Everolimus-induced hyperpermeability of endothelial cells causes lung injury.

The mammalian target of rapamycin (mTOR) inhibitors, everolimus (but not dactolisib), is frequently associated with lung injury in clinical therapies. However, the underlying mechanisms remain unclear. Endothelial cell barrier dysfunction plays a major role in the pathogenesis of the lung injury. This study hypothesizes that everolimus increases pulmonary endothelial permeability, which leads to lung injury. We tested the effects of everolimus on human pulmonary microvascular endothelial cell (HPMEC) permeability and a mouse model of intraperitoneal injection of everolimus was established to investigate the effect of everolimus on pulmonary vascular permeability. Our data showed that everolimus increased human pulmonary microvascular endothelial cell (HPMEC) permeability which was associated with MLC phosphorylation and F-actin stress fiber formation. Furthermore, everolimus induced an increasing concentration of intracellular calcium Ca2+ leakage in HPMECs and this was normalized with ryanodine pretreatment. In addition, ryanodine decreased everolimus-induced phosphorylation of PKCα and MLC, and barrier disruption in HPMECs. Consistent with in vitro data, everolimus treatment caused a visible lung-vascular barrier dysfunction, including an increase in protein in BALF and lung capillary-endothelial permeability, which was significantly attenuated by pretreatment with an inhibitor of PKCα, MLCK, and ryanodine. This study shows that everolimus induced pulmonary endothelial hyper-permeability, at least partly, in an MLC phosphorylation-mediated EC contraction which is influenced in a Ca2+-dependent manner and can lead to lung injury through mTOR-independent mechanisms.

Sodium butyrate enhances titanium nail osseointegration in ovariectomized rats by inhibiting the PKCα/NOX4/ROS/NF-κB pathways.

BACKGROUND: Elevated levels of oxidative stress as a consequence of estrogen deficiency serve as a key driver of the onset of osteoporosis (OP). In addition to increasing the risk of bone fractures, OP can reduce the bone volume proximal to titanium nails implanted to treat these osteoporotic fractures, thereby contributing to titanium nail loosening. Sodium butyrate (NaB) is a short-chain fatty acid produced by members of the gut microbiota that exhibits robust antioxidant and anti-inflammatory properties. METHODS: OP fracture model rats parameters including bone mineral density (BMD), new bone formation, and the number of bonelets around the implanted nail were analyzed via micro-CT scans, H&E staining, and Masson's staining. The protective effects of NaB on such osseointegration and the underlying mechanisms were further studied in vitro using MC3T3-E1 cells treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce oxidative stress. Techniques including Western immunoblotting, electron microscopy, flow cytometry, alkaline phosphatase (ALP) staining, and osteoblast mineralization assays were employed to probe behaviors such as reactive oxygen species production, mineralization activity, ALP activity, protein expression, and the ability of cells to attach to and survive on titanium plates. RESULTS: NaB treatment was found to enhance ALP activity, mineralization capacity, and Coll-I, BMP2, and OCN expression levels in CCCP-treated MC3T3-E1 cells, while also suppressing PKC and NF-κB expression and enhancing Nrf2 and HO-1 expression in these cells. NaB further suppressed intracellular ROS production and malondialdehyde levels within the cytosol while enhancing superoxide dismutase activity and lowering the apoptotic death rate. In line with these results, in vivo work revealed an increase in BMD in NaB-treated rats that was associated with enhanced bone formation surrounding titanium nails. CONCLUSION: These findings indicate that NaB may represent a valuable compound that can be postoperatively administered to aid in treating OP fractures through the enhancement of titanium nail osseointegration.

Selenomethionine promotes ANXA2 phosphorylation for proliferation and protein synthesis of myoblasts and skeletal muscle growth.

