Protein kinase C prevents oligodendrocyte differentiation: modulation of actin cytoskeleton and cognate polarized membrane traffic

In a previous study, we showed that activation of protein kinase C (PKC) prevents oligodendrocyte differentiation at the pro‐oligodendrocyte stage. The present study was undertaken to identify downstream targets of PKC action in oligodendrocyte progenitor cells. Activation of PKC induced the predominant phosphorylation of an 80‐kD protein, identified as myristoylated alanine‐rich C‐kinase substrate (MARCKS). Upon phosphorylation, MARCKS is translocated from the plasma membrane to the cytosol. Furthermore, PKC activation perturbed the organization of the actin cytoskeleton, causing a redistribution of actin filaments to the submembranous or cortical actin cytoskeleton. As a consequence, transport of a protein traffic marker, the vesicular stomatitis virus glycoprotein, from the trans‐Golgi network to the plasma membrane becomes perturbed. The effect of disruption of the actin filament network by cytochalasin D perfectly matched the effect of PKC. These data thus favor the existence of a causal relationship between actin rearrangement and docking and/or fusion of proteins to the plasma membrane. Interestingly, neither in control cells nor in PKC‐activated cells did another protein traffic marker, influenza hemagglutinin (HA), reach the cell surface. However, an eminent and specific accumulation of HA just underneath the plasma membrane became apparent upon PKC activation. Yet, this effect could not be simulated by cytochalasin D treatment. Therefore, these observations imply that although MARCKS represents a prominent PKC target site in regulating differentiation, another target involves the differential control of cognate polarized trafficking pathways, which are apparently operating in oligodendrocyte progenitor cells. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 385–398, 1999     

Protein kinase C and the regulation of the actin cytoskeleton

Protein kinase C (PKC) isoforms are central components in intracellular networks that regulate a vast number of cellular processes. It has long been known that in most cell types, one or more PKC isoforms influences the morphology of the F-actin cytoskeleton and thereby regulates processes that are affected by remodelling of the microfilaments. These include cellular migration and neurite outgrowth. This review focuses on the role of classical and novel PKC isoforms in migration and neurite outgrowth, and highlights some regulatory steps that may be of importance in the regulation by PKC of migration and neurite outgrowth. Many studies indicate that integrins are crucial mediators both upstream and downstream of PKC in inducing morphological changes. Furthermore, a number of PKC substrates, directly associated with the microfilaments, such as MARCKS, GAP43, adducin, fascin, ERM proteins and others have been identified. Their potential role in PKC effects on the cytoskeleton is discussed.     

Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton

The cytoskeleton is required for multiple cellular events including endocytosis and the transfer of cargo within the endocytic system. Polarized epithelial cells are capable of endocytosis at either of their distinct apical or basolateral plasma membrane domains. Actin plays a role in internalization at both cell surfaces. Microtubules and actin are required for efficient transcytosis and delivery of proteins to late endosomes and lysosomes. Microtubules are also important in apical recycling pathways and, in some polarized cell types, basolateral recycling requires actin. The microtubule motor proteins dynein and kinesin and the class I unconventional myosin motors play a role in many of these trafficking steps. This review examines the endocytic pathways of polarized epithelial cells and focuses on the emerging roles of the actin cytoskeleton in these processes.     

Protein kinase C inhibitor H-7 alters the actin cytoskeleton of cultured cells

The effects of the protein kinase C inhibitor H-7 on the actin cytoskeleton of cultured cells (Swiss 3T3 and PTK2) are described. As documented by fluorescence microscopy and the higher-resolution technique of photoelectron microscopy, the effects are rapid and dramatic; exposure to 30 μM H-7 in culture medium for less than 6 min is sufficient to induce a significant reduction in the numbers and thickness of actin microfilament bundles and alterations in the morphology of cell-cell boundaries in PTK2 cells. One-hour exposure to 30 μM H-7 results in nearly complete depletion of normal actin microfilament bundles from all of the cell types examined, without dramatic changes in overall cell shape. The intermediate filament and microtubule cytoskeletal networks did not appear to be affected to any extent over the times and doses examined. Forty-five minutes of exposure of Swiss 3T3 cells to 200 μM of either HA1004 (which is comparable to H-7 with respect to inhibition of cyclic nucleotide dependent kinases) or to the protein kinase C inhibitor sangivamycin did not induce the actin alterations characteristic of H-7. In addition, depletion of protein kinase C from Swiss 3T3 cells by means of phorbol ester-induced down-regulation did not prevent the effects of H-7 on the actin cytoskeleton. These results demonstrate that the protein kinase C inhibitor H-7 has a specific and rapid effect on the actin cytoskeleton, and furthermore H-7 may have biochemical effects beyond those mediated by inhibition of protein kinase C or the cyclic nucleotide dependent kinases.     

