Retinoic acid-induced differentiation into astrocytes and glutamatergic neurons is associated with expression of functional and activable phospholipase D

Phospholipase D (PLD) activity in mammalian cells has been associated with cell proliferation and differentiation. Here, we investigated the expression of PLD during differentiation of pluripotent embryonal carcinoma cells (P19) into astrocytes and neurons. Retinoic acid (RA)-induced differentiation increased PLD1 and PLD2 mRNA levels and PLD activity that was responsive to phorbol myristate acetate. Various agonists of membrane receptors activated PLD in RA-differentiated cells. Glutamate was a potent activator of PLD in neurons but not in astrocytes, whereas noradrenaline and carbachol increased PLD activity only in astrocytes. P19 neurons but not astrocytes released glutamate in response to a depolarizing stimulus, confirming the glutamatergic phenotype of these neurons. These results indicate upregulation of PLD gene expression associated with RA-induced neural differentiation.     

Differential regulation of apoptosis by caspase-mediated cleavage of phospholipase D isozymes

Phospholipase D (PLD) has been implicated in survival and anti-apoptosis, but the molecular mechanism by which it responds to apoptotic stimuli is poorly unknown. Here, we demonstrate that cleavage of PLD isozymes as specific substrates of caspase differentially regulates apoptosis. PLD1 is cleaved at one internal site (DDVD₅₄₅S) and PLD2 is cleaved at two or three sites (PTGD₁₃ELD₁₆S and DEVD₂₈T) in the front of N-terminus. Cleavage of PLD was endogenously detected in post-mortem Alzheimer brain together with activated caspase-3, suggesting the physiological relevance. The cleavage of PLD1 but not PLD2 might act as an inactivating process since PLD1 but not PLD2 activity is significantly decreased during apoptosis, suggesting that differential cleavage of PLD isozymes could affect its enzymatic activity. Moreover, caspase-resistant mutant of PLD1 showed more potent anti-apoptotic capacity than that of wild type PLD1, whereas PLD2 maintained anti-apoptotic potency in spite of its cleavage during apoptosis. Moreover, PLD2 showed more potent anti-apoptotic effect than that of PLD1 in overexpression and knockdown experiments, suggesting that difference in anti-apoptotic potency between PLD1 and PLD2 might be due to its intrinsic protein property. Taken together, our results demonstrate that differential cleavage pattern of PLD isozymes by caspase might affect its enzymatic activity and anti-apoptotic function.     

Over-expression of phospholipase D isozymes down-regulates protein kinase CKII activity via proteasome-dependent CKIIβ degradation in NIH3T3 cells

Over-expression of phospholipase D (PLD) 1 or PLD2 down-regulated CKII activity in NIH3T3 cells. The same results were found with catalytically inactive mutants of PLD isozymes, indicating that the catalytic activity of PLD is not required for PLD-mediated CKII inhibition. Consistent with this, 1-butanol did not alter CKII activity. The reduction in CKII activity in PLD-over-expressing NIH3T3 cells was due to reduced protein level, but not mRNA level, of the CKIIβ subunit. This PLD-induced CKIIβ degradation was mediated by ubiquitin-proteasome machinery, but MAP kinase and mTOR were not involved in CKIIβ degradation. PLD isozymes interacted with the CKIIβ subunit. Immunocyto-chemical staining revealed that PLD and CKIIβ colocalize in the cytoplasm of NIH3T3 cells, especially in the perinuclear region. PLD binding to CKIIβ inhibited CKIIβ autophosphory-lation, which is known to be important for CKIIβ stability. In summary, the current data indicate that PLD isozymes can down-regulate CKII activity through the acceleration of CKIIβ degradation by ubiquitin-proteasome machinery.     