Selenomethionine (Se-Met) has many beneficial effects on higher animals and human, and can regulate cellular physiology through distinct signaling pathways. However, the role and molecular mechanism of Se-Met in skeletal muscle growth remains unclear. In this study, we observed the effects of Se-Met on C2C12 myoblasts and skeletal muscle growth of mice, and explored the corresponding molecular mechanism. Se-Met affected proliferation and protein synthesis of C2C12 myoblasts in a hormesis type of relationship, and had an optimal stimulatory effect at 50 µM concentration. Se-Met also affected mTOR, ANXA2, and PKCα phosphorylation in the same manner. ANXA2 knockdown blocked the stimulation of Se-Met on cell proliferation and protein synthesis and inhibition of Se-Met on autophagy of C2C12 myoblasts. Western blotting analysis showed that PI3K inhibition blocked the stimulation of Se-Met on mTOR phosphorylation. ANXA2 knockdown further blocked the stimulation of Se-Met on PI3K and mTOR phosphorylation. Point mutation experiment showed that ANXA2 mediated the stimulation of Se-Met on the PI3K-mTOR signaling through phosphorylation at Ser26. PKCα interacted with ANXA2, and PKCα knockdown blocked the stimulation of Se-Met on ANXA2 phosphorylation at Ser26. Se-Met addition (7.5mg/kg diet, 4 weeks) increased mouse carcass weight, promoted gastrocnemius skeletal muscle growth and ANXA2 and mTOR phosphorylation in this tissue. Collectively, our findings reveal that Se-Met can promote proliferation and protein synthesis of myoblasts and skeletal muscle growth through ANXA2 phosphorylation.

Titanium dioxide nanotubes increase purinergic receptor P2Y6 expression and activate its downstream PKCα-ERK1/2 pathway in bone marrow mesenchymal stem cells under osteogenic induction.

Titanium dioxide (TiO2) nanotubes can improve the osseointegration of pure titanium implants, but this exact mechanism has not been fully elucidated. The purinergic receptor P2Y6 is expressed in bone marrow mesenchymal stem cells (BMSCs) and participates in the regulation of bone metabolism. However, it is unclear as to whether P2Y6 is involved in the osteogenic differentiation of BMSCs induced by TiO2 nanotubes. TiO2 nanotubes were prepared on the surface of titanium specimens using the anodizing method and characterized their features. Quantitative reverse transcriptase polymerase chain reaction and western blotting were used to detect the expression of P2Y6, markers of osteogenic differentiation, and PKCα-ERK1/2. A rat femoral defect model was established to evaluate the osseointegration effect of TiO2 nanotubes combined with P2Y6 agonists. The results showed that the average inner diameter of the TiO2 nanotubes increased with an increase in voltage (voltage range of 30-90V), and the expression of P2Y6 in BMSCs could be upregulated by TiO2 nanotubes in osteogenic culture. Inhibition of P2Y6 expression partially inhibited the osteogenic effect of TiO2 nanotubes and downregulated the activity of the PKCα-ERK1/2 pathway. When using in vitro and in vivo experiments, the osteogenic effect of TiO2 nanotubes when combined with P2Y6 agonists was more pronounced. TiO2 nanotubes promoted the P2Y6 expression of BMSCs during osteogenic differentiation and promoted osteogenesis by activating the PKCα-ERK1/2 pathway. The combined application of TiO2 nanotubes and P2Y6 agonists may be an effective new strategy to improve the osseointegration of titanium implants. STATEMENT OF SIGNIFICANCE: Titanium dioxide (TiO2) nanotubes can improve the osseointegration of pure titanium implants, but this exact mechanism has not been fully elucidated. The purinergic receptor P2Y6 is expressed in bone marrow mesenchymal stem cells (BMSCs) and participates in the regulation of bone metabolism. However, it is unclear as to whether P2Y6 is involved in the osteogenic differentiation of BMSCs induced by TiO2 nanotubes. For the first time, this study revealed the relationship between TiO2 nanotubes and purine receptor P2Y6, and further explored its mode of action, which may provide clues as to the regulatory role of TiO2 nanotubes on osteogenic differentiation of BMSCs. These findings will help to develop novel methods for guiding material design and biosafety evaluation of nano implants.

