol and membrane structures. The results reported here show that purified cytosolic Tyr-protein kinase activity, assayed on added substrates such as poly(Glu,Tyr)4:1 and isolated chymotryptic fragments of band 3 cytoplasmic domain (cdb3), is potently inhibited by PIP and even more by PIP2. Similar inhibitory effects are displayed by these polyphosphoinositides also on the endogenous Tyr-phosphorylation of band 3, when they are added to the isolated native membranes, thus suggesting their involvement in regulating in-vivo Tyr-phosphorylation of membrane proteins.     

The distribution of aquaporin subtypes (PIP1, PIP2 and gamma-TIP) is tissue dependent in soybean (Glycine max) root nodules

BACKGROUND AND AIMS The inner cortical cells (IC-cells) of legume root nodules have been previously shown to regulate the resistance to nodule O2 diffusion by a rapid contraction/expansion mechanism, which controls the volume of intercellular spaces and their occlusion by a liquid phase. The expression of aquaporins in IC-cells was also found to be involved in this nodule O2 diffusion mechanism. The aim of this study was to compare the expression of plasma membrane intrinsic proteins (PIP) aquaporin isoforms with tonoplast intrinsic protein (gamma-TIP) in both IC-cells and adjacent cell types. METHODS: Using immunogold labelling in ultra-thin sections of Glycine max nodules, the expression of two PIP isoforms was observed and compared with the gamma-TIP pattern. KEY RESULTS: The plasma membrane aquaporins PIP1 and PIP2 were expressed more in IC-cells and endodermis than in pericycle and infected cells. The tonoplast aquaporin gamma-TIP has shown a distribution pattern similar to that of the PIPs. CONCLUSIONS: PIPs and gamma-TIP aquaporins are highly expressed in both plasmalemma and tonoplast of nodule IC-cells. This distribution is consistent with the putative role of water fluxes associated with the regulation of nodule conductance to O2 diffusion and the subsequent ATP-dependent nitrogenase activity. In the endodermis, these aquaporins might also be involved in nutrient transport between the infected zone and vascular traces.     

PIP kinase Igamma is the major PI(4,5)P(2) synthesizing enzyme at the synapse

Disruption of the presynaptically enriched polyphosphoinositide phosphatase synaptojanin 1 leads to an increase of clathrin-coated intermediates and of polymerized actin at endocytic zones of nerve terminals. These changes correlate with elevated levels of PI(4,5)P(2) in neurons. We report that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), a major brain PI(4)P 5-kinase, is concentrated at synapses. Synaptojanin 1 and PIPKIgamma antagonize each other in the recruitment of clathrin coats to lipid membranes. Like synaptojanin 1 and other proteins involved in endocytosis, PIPKIgamma undergoes stimulation-dependent dephosphorylation. These results implicate PIPKIgamma in the synthesis of a PI(4,5)P(2) pool that acts as a positive regulator of clathrin coat recruitment and actin function at the synapse.     

Effect of insulin on the incorporation of3H-inositol into the inositol phospholipids (PI,PIP, PIP2) and Glycosyl-phoshatidylinositols (GPIs) of Tetrahymena pyriformis

The unicellular tetrahymena contains inositol phospholipids (PI, PIP, PIP₂) and GPIs. Treatment with 10^–5M insulin decreases the total³H-inositol incorporation and incorporation into PI. 24 h after 10^–6M insulin treatment there is an elevation of these parameters. Second treatment with 10^–6M insulin doubles and 10^–5M decreases these levels. This means that the effect on phosphoinositide turnover by insulin in Tetrahymena is rather concentration dependent. Inositol incorporation into GPIs is also influenced by insulin.     

Cleavage furrow organization requires PIP(2)-mediated recruitment of anillin

Cell division is achieved by a plasma membrane furrow that must ingress between the segregating chromosomes during anaphase [1-3]. The force that drives furrow ingression is generated by the actomyosin cytoskeleton, which is linked to the membrane by an as yet undefined molecular mechanism. A key component of the membrane furrow is anillin. Upon targeting to the furrow through its pleckstrin homology (PH) domain, anillin acts as a scaffold linking the actomyosin and septin cytoskeletons to maintain furrow stability (reviewed in [4, 5]). We report that the PH domain of anillin interacts with phosphatidylinositol phosphate lipids (PIPs), including PI(4,5)P(2), which is enriched in the furrow. Reduction of cellular PI(4,5)P(2) or mutations in the PH domain of anillin that specifically disrupt the interaction with PI(4,5)P(2), interfere with the localization of anillin to the furrow. Reduced expression of anillin disrupts symmetric furrow ingression that can be restored by targeting ectopically expressed anillin to the furrow using an alternate PI(4,5)P(2) binding module, a condition where the septin cytoskeleton is not recruited to the plasma membrane. These data demonstrate that the anillin PH domain has two functions: targeting anillin to the furrow by binding to PI(4,5)P(2) to maintain furrow organization and recruiting septins to the furrow.     

Domain analysis of cortexillin I: actin-bundling, PIP(2)-binding and the rescue of cytokinesis

Cortexillins are actin-bundling proteins that form a parallel two-stranded coiled-coil rod. Actin-binding domains of the alpha-actinin/spectrin type are located N-terminal to the rod and unique sequence elements are found in the C-terminal region. Domain analysis in vitro revealed that the N-terminal domains are not responsible for the strong actin-filament bundling activity of cortexillin I. The strongest activity resides in the C-terminal region. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) suppresses this bundling activity by binding to a C-terminal nonapeptide sequence. These data define a new PIP(2)-regulated actin-bundling site. In vivo the PIP(2)-binding motif enhances localization of a C-terminal cortexillin I fragment to the cell cortex and improves the rescue of cytokinesis. This motif is not required, however, for translocation to the cleavage furrow. A model is presented proposing that cortexillin translocation is based on a mitotic cycle of polar actin polymerization and midzone depolymerization.     