Caspase cleavage of phospholipase D1 in vitro alters its regulation and reveals a novel property of the "loop" region

Phospholipase D (PLD) has been implicated in mediating vesicular transport, mitosis, differentiation and apoptosis. The product of PLD activity, phosphatidic acid (PA) has mitogenic potential and elevated PLD expression has been detected in many tumor cell lines. Several reports have demonstrated that distinct PLD domains regulate its activity and that truncated forms of PLD retain enzymatic activity. We hypothesized that during apoptosis caspase cleavage of PLDs could result in modification of their activities. To test this idea, we have used in vitro translation of PLD1 and PLD2 which generated active enzymes exhibiting properties mimicking those of the endogenous proteins. Here we demonstrate that PLD1 was rapidly cleaved in vitro by caspases-8, -3 and -7. In contrast, PLD2 cleavage was delayed and its activity was unaffected by incubation with caspase-3. Significantly, following caspase cleavage the response of PLD1 to regulatory stimuli was altered; it was no longer activated by PKC and instead exhibited an increased activity in response to small GTPases. Notably, this enhanced activity was due to cleavage of PLD1 in the "loop" domain, a region previously associated with negative regulatory function. Thus our data have identified a novel regulatory domain in PLD1.     

Overexpression of phospholipase D suppresses taxotere-induced cell death in stomach cancer cells

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid (PA) and choline. There are at least two PLD isozymes, PLD1 and PLD2. Genetic and pharmacological approaches implicate both PLD isozymes in a diverse range of cellular processes, including receptor signaling, membrane transport control, and actin cytoskeleton reorganization. Several recent studies reported that PLD has a role in signaling pathways that oppose apoptosis and promote cell survival in cancer. In this study, we examined the role of PLD in taxotere-induced apoptosis in stomach cell lines; normal stomach (NSC) and stomach cancer cells (SNU 484). Taxotere treatment resulted in increase of PLD activity. To confirm the role of PLD in taxotere-induced apoptosis, PLDs were transfected into SNU 484 cells. Overexpression of PLD isozymes resulted in inhibition of taxotere-induced apoptotic cell death, evidenced by decreased degradation of chromosomal DNA, and increased cell viability. Concurrently, Bcl-2 expression was upregulated, and taxotere-induced activation of procaspase 3 was inhibited after PLD's transfection. However, when PLD was selectively inhibited by specific siRNA-PLD1 or -PLD2, taxotere-induced apoptosis was exacerbated in SNU 484 cells. On top of this, PA -- the product of PLDs, also resulted in upregulation of Bcl-2 in SNU 484. Although PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of phospholipase A(2) (PLA(2)), increased Bcl-2 expression by PA was not abrogated by propranolol, an inhibitor of PA phospholyhydrolase (PAP). Taken together, PLD1 and PLD2 are closely related with Bcl-2 expression together with PLA(2), but not with PAP, during taxotere-induced apoptosis in SNU 484 cells.     

Dimerization of phospholipase d isozymes.

Two mammalian phospholipase D (PLD) isozymes (PLD1 and PLD2) have been reported. In this study, we differentially tagged these isozymes with enhanced green fluorescent protein (EGFP-rPLD1 and EGFP-rPLD2) or Xpress peptide epitope (Xpress-rPLD1 and Xpress-rPLD2) to examine the association between these isozymes. Overexpressed EGFP-rPLD1 coimmunoprecipitated with Xpress-rPLD1 using anti-Xpress antibody. However, the coimmunoprecipitation was independent of the activity of rPLD1. Xpress-rPLD2 also bound to EGFP-rPLD1 although the binding was less efficient than observed with Xpress-rPLD1. The association between rPLD2 and rPLD1 was confirmed by coimmunoprecipitation of EGFP-rPLD2 with Xpress-rPLD1. EGFP-rPLD2 also bound to Xpress-rPLD2 as shown by coimmunoprecipitation. Immunofluorescence staining of COS-7 cells coexpressing EGFP-rPLDs and Xpress-rPLDs showed that the PLD isozymes colocalized in the perinuclear and plasma membrane regions, suggesting that they could associate in a cellular setting. These results suggest that rPLD1 and rPLD2 can exist as homodimers and can form heterodimers.     

Leishmania major: differential regulation of the surface metalloprotease in amastigote and promastigote stages.