Luteolin alleviates inorganic mercury-induced liver injury in quails by resisting oxidative stress and promoting mercury ion excretion.

BackgroundInorganic mercury is a well-known toxic substance that can cause oxidative stress and liver damage. Luteolin (Lut) is a kind of natural antioxidant, which is widely found in plants. Therefore, we focused on exploring the alleviative effect of Lut on liver injury induced by mercuric chloride (HgCl2), and the potential molecular mechanism of eliminating mercury ions in quails.Methods and resultsTwenty-one-day-old male quails were randomly split into four groups: control group, Lut group, HgCl2 group, and HgCl2 + Lut group. The test period was 12 weeks. The results showed that Lut could significantly ameliorate oxidative stress, the release of inflammatory factors, and liver damage caused by HgCl2, and reduce the accumulation of Hg2+ in quail liver. Furthermore, Lut evidently increased the levels of protein kinase C α (PKCα), nuclear factor-erythroid-2-related factor 2 (Nrf2), and its downstream proteins, and inhibited nuclear factor-kappaB (NF-κB) production in the liver of quails treated by HgCl2.ConclusionsTo sum up, our results suggest that Lut not only reduces the levels of oxidative stress and inflammation, but also promotes the excretion of Hg2+ by promoting the PKCα/Nrf2 signaling pathway to alleviate HgCl2-induced liver injury in quails.

Protective effect of ebselen on bleomycin-induced lung fibrosis: analysis of the molecular mechanism of lung fibrosis mediated by oxidized diacylglycerol.

The molecular mechanisms underlying the development of pulmonary fibrosis remain unknown, and effective treatments have not yet been developed. It has been shown that oxidative stress is involved in lung fibrosis. Oxidized diacylglycerol (DAG) produced by oxidative stress is thought to play an important role in lung fibrosis. This study assessed the effect of oxidized DAG in an animal model of pulmonary fibrosis induced by aspiration of bleomycin (BLM) into the lungs. The inhibitory effect of ebselen on pulmonary fibrosis was also investigated. In lung fibrotic tissue induced by BLM, an increase in lipid peroxides and collagen accumulation was observed. Moreover, the levels of oxidized DAG, which has strong protein kinase C (PKC) activation activity, were significantly increased over time following the administration of BLM. Western blotting showed that phosphorylation of PKCα and δ isoforms was increased by BLM. Oral administration of ebselen significantly suppressed the increase in oxidized DAG induced by BLM and improved lung fibrosis. PKCα and δ phosphorylation were also significantly inhibited. The mRNA expression of α-smooth muscle actin and collagen I (marker molecules for fibrosis), as well as the production of transforming growth factor-ß and tumor necrosis factor-α(a potentially important factor in the fibrotic process), were increased by BLM and significantly decreased by ebselen. The administration of BLM may induce lipid peroxidation in lung tissue, while the oxidized DAG produced by BLM may induce overactivation of PKCα and δ, resulting in the induction of lung fibrosis.

Neuroprotective effects of donepezil against Aß25-35-induced neurotoxicity.