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane

The ATP-sensitive K(+) (K(ATP)) channels in both sarcolemmal (sarcK(ATP)) and mitochondrial inner membrane (mitoK(ATP)) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcK(ATP) contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoK(ATP) remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing K(ATP) into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing K(ATP) channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing K(ATP) channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional K(ATP) channels in mitochondria was significantly increased by PMA, as shown by K(ATP)-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing K(ATP) channels was blocked by a selective PKCepsilon inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the K(ATP) channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKCepsilon. PKC isoform-regulated mitochondrial import of K(ATP) channels may have significant implication in cardioprotection of IPC.     

Regulation of calcium-dependent [3H]noradrenaline release from rat cerebrocortical synaptosomes by protein kinase C and modulation of the actin cytoskeleton.

The effects that active phorbol esters, staurosporine, and changes in actin dynamics, might have on Ca2+ -dependent exocytosis of [3H]-labelled noradrenaline, induced by either membrane-depolarizing agents or a Ca2+ ionophore, have been examined in isolated nerve terminals in vitro. Depolarization-induced openings of voltage-dependent Ca2+ channels with 30 mM KCl or 1 mM 4-aminopyridine induced limited exocytosis of [3H]noradrenaline, presumably from a readily releasable vesicle pool. Application of the Ca2+ ionophore calcimycin (10 microM) induced more extensive [3H]noradrenaline release, presumably from intracellular reserve vesicles. Stimulation of protein kinase C with phorbol 12-myristate,13-acetate increased release evoked by all secretagogues. Staurosporine (1 microM) had no effect on depolarization-induced release, but decreased ionophore-induced release and reversed all effects of the phorbol ester. When release was induced by depolarization, internalization of the actin-destabilizing agent DNAase I into the synaptosomes gave a slight increase in [3H]NA release and strongly increased the potentiating effect of the phorbol ester. In contrast, when release was induced by the Ca2+ ionophore, DNAase I had no effect, either in the absence or presence of phorbol ester. The results indicate that depolarization of noradrenergic rat synaptosomes induces Ca2+ -dependent release from a releasable pool of staurosporine-insensitive vesicles. Activation of protein kinase C increases this release by staurosporine-sensitive mechanisms, and destabilization of the actin cytoskeleton further increases this effect of protein kinase C. In contrast, ionophore-induced noradrenaline release originates from a pool of staurosporine-sensitive vesicles, and although activation of protein kinase C increases release from this pool, DNAase I has no effect and also does not change the effect of protein kinase C. The results support the existence of two functionally distinct pools of secretory vesicles in noradrenergic CNS nerve terminals, which are regulated in distinct ways by protein kinase C and the actin cytoskeleton.     

Cell migration: GAPs between membrane traffic and the cytoskeleton

During cell migration, coordination between membrane traffic, cell substrate adhesion and actin reorganization is required for protrusive activity to occur at the leading edge. Actin organization is regulated by Rho family GTPases and, with a contribution from the endocytic cycle, serves to extend the cell front. The details of the molecular mechanisms that direct membrane traffic at sites of adhesion and rearrange actin at the cell edge are still unknown. However, recent findings show that a number of multi-domain proteins characterized by an ArfGAP domain interact with both actin-regulating and integrin-binding proteins, as well as affecting Rac-mediated protrusive activity and cell migration. Some of these proteins have been shown to localize to endocytic compartments and to have a role in regulating endocytosis. Given the participation of Arf proteins in regulating membrane traffic, one appealing hypothesis is that the ArfGAPs act as molecular devices that coordinate membrane traffic and cytoskeletal reorganization during cell motility.     

Protein kinase activities associated with actin cytoskeleton in the oocytes and eggs of African clawed frog

Protein phosphorylation with specific protein kinases plays the key role in the regulation of meiotic maturation of oocytes. However, little is known about the contribution of kinases to the temporal and positional regulation of the cytoskeleton rearrangement in maturing oocytes, including the actin cytoskeleton. In order to study a relationship between the kinase activities and actin cytoskeleton rearrangement, we analyzed protein phosphorylation in the isolated actin cytoskeleton of Xenopus laevis oocytes. Analysis of the full grown oocytes and eggs injected with [gamma-32P] "P has revealed phosphorylation of many proteins associated with the actin cytoskeleton and shown the appearance of three additional major phosphoproteins, 20, 43, and 69 kDa, during oocyte maturation. A significant number of these phosphoproteins were also found after incubation of the isolated cytoskeleton with [gamma-32P] "P in vitro, thus confirming that the kinases modifying these substrates are also specifically associated with actin. The in vivo and in vitro kinase activities were also stimulated during maturation. Analysis of kinase self-phosphorylation in situ and protein phosphorylation in solutions and substrate containing gels revealed a set of actin-associated kinases, including cAMP- and Ca(2+)-dependent kinases, as well as MAP, p34cdc2, and tyrosine kinase activities. Their level was the highest in the eggs. The involvement of kinases in the actin cytoskeleton rearrangement during oocyte maturation is discussed.     