During its life cycle, the protozoan parasite Leishmania major alternates from an intracellular amastigote form in the mammalian host to a flagellated promastigote form in the insect vector. The expression of the surface metalloprotease (PSP) during differentiation in vitro was investigated by Western and Northern blots, by immunoprecipitation of cells metabolically labeled with [35S]methionine or labeled at the surface with radioactive iodine, and by quantification of the proteolytic activity in substrate-containing polyacrylamide gels. We report that the surface metalloprotease is down-regulated at both the mRNA and the protein level in amastigotes, where it represents less than 1% of the equivalent proteolytic activity detected in promastigotes. A significant amount of mRNA is detected 4 hr after the onset of differentiation. The expression of the protease begins at that time and reaches steady state 8 hr later. The synthesis of PSP precedes the complete morphological differentiation to the promastigote stage and the appearance of the lipophosphoglycan, another major promastigote surface component. In contrast to PSP, a family of mercaptoethanol-activated proteases present in the amastigote exists only at a reduced level in the promastigote. The confinement of the surface metalloprotease to the insect stage of the parasite suggests that it has no physiological function in the parasitism maintenance of mammalian host macrophages.     

Small stress protein Hsp27 accumulation during dopamine-mediated differentiation of rat olfactory neurons counteracts apoptosis

The small stress protein Hsp27 is expressed during mammalian neural development. We have analyzed the role of this protein in immortalized rat olfactory neuroblasts. In the presence of dopamine a fraction of these cells differentiate into neurons while the remaining cells undergo apoptosis. We report here that the dopamine induced differentiation and apoptosis are associated with a transient and specific accumulation of Hsp27. Moreover, transfection experiments have shown that Hsp27 overexpression drastically decreases the fraction of cells undergoing apoptosis. In contrast, reduction of the endogenous level of Hsp27 led to abortion of differentiation and, therefore, drastically increased the number of apoptotic cells. Furthermore, in the normal cell population we show that Hsp27 accumulation takes place only in differentiating cells that were not undergoing apoptosis. We therefore conclude that Hsp27 may represent a key protein that controls the decision of olfactory precursor cells to undergo either differentiation or cell death.     

Phospholipase D1 facilitates second-phase myoblast fusion and skeletal muscle regeneration

Myoblast differentiation and fusion is a well-orchestrated multistep process that is essential for skeletal muscle development and regeneration. Phospholipase D1 (PLD1) has been implicated in the initiation of myoblast differentiation in vitro. However, whether PLD1 plays additional roles in myoblast fusion and exerts a function in myogenesis in vivo remains unknown. Here we show that PLD1 expression is up-regulated in myogenic cells during muscle regeneration after cardiotoxin injury and that genetic ablation of PLD1 results in delayed myofiber regeneration. Myoblasts derived from PLD1-null mice or treated with PLD1-specific inhibitor are unable to form mature myotubes, indicating defects in second-phase myoblast fusion. Concomitantly, the PLD1 product phosphatidic acid is transiently detected on the plasma membrane of differentiating myocytes, and its production is inhibited by PLD1 knockdown. Exogenous lysophosphatidylcholine, a key membrane lipid for fusion pore formation, partially rescues fusion defect resulting from PLD1 inhibition. Thus these studies demonstrate a role for PLD1 in myoblast fusion during myogenesis in which PLD1 facilitates the fusion of mononuclear myocytes with nascent myotubes.     

Role of phospholipase D1 in neurite outgrowth of neural stem cells

Employing neural stem cells from the brain cortex of E12 rat embryos, we investigated the possible role of phospholipase D (PLD) in the synaptogenesis and neurite formation of neural cells during differentiation. Expression level of PLD1 increased during neuronal differentiation of the neural stem cells, resulting in increased PLD activity. Expression level of synapsin I, a marker of synaptogenesis, also increased as the differentiation of neural stem cells progressed. To figure out the effect of PLD on synapsin I expression, we treated the neural stem cells with phorbol myristate acetate (PMA) to stimulate PLD activity. Increased PLD activity induced by PMA treatment resulted in elevated synapsin I expression and neurite outgrowth during neuronal differentiation. To further confirm the role of PLD in neurite outgrowth, we transfected the dominant-negative form of rat PLD1 cDNA (DN-rPLD1) into neural stem cells to downregulate PLD activity. Overexpression of DN-rPLD1 showed the complete inhibition of neurite outgrowth of neural stem cells under differentiation condition. While transfection of DN-rPLD1 did not affect the synapsin I expression, overexpression of rPLD1 resulted in increased synapsin I expression of the neural cells. These results suggest that PLD1 plays a critical role in neurite outgrowth during differentiation of the neural stem cells. In conclusion, this is the first evidence to show that PLD1 acts as an important regulator of neurite outgrowth in neural stem cell by promoting neuronal differentiation via increase of synapsin I expression.     