PURPOSE: The purpose of this study was to investigate the neuroprotective effect of donepezil against ß-amyloid25-35 (Aß25-35)-induced neurotoxicity and the possible mechanism. METHODS: PC12 cells were conventionally cultured. Serial concentrations of Aß25-35 and donepezil (0, 0.5, 1, 5, 10, 20 and 50 µmol/L) were added to the PC12 cells, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) staining was performed to detect the effects of these treatments on PC 12 viability. The PC 12 cells were pretreated with 1, 5, 10, 20 or 50 µmol/L donepezil two hours before 20 µmol/L Aß25-35 was added to pretreatment groups A, B, C, D and E. Normal control group I and the 20 µmol/L Aß25-35-treated group were selected. An MTT assay was used to detect PC12 cell viability, and the level of lactate dehydrogenase (LDH) was determined. PC12 cells were pretreated with 10 µmol/L GF109203X (a protein kinase C [PKC] antagonist) 30 min before 10 µmol/L donepezil was added to pretreatment group F, and normal control group II, the 10 µmol/L GF109203X-treated group and the 10 µmol/L donepezil-treated group were chosen. The expression of phosphorylation-PKC (P-PKC) and its major substrate phosphorylated myristoylated alanine-rich protein C kinase substrate (P-MARCKS) was measured by Western blotting. The effects of donepezil on the subcellular distribution of the PKCα and PKCε isoforms were detected by immunofluorescence staining. RESULTS: Treatment with Aß25-35 (5, 10, 20 or 50 µmol/L) for 24 h significantly (P < 0.05) decreased PC 12 cell viability in a dose-dependent manner. Compared with the PC12 cells in the control group, those in the 20 µmol/L Aß25-35-treated group exhibited lower viability but higher LDH release. Compared with the 20 µmol/L Aß25-35-treated group, pretreatment groups B, C, D and E exhibited significantly (P < 0.05) increased cell viability but significantly (P < 0.05) decreased LDH release. Western blotting demonstrated that compared with control, 10 µmol/L donepezil promoted PKC and MARCKS phosphorylation and that the expression of P-PKC and P-MARCKS in pretreatment group F was significantly (P < 0.05) lower than that in the donepezil-treated group. Immunofluorescence staining revealed that the PKCα and PKCε isoforms were located mainly in the cytoplasm of PC12 control cells, whereas donepezil increased the expression of the PKCα and PKCε isoforms in the membrane fraction. The Western blot results showed that donepezil altered the subcellular distribution of the PKCα and PKCε isoforms by decreasing their expression in the cytosolic fraction but increasing their expression in the membrane fraction. CONCLUSION: Donepezil can antagonize Aß25-350-induced neurotoxicity in PC 12 cells, and PKC activation may account for the neuroprotective effect of donepezil.

Novel Smooth Muscle Ca2+-Signaling Nanodomains in Blood Pressure Regulation.

BACKGROUND: Ca2+ signals in smooth muscle cells (SMCs) contribute to vascular resistance and control blood pressure. Increased vascular resistance in hypertension has been attributed to impaired SMC Ca2+ signaling mechanisms. In this regard, transient receptor potential vanilloid 4 (TRPV4SMC) ion channels are a crucial Ca2+ entry pathway in SMCs. However, their role in blood pressure regulation has not been identified. METHODS: We used SMC-specific TRPV4-/- (TRPV4SMC-/-) mice to assess the role of TRPV4SMC channels in blood pressure regulation. We determined the contribution of TRPV4SMC channels to the constrictor effect of α1 adrenergic receptor (α1AR) stimulation and elevated intraluminal pressure: 2 main physiologic stimuli that constrict resistance-sized arteries. The contribution of spatially separated TRPV4SMC channel subpopulations to elevated blood pressure in hypertension was evaluated in angiotensin II-infused mice and patients with hypertension. RESULTS: We provide first evidence that TRPV4SMC channel activity elevates resting blood pressure in normal mice. α1AR stimulation activated TRPV4SMC channels through PKCα (protein kinase Cα) signaling, which contributed significantly to vasoconstriction and blood pressure elevation. Intraluminal pressure-induced TRPV4SMC channel activity opposed vasoconstriction through activation of Ca2+-sensitive K+ (BK) channels, indicating functionally opposite pools of TRPV4SMC channels. Superresolution imaging of SMCs revealed spatially separated α1AR:TRPV4 and TRPV4:BK nanodomains in SMCs. These data suggest that spatially separated α1AR-TRPV4SMC and intraluminal pressure-TRPV4SMC-BK channel signaling have opposite effects on blood pressure, with α1AR-TRPV4SMC signaling dominating under resting conditions. Furthermore, in patients with hypertension and a mouse model of hypertension, constrictor α1AR-PKCα-TRPV4 signaling was upregulated, whereas dilator pressure-TRPV4-BK channel signaling was disrupted, thereby increasing vasoconstriction and elevating blood pressure. CONCLUSIONS: Our data identify novel smooth muscle Ca2+-signaling nanodomains that regulate blood pressure and demonstrate their impairment in hypertension.