Protein kinase C regulates vascular calcification via cytoskeleton reorganization and osteogenic signaling

Vascular calcification is an active cell-mediated process that reduces elasticity of blood vessels and increases blood pressure. Until now, the molecular basis of vascular calcification has not been fully understood. We previously reported that microtubule disturbances mediate vascular calcification. Here, we found that protein kinase C (PKC) signaling acted as a novel coordinator between cytoskeletal changes and hyperphosphatemia-induced vascular calcification. Phosphorylation and expression of both PKCα and PKCδ decreased during inorganic phosphate (Pi)-induced vascular smooth muscle cell (VSMC) calcification. Knockdown of PKC isoforms by short interfering RNA as well as PKC inactivation by Go6976 or rottlerin treatment revealed that specific inhibition of PKCα and PKCδ accelerated Pi-induced calcification both in VSMCs and ex vivo aorta culture through upregulation of osteogenic signaling. Additionally, inhibition of PKCα and PKCδ induced disassembly of microtubule and actin, respectively. In summary, our results indicate that cytoskeleton perturbation via PKCα and PKCδ inactivation potentiates vascular calcification through osteogenic signal induction.     

Protein kinase C induces actin reorganization via a Src- and Rho-dependent pathway

We have investigated the mechanism of PKC-induced actin reorganization in A7r5 vascular smooth muscle cells. PKC activation by 12-O-tetradecanoylphorbol-13-acetate induces the disassembly of actin stress fibers concomitant with the appearance of membrane ruffles. PKC also induces rapid tyrosine phosphorylation in these cells. As we could show, utilizing the Src-specific inhibitor PP2 and a kinase-deficient c-Src mutant, actin reorganization is dependent on PKC-induced Src activation. Subsequently, the activity of the small G-protein RhoA is decreased, whereas Rac and Cdc42 activities remain unchanged. Disassembly of actin stress fibers could also be observed using the Rho kinase-specific inhibitor Y-27632, indicating that the decrease in RhoA activity on its own is responsible for actin reorganization. In addition, we show that tyrosine phosphorylation of p190RhoGAP is increased upon 12-O-tetradecanoylphorbol-13-acetate stimulation, directly linking Src activation to a decrease in RhoA activity. Our data provide substantial evidence for a model elucidating the molecular mechanisms of PKC-induced actin rearrangements.     

Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.     

Protein kinase C modulation in apoptotic rat thymocytes: an ultrastructural analysis

Numerous events in the cell, such as gene expression, cell growth and metabolism are regulated by signal transduction pathways involving protein kinase C (PKC). Recent data indicate that a PKC-dependent mechanism also underlies the apoptotic death of cells induced by glucocorticoid hormones. In this report we have analysed the changes of PKC during dexamethasone-induced apoptosis in thymocytes by means of immunocytochemical and immunochemical analysis. The data obtained show an increase and intracellular movement of protein kinase C, which is translocated to the nucleus and linked to the nuclear matrix during the apoptotic process.     

Protein kinase C: properties and possible role in cellular division and differentiation

Protein kinase C was first described some eight years ago. Recent results indicate that this kinase may have a crucial role in signal transduction for substances involved in cellular differentiation and division. Protein kinase C is activated by attachment to plasma membranes, in the presence of calcium and diacylglycerol. The activator is produced in the membrane following the signal-induced breakdown of phosphoinositides. Tumor promoters, such as phorbol ester, can substitute for diacylglycerol. The recent findings that: tyrosine kinases might be involved in the phosphoinositide turnover and, phosphorylation of growth factor receptors by protein kinase C regulates some of their functions, indicate more and more clearly that this kinase is involved in the control of cell growth division and differentiation. Purification procedures, properties and mechanisms of regulation will be summarized and discussed.     