The C-terminal domain of PLD2 participates in degradation of protein kinase CKII β subunit in human colorectal carcinoma cells

Elevated phospholipase D (PLD) expression prevents cell cycle arrest and apoptosis. However, the roles of PLD isoforms in cell proliferation and apoptosis are incompletely understood. Here, we investigated the physiological significance of the interaction between PLD2 and protein kinase CKII (CKII) in HCT116 human colorectal carcinoma cells. PLD2 interacted with the CKIIβ subunit in HCT116 cells. The C-terminal domain (residues 578-933) of PLD2 and the N-terminal domain of CKIIβ were necessary for interaction between the two proteins. PLD2 relocalized CKIIβ to the plasma membrane area. Overexpression of PLD2 reduced CKIIβ protein level, whereas knockdown of PLD2 led to an increase in CKIIβ expression. PLD2-induced CKIIβ reduction was mediated by ubiquitin-dependent degradation. The C-terminal domain of PLD2 was sufficient for CKIIβ degradation as the catalytic activity of PLD2 was not required. Taken together, the results indicate that the C-terminal domain of PLD2 can regulate CKII by accelerating CKIIβ degradation in HCT116 cells.     

Phospholipase D2 activity suppresses hydrogen peroxide-induced apoptosis in PC12 cells

Phospholipase D (PLD) plays an important role as an effector in the membrane lipid-mediated signal transduction. However, the precise physiological functions of PLD are not yet well understood. In this study, we examined the role of PLD activity in hydrogen peroxide (H(2)O(2))-induced apoptosis in rat pheochromocytoma (PC12) cells. Treatment of PC12 cells with H(2)O(2) resulted in induction of apoptosis in these cells, which is accompanied by the activation of PLD. This H(2)O(2)-induced apoptosis was enhanced remarkably when phosphatidic acid production by PLD was selectively inhibited by pretreating the PC12 cells with 1-butanol. Expression of PLD2, but not of PLD1, correlated with increased H(2)O(2)-induced PLD activity in a concentration- and time-dependent manner. Concomitant with PLD activation, the PLD2 activity suppressed H(2)O(2)-induced apoptosis in PC12 cells. Expression of PLD2 lipase-inactive mutant (K758R) had no effect on either PLD activity or apoptosis. PLD2 activity also suppressed H(2)O(2)-induced cleavage and activation of caspase-3. Taken together, the results suggest that PLD2 activity is specifically up-regulated by H(2)O(2) in PC12 cells and that it plays a suppressive role in H(2)O(2)-induced apoptosis.     

Phorbol myristate acetate-induced Egr-1 expression is suppressed by phospholipase D isozymes in human glioma cells

Early growth response-1 (Egr-1) is involved in the regulation of cell growth. Here, we found that overexpression of phospholipase D (PLD) isozymes decreased tumor promoter phorbol myristate acetate (PMA)-induced Egr-1 expression and transactivation in glioma cells. Suppression of PMA-induced Egr-1 was dependent on the expression level of PLD isozymes. Overexpression of catalytically inactive PLD, treatment with PA, and prevention of PA dephosphorylation by 1-propranolol significantly suppressed PMA-induced Egr-1 expression. PLD-induced suppression of Egr-1 was reversed by inhibition of phosphatidylinositol 3-kinase (PI3K). Taken together, these results suggest that elevated expression and activity of PLD attenuate PMA-induced Egr-1 expression via PI3K pathway.     