[Effects of typical PKC subtypes on the proliferation of mouse pulmonary artery smooth muscle cells and the expression of ERK1/2 and Akt induced by hypoxia].

Objective: To study the effects of specific isoforms of classic protein kinase C (cPKCs) on hypoxia-induced proliferation and the expression of ERK1/2 and Akt using drug intervention or virus transfection in vitro. Methods: Dynal MPC-1 magnetic particle concentrator was used to separate iron-containing pulmonary arterioles fragments, and the pulmonary artery smooth muscle cells (PASMCs) were primary cultured and identified. The cells were intervened by PKC agonist (PMA), PKCα inhibitor (safingol), PKCßⅠ inhibitor (Go6976) and PKCßⅡ inhibitor (LY333531) respectively, and the changes in protein expressions of cPKCs, and the phosphorylation levels of ERK1/2 and Akt were observed by immunoblotting under the condition of normal oxygen or hypoxia. The lentiviral vectors of PKCα and PKCß were used to specifically knock-down the activity of target genes by virus transfection techniques, and Western blotting was used to observe the protein expressions of cPKCs, and the phosphorylation levels of ERK1/2 and Akt in hypoxia-induced PASMCs in mice. Results: With Brdu method, the proliferation of PASMCs induced by hypoxia was significantly inhibited by safingol, Go6976 and LY333531 by inhibiting cPKCα, ßⅠ and ßⅡ respectively. Compared with the hypoxic control group, the rates of Brdu positive cells were (7.35±0.26)% vs (11.28±0.43)%, (3.76±0.25)% vs (7.98±0.28)% and (4.12±0.46)% vs (7.78±0.53)%. We also observed that PMA could significantly promote the proliferation of PASMCs under normoxic condition. Compared with the normoxia control group, the Brdu-positive cell rates were (9.65±0.47)% vs (6.34±0.52)%, (9.34±0.38)% vs (5.42±0.21)% and (7.78±0.53)% vs (4.12±0.46)%. In addition, after transfection with PKCα or PKCß lentiviral vector, the proliferation of PASMCs was significantly lower in hypoxia transfection group than in the control group. The rates of Brdu positive cells were (3.58±0.54)% vs (5.97±0.63)%, respectively. Using Western blotting, we also observed that after being inhibited by safingol, Go6976 and LY333531 respectively, the phosphorylation levels of ERK1/2 and Akt in PASMCs induced by hypoxia was significantly lower than the control group. After using safingol, the phosphorylation levels of ERK1/2 and Akt were (0.56±0.07) vs (1.08±0.13) and (0.49±0.04) vs (0.97±0.08). After using Go6976, the phosphorylation levels of ERK1/2 and Akt were (0.41±0.09) vs (0.79±0.10) and (0.48±0.09) vs (0.82±0.16), after using LY333531, the phosphorylation levels of ERK1/2 and Akt were (0.42±0.03) vs (0.87±0.06) and (0.34±0.07) vs (0.78±0.05). While PMA could promote the phosphorylation levels of ERK1/2 and Akt under normoxic condition, 1.25±0.12 vs 0.41±0.07 and 0.98±0.06 vs 0.37±0.08, respectively. Using transfection technique to specifically knock down the expression of cPKCα and ß, we found that under hypoxic conditions, transfection of PASMCs could significantly lower the phosphorylation levels of ERK1/2, its phosphorylation level was 0.29±0.06 vs 0.76±0.05, with no evident change in the phosphorylation levels of Akt. Conclusions: Hypoxia may lead to phosphorylation of ERK1/2 by promoting the protein expression of cPKCα, cPKCßⅠ and cPKCßⅡ respectively, which eventually induces abnormal proliferation of PASMCs from the distal pulmonary arteries, participating in the development of hypoxic pulmonary hypertension (HPH) of the mice. Regulation of the expression of cPKCα, cPKCßⅠ and cPKCßⅡ may help to attenuate the formation of pulmonary vascular remodeling. Target therapy based on cPKCs is expected to be a new direction for HPH therapy in the future.