PIP(2)-PDZ domain binding controls the association of syntenin with the plasma membrane

PDZ proteins organize multiprotein signaling complexes. According to current views, PDZ domains engage in protein-protein interactions. Here we show that the PDZ domains of several proteins bind phosphatidylinositol 4,5-bisphosphate (PIP(2)). High-affinity binding of syntenin to PIP(2)-containing lipid layers requires both PDZ domains of this protein. Competition and mutagenesis experiments reveal that the protein and the PIP(2) binding sites in the PDZ domains overlap. Overlay assays indicate that the two PDZ domains of syntenin cooperate in binding to cognate peptides and PIP(2). Experiments on living cells demonstrate PIP(2)-dependent and peptide-dependent modes of plasma membrane association of the PDZ domains of syntenin. These observations suggest that local changes in phosphoinositide concentration control the association of PDZ proteins with their target receptors at the plasma membrane.     

Arabidopsis SNAREs SYP61 and SYP121 Coordinate the Trafficking of Plasma Membrane Aquaporin PIP2;7 to Modulate the Cell Membrane Water Permeability

Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane.     

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Phosphatidylinositol (4,5)-bisphosphate (PIP₂) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP₂ on the open state stabilization. A similar effect of PIP₂ on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K⁺ channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP₂ effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP₂ exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations.     

Activation of inwardly rectifying potassium (Kir) channels by phosphatidylinosital-4,5-bisphosphate (PIP2): interaction with other regulatory ligands

All members of the inwardly rectifying potassium channels (Kir1-7) are regulated by the membrane phospholipid, phosphatidylinosital-4,5-bisphosphate (PIP₂). Some are also modulated by other regulatory factors or ligands such as ATP and G-proteins, which give them their common names, such as the ATP sensitive potassium (K_ATP) channel and the G-protein gated potassium channel. Other more non-specific regulators include polyamines, kinases, pH and Na⁺ ions. Recent studies have demonstrated that PIP₂ acts cooperatively with other regulatory factors to modulate Kir channels. Here we review how PIP₂ and co-factors modulate channel activities in each subfamily of the Kir channels.     

A Reducing Milieu Renders Cofilin Insensitive to Phosphatidylinositol 4,5-Bisphosphate (PIP2) Inhibition

Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP₂). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP₂- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP₂. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP₂ inhibition, resulting in enhanced actin dynamics.     

New EMBO members' review: actin cytoskeleton regulation through modulation of PI(4,5)P(2) rafts

The phosphoinositide lipid PI(4,5)P(2) is now established as a key cofactor in signaling to the actin cytoskeleton and in vesicle trafficking. PI(4,5)P(2) accumulates at membrane rafts and promotes local co-recruitment and activation of specific signaling components at the cell membrane. PI(4,5)P(2) rafts may thus be platforms for local regulation of morphogenetic activity at the cell membrane. Raft PI(4,5)P(2) is regulated by lipid kinases (PI5-kinases) and lipid phosphatases (e.g. synaptojanin). In addition, GAP43-like proteins have recently emerged as a group of PI(4,5)P(2) raft-modulating proteins. These locally abundant proteins accumulate at inner leaflet plasmalemmal rafts where they bind to and co-distribute with PI(4,5)P(2), and promote actin cytoskeleton accumulation and dynamics. In keeping with their proposed role as positive modulators of PI(4,5)P(2) raft function, GAP43-like proteins confer competence for regulated morphogenetic activity on cells that express them. Their function has been investigated extensively in the nervous system, where their expression promotes neurite outgrowth, anatomical plasticity and nerve regeneration. Extrinsic signals and intrinsic factors may thus converge to modulate PI(4,5)P(2) rafts, upstream of regulated activity at the cell surface.     

Hydrolyzable ATP and PIP(2) modulate the small-conductance K+ channel in apical membranes of rat cortical-collecting duct (CCD)

The small-conductance K+ channel (SK) in the apical membrane of the cortical-collecting duct (CCD) is regulated by adenosine triphosphate (ATP) and phosphorylation-dephosphorylation processes. When expressed in Xenopus oocytes, ROMK, a cloned K+ channel similar to the native SK channel, can be stimulated by phosphatidylinositol bisphosphate (PIP2), which is produced by phosphoinositide kinases from phosphatidylinositol. However, the effects of PIP2 on SK channel activity are not known. In the present study, we investigated the mechanism by which hydrolyzable ATP prevented run-down of SK channel activity in excised apical patches of principal cells from rat CCD. Channel run-down was significantly delayed by pretreatment with hydrolyzable Mg-ATP, but ATP gamma S and AMP-PNP had no effect. Addition of alkaline phosphatase also resulted in loss of channel activity. After run-down, SK channel activity rapidly increased upon addition of PIP2. Exposure of inside-out patches to phosphoinositide kinase inhibitors (LY294002, quercetin or wortmannin) decreased channel activity by 74% in the presence of Mg-ATP. PIP2 added to excised patches reactivated SK channels in the presence of these phosphoinositide kinase inhibitors. The protein kinase A inhibitor, PKI, reduced channel activity by 36% in the presence of Mg-ATP. PIP2 was also shown to modulate the inhibitory effects of extracellular and cytosolic ATP. We conclude that both ATP-dependent formation of PIP2 through membrane-bound phosphoinositide kinases and phosphorylation of SK by PKA play important roles in modulating SK channel activity.