Regulation of chondrocyte differentiation by the actin cytoskeleton and adhesive interactions

Chondrocyte differentiation is a multi-step process characterized by successive changes in cell morphology and gene expression. In addition to tight regulation by numerous soluble factors, these processes are controlled by adhesive events. During the early phase of the chondrocyte life cycle, cell-cell adhesion through molecules such as N-cadherin and neural cell adhesion molecule (N-CAM) is required for differentiation of mesenchymal precursor cells to chondrocytes. At later stages, for example in growth plate chondrocytes, adhesion signaling from extracellular matrix (ECM) proteins through integrins and other ECM receptors such as the discoidin domain receptor (DDR) 2 (a collagen receptor) and Annexin V is necessary for normal chondrocyte proliferation and hypertrophy. Cell-matrix interactions are also important for chondrogenesis, for example through the activity of CD44, a receptor for Hyaluronan and collagens. The roles of several signaling molecules involved in adhesive signaling, such as integrin-linked kinase (ILK) and Rho GTPases, during chondrocyte differentiation are beginning to be understood, and the actin cytoskeleton has been identified as a common target of these adhesive pathways. Complete elucidation of the pathways connecting adhesion receptors to downstream effectors and the mechanisms integrating adhesion signaling with growth factor- and hormone-induced pathways is required for a better understanding of physiological and pathological skeletal development.     

Effect of Rho-associated kinase inhibition on actin cytoskeleton structure and calcium response in glioma C6 cells

The role of actin cytoskeleton functional state in glioma C6 cell morphology and calcium signaling was investigated through modification of myosin II activity by blocking Rho-associated kinase with the specific inhibitor Y-27632. Treatment of glioma C6 cells with ROCK inhibitor resulted in actin cytoskeleton reorganization and also in the changed shape and distribution of mitochondria. Changes in the distribution of ER, the main calcium store in glioma C6 cells, were not visible. The inhibition of myosin II activity influences the first phase of calcium signaling evoked by agonist, and both phases of thapsigargin-evoked calcium response. We suggest that the observed increase in Ca2+ release from intracellular stores induced by IP3 formation as well as inhibition of SERCA ATPase is at least in part related to severely affected mitochondria. Enhancement of capacitative calcium entry evoked by thapsigargin is probably associated with the reorganization of the acto-myosin II system. ATP-induced calcium response presents no changes in the second phase. We observed that ATP stimulation of Y-27632 pretreated cells leads to immediate morphological rearrangement of glioma C6 cells. It is a consequence of actin cytoskeleton reorganization: formation of stress fibers and relocation of phosphorylated myosin II to actin filaments. It seems that the agonist-evoked strong calcium signal may be sufficient for myosin II activation and the stress fiber organization. This is the first work showing the dependence between the functional state of the acto-myosin II system and calcium signaling stressing the reversible character of this relationship.     

Effect of alkaloid sanguinarine and a pharmaceutical preparation ukrain on modulation of vesicular membrane fusion and actin cytoskeleton of macrophages

A study was made of modulations of lysosome-phagosome fusion process and of fibrillar actin content in mouse peritoneal macrophages by an antitumor alkaloid sanguinarine and a derivative drug Ukrain. In addition, effects of these substances on in vitro polymerization of monomeric globular actin from rabbit muscle were investigated. Sanguinarine and Ukrain stimulated lysosome-phagosome fusion and increased the content of polymerized fibrillar form of actin in mouse macrophages. Effects of these substances were enhanced at their higher concentrations. Both sanguinarine and Ukrain induced in vitro polymerization of globular actin from rabbit muscle. A possible role of sanguinarine and Ukrain in changing vesicular membrane states during intracellular membrane interaction in lysosome-phagosome fusion process was discussed. The influence of these substances on actin polymerization and actin cytoskeleton rearrangement was evaluated. It could be supposed that sanguinarine and Ukrain may alter intracellular membrane transport.     

Stretch-dependent growth and differentiation in vascular smooth muscle: role of the actin cytoskeleton

The smooth muscle cells in the vascular wall are constantly exposed to distending forces from the intraluminal pressure. A rise in blood pressure triggers growth of the vessel wall, which is characterized primarily by hypertrophy of smooth muscle cells with maintained differentiation in a contractile phenotype. Growth factor stimulation of dissociated smooth muscle cells, on the other hand, causes proliferative growth with loss of contractility. This type of response is also found in neointima development following angioplasty and in atherosclerotic lesions. An intact tissue environment is therefore critical for preserved differentiation. Recent advances point to a role of actin polymerization in the expression of smooth muscle differentiation marker genes, in concert with serum response factor (SRF) and cofactors, such as myocardin. Stretch of intact venous smooth muscle activates Rho and inhibits the actin filament severing factor cofilin, resulting in increased actin polymerization. Concomitantly, the rates of synthesis of SRF-regulated differentiation markers, such as SM22alpha, calponin, and alpha-actin, are increased. This increase in differentiation signals is parallel with activation of the mitogen-activated protein (MAP) kinase pathway. Thus stretch-induced growth in a maintained contractile phenotype occurs by dual activation of signal pathways regulating both growth and differentiation. A current challenge is to identify sites of crosstalk between these pathways in intact smooth muscle tissue.