Maprotiline Ameliorates High Glucose-Induced Dysfunction in Renal Glomerular Endothelial Cells.

Maprotiline is an antidepressant that has been found to cause hypoglycemia. However, the effect of maprotiline on diabetic nephropathy (DN) has not been investigated. Here, we explored the effect of maprotiline on human renal glomerular endothelial cells (HRGECs) in response to high glucose (HG) stimulation. We found that maprotiline attenuated HG-induced oxidative stress in HRGECs with decreased reactive oxygen species production and increased superoxide dismutase activity. Maprotiline repressed the HG-induced expression of cyclooxygenases 2 at both mRNA and protein levels in HRGECs. The increased thromboxane B2 level and decreased 6-keto-prostaglandin F1α level induced by HG were significantly attenuated by maprotiline treatment. Maprotiline also prevented the HG-induced increase in the permeability of HRGECs and the decrease in the zonula occludens-1 expression and downregulated HG-induced increase in the expression of protein kinase C-α (PKC-α) in HRGECs. This protective effect of maprotiline on HG-induced HRGECs dysfunction was abolished by overexpression of PKC-α. In conclusion, maprotiline displayed a protective effect on HG-challenged HRGECs, which was mediated by the regulation of PKC-α. These findings provide further evidence for the potential use of maprotiline for the treatment of DN.

Activation of GPR75 Signaling Pathway Contributes to the Effect of a 20-HETE Mimetic, 5,14-HEDGE, to Prevent Hypotensive and Tachycardic Responses to Lipopolysaccharide in a Rat Model of Septic Shock.

ABSTRACT: The orphan receptor, G protein-coupled receptor (GPR) 75, which has been shown to mediate various effects of 20-hydroxyeicosatetraenoic acid (20-HETE), is considered as a therapeutic target in the treatment of cardiovascular diseases in which changes in the production of 20-HETE play a key role in their pathogenesis. Our previous studies showed that 20-HETE mimetic, N -(20-hydroxyeicosa-5[Z],14[Z]-dienoyl)glycine (5,14-HEDGE), protects against vascular hyporeactivity, hypotension, tachycardia, and arterial inflammation induced by lipopolysaccharide (LPS) in rats. This study tested the hypothesis that the GPR75 signaling pathway mediates these effects of 5,14-HEDGE in response to systemic exposure to LPS. Mean arterial pressure reduced by 33 mm Hg, and heart rate increased by 102 beats/min at 4 hours following LPS injection. Coimmunoprecipitation studies demonstrated that (1) the dissociation of GPR75/Gα q/11 and GPR kinase interactor 1 (GIT1)/protein kinase C (PKC) α, the association of GPR75/GIT1, large conductance voltage and calcium-activated potassium subunit ß (MaxiKß)/PKCα, MaxiKß/proto-oncogene tyrosine-protein kinase (c-Src), and epidermal growth factor receptor (EGFR)/c-Src, MaxiKß, and EGFR tyrosine phosphorylation were decreased, and (2) the association of GIT1/c-Src was increased in the arterial tissues of rats treated with LPS. The LPS-induced changes were prevented by 5,14-HEDGE. N -[20-Hydroxyeicosa-6( Z ),15( Z )-dienoyl]glycine, a 20-HETE antagonist, reversed the effects of 5,14-HEDGE in the arterial tissues of LPS-treated rats. Thus, similar to 20-HETE, by binding to GPR75 and activating the Gα q/11 /PKCα/MaxiKß, GIT1/PKCα/MaxiKß, GIT1/c-Src/MaxiKß, and GIT1/c-Src/EGFR signaling pathways, 5,14-HEDGE may exert its protective effects against LPS-induced hypotension and tachycardia associated with vascular hyporeactivity and arterial inflammation.