RhoA-mediated Phospholipase D1 signaling is not required for the formation of stress fibers and focal adhesions

The small GTPase RhoA regulates a wide spectrum of cellular functions including transformation and cytoskeletal reorganization. A large number of proteins have been identified as targets of RhoA, but their specific roles in these processes are not clear. Phospholipase D (PLD) was shown to be one such target several years ago; more recent work from our laboratory and others has demonstrated that of the two mammalian PLD isozymes, PLD1 but not PLD2 is activated by RhoA and this activation proceeds through direct binding both in vitro and in vivo. In this study, using a series of RhoA mutants, we have defined a PLD1-specific interacting site on RhoA composed of the residues Asn41, Trp58 and Asp76, using the yeast two-hybrid system, co-immunoprecipitation, and a PLD in vivo assay. The results further substantiate our previous finding that RhoA activates PLD1 through direct interaction. These mutants were then used to investigate the role of PLD1 in the cytoskeletal reorganization stimulated by RhoA signaling. Our results show that PLD1 is not required for the RhoA-mediated stress fiber and focal adhesion formation. The lack of importance of PLD1 signaling in RhoA-mediated cytoskeletal reorganization is further supported by the observation that PLD1 depletion using an shRNA approach and tetracycline-induced overexpression of the wild-type and the catalytically inactive mutant of PLD1 in stable cell lines do not alter stress fiber and focal adhesion formation.     

Potent direct inhibition of mammalian phospholipase D isoenzymes by calphostin-c

Calphostin-c inhibits protein kinase C (PKC) isoenzymes by covalent modification of the lipid binding regulatory domain. Exposure of cells to calphostin-c elicits PKC independent effects including disruption of intracellular transport, growth inhibition, and stimulation of apoptosis suggesting actions at additional targets. Phospholipase D (PLD) enzymes are targets for activation by PKC. We have investigated the PKC isoenzyme selectivity for activation of two mammalian PLD enzymes, PLD1 and PLD2, by PKC. We examined the sensitivity of this process to widely used PKC inhibitors and report the surprising finding that calphostin-c is a potent direct inhibitor of PLD1 and PLD2. In vitro, calphostin-c inhibits activity of both PLD1 and PLD2 with an IC(50) of approximately 100 nM. Inhibition is not overcome by protein and lipid activators of these enzymes and does not involve blockade of phosphatidylinositol 4,5-bisphosphate-dependent PLD binding to substrate containing liposomes. Studies using a series of deletion and point mutants of the enzymes suggest that calphostin-c targets the PLD catalytic domain. Inhibition of PLD by calphostin-c in vitro involves stable and apparently irreversible modification of the enzyme. Activity of both PLD1 and PLD2 can be inhibited by calphostin-c treatment of intact cells in a manner that is independent of upstream actions of PKC. Our results suggest that inhibition of PLD1 and PLD2 may explain some of the PKC-independent effects of calphostin-c observed when the compound is applied to intact cells.     

Effects of phospholipase D during cultured osteoblast mineralization and bone formation

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos-2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos-2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1-selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high-resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.     

Up-regulation of cyclooxygenase-2 by cobalt chloride-induced hypoxia is mediated by phospholipase D isozymes in human astroglioma cells

Cyclooxygenase-2 (COX-2) is an isoform of prostaglandin H synthase induced by hypoxia and has been implicated in the growth and progression of a variety of human cancers. In the present study, we investigated the role of phospholipase D (PLD) isozymes in cobalt chloride (CoCl(2))-induced hypoxia-driven COX-2 expression in U87 MG human astroglioma cells. CoCl(2) stimulated PLD activity and synthesis of COX-2 protein in a dose and time-dependent manner. Moreover, elevated expression of PLD1 and PLD2 increased hypoxia-induced COX-2 expression and prostaglandin E2 (PGE(2)) production. Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed CoCl(2)-induced COX-2 expression and PGE(2) formation. In addition, evidence that PLD activity was involved in the stimulation of COX-2 expression was provided by the observations that overexpression of wild type PLD isozymes, but not catalytically inactive PLD isozymes, stimulated CoCl(2)-induced COX-2 expression and PGE(2) production. PLD1 enhanced COX-2 expression by CoCl(2) via reactive oxygen species (ROS), p38 MAPK kinase, PKC-delta, and PKA, but not ERK, whereas PLD2 enhanced CoCl(2)-induced COX-2 expression via ROS and p38 MAPK, but not ERK, PKC-delta, and PKA. Differential regulation of COX-2 expression mediated through PLD isozymes was comparable with that of CoCl(2)-induced PLD activity in these two PLD isozymes. Taken together, our results demonstrate for the first time that PLD1 and PLD2 isozymes enhance CoCl(2)-induced COX-2 expression through differential signaling pathways in astroglioma cells.