Dexmedetomidine modulates mitochondrial dynamics to protect against endotoxin-induced lung injury via the protein kinase C-ɑ/haem oxygenase-1 signalling pathway.

BACKGROUND: Endotoxin-induced acute lung injury (ALI) has a high mortality rate, and there are limited effective treatment options available. The aim of the present study was to identify if dexmedetomidine could regulate mitochondrial fusion and fission through the protein kinase C (PKC)-α/haem oxygenase (HO)-1 pathway to protect against endotoxin-induced ALI. MATERIALS AND METHODS: Dexmedetomidine was administered by intraperitoneal injection once daily for three days prior to induction of lung injury to mice. Mice in the PKC-α inhibitor group received dexmedetomidine by intraperitoneal injection 1 h after each chelerythrine injection, and lipopolysaccharide was injected 1 h after the last dose of dexmedetomidine. The lung wet/dry weight ratio, oxidative stress, inflammatory response, and expression levels of PKC-α, Nrf2, HO-1, Mfn1, Mfn2, OPA1, Drp1, and Fis1 were determined. RESULTS: Dexmedetomidine administration attenuated lung oxidative stress, decreased inflammatory cytokines secretion, and downregulated the expression levels of Drp1 and Fis1. Moreover, dexmedetomidine increased levels of Mfn1, Mfn2, and OPA1, and alleviated endotoxin-induced lung injury. Administration of chelerythrine partially reversed the pneumoprotective effects of dexmedetomidine. CONCLUSIONS: Dexmedetomidine may activate the PKC-ɑ/HO-1 pathway to increase the expression of Mfn1, Mfn2, and OPA1, while decreasing Drp1 and Fis1 expression, thereby reduce endotoxin-induced acute lung injury.

PKCα-mediated phosphorylation of the diacylglycerol kinase ζ MARCKS domain switches cell migration modes by regulating interactions with Rac1 and RhoA.

Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.

Modulation of Bax mitochondrial insertion and induced cell death in yeast by mammalian protein kinase Cα.

Protein kinase Cα (PKCα) is a classical PKC isoform whose involvement in cell death is not completely understood. Bax, a major member of the Bcl-2 family, is required for apoptotic cell death and regulation of Bax translocation and insertion into the outer mitochondrial membrane is crucial for regulation of the apoptotic process. Here we show that PKCα increases the translocation and insertion of Bax c-myc (an active form of Bax) into the outer membrane of yeast mitochondria. This is associated with an increase in cytochrome c (cyt c) release, reactive oxygen species production (ROS), mitochondrial network fragmentation and cell death. This cell death process is regulated, since it correlates with an increase in autophagy but not with plasma membrane permeabilization. The observed increase in Bax c-myc translocation and insertion by PKCα is not due to Bax c-myc phosphorylation, and the higher cell death observed is independent of the PKCα kinase activity. PKCα may therefore have functions other than its kinase activity that aid in Bax c-myc translocation and insertion into mitochondria. Together, these results give a mechanistic insight on apoptosis regulation by PKCα through regulation of Bax insertion into mitochondria.

PKCalpha-induced drug resistance in pancreatic cancer cells is associated with transforming growth factor-beta1.

BACKGROUND: Drug resistance remains a great challenge in the treatment of pancreatic cancer. The goal of this study was to determine whether TGF-beta1 is associated with drug resistance in pancreatic cancer. METHODS: Pancreatic cancer BxPC3 cells were stably transfected with TGF-beta1 cDNA. Cellular morphology and cell cycle were determined and the suppressive subtracted hybridization (SSH) assay was performed to identify differentially expressed genes induced by TGF-beta1. Western blotting and immunohistochemistry were used to detect expression of TGF-beta1-related genes in the cells and tissue samples. After that, the cells were further treated with an anti-cancer drug (e.g., cisplatin) after pre-incubated with the recombinant TGF-beta1 plus PKCalpha inhibitor Gö6976. TGF-beta1 type II receptor, TbetaRII was also knocked down using TbetaRII siRNA to assess the effects of these drugs in the cells. Cell viability was assessed by MTT assay. RESULTS: Overexpression of TGF-beta1 leads to a markedly increased invasion potential but a reduced growth rate in BxPC3 cells. Recombinant TGF-beta1 protein increases expression of PKCalpha in BxPC3 cells, a result that we confirmed by SSH. Moreover, TGF-beta1 reduced the sensitivity of BxPC3 cells to cisplatin treatment, and this was mediated by upregulation of PKCalpha. However, blockage of PKCalpha with Gö6976 and TbetaRII with siRNA reversed the resistance of BxPC3 cells to gemcitabine, even in the presence of TGF-beta1. Immunohistochemical data show that pancreatic cancers overexpress TGF-beta1 and P-gp relative to normal tissues. In addition, TGF-beta1 expression is associated with P-gp and membranous PKCalpha expression in pancreatic cancer. CONCLUSIONS: TGF-beta1-induced drug resistance in pancreatic cancer cells was associated with PKCalpha expression. The PKCalpha inhibitor Gö6976 could be a promising agent to sensitize pancreatic cancer cells to chemotherapy.

Excision of titin's cardiac PEVK spring element abolishes PKCalpha-induced increases in myocardial stiffness.

Protein kinase C-alpha (PKCalpha) was recently reported to increase myocardial stiffness, an effect that was proposed to be due to phosphorylation of two highly conserved sites (S11878 and S12022) within the proline-glutamic acid-valine-lysine (PEVK) rich spring element of titin. To test this proposal we investigated the effect of PKCalpha on phosphorylation and passive stiffness in a mouse model lacking the titin exons that contain these two phosphorylation sites, the PEVK knockout (KO). We used skinned, gelsolin-extracted, left ventricular myocardium from wildtype and PEVK KO mice. Consistent with previous work we found that PKCalpha increased passive stiffness in the WT myocardium by 27+/-6%. Importantly, this effect was completely abolished in KO myocardium. In addition, increases in the elastic and viscous moduli at a wide range of frequencies (properties important in diastolic filling) following PKCalpha incubation (27+/-3% and 20+/-4%, respectively) were also ablated in the KO. Back phosphorylation assays showed that titin phosphorylation following incubation with PKCalpha was significantly reduced by 36+/-12% in skinned PEVK KO myocardial tissues. The remaining phosphorylation in the KO suggests that PKCalpha sites exist in the titin molecule outside the PEVK region; these sites are not involved in increasing passive stiffness. Our results firmly support that the PEVK region of cardiac titin is phosphorylated by PKCalpha and that this increases passive tension. Thus, the PEVK spring element is the critical site of PKCalpha's involvement in passive myocardial stiffness.

Activation of ERK1/2 by protein kinase C-alpha in response to hydrogen peroxide-induced cell death in human gingival fibroblasts.

Hydrogen peroxide (H(2)O(2)) increases protein tyrosine phosphorylation of numerous proteins in human gingival fibroblasts (HGFs). Two main proteins, with an apparent molecular weight of 44 and 42kDa, were phosphorylated after hydrogen peroxide stimulation of the human gingival fibroblasts. Further analysis identified these two proteins as ERK1/2. Maximum phosphorylation was detected at 10min post-H(2)O(2) treatment. Pretreatment with an MEK inhibitor, PD98059, inhibited H(2)O(2)-stimulated ERK1/2 phosphorylation in a dose-dependent manner. Treatment with H(2)O(2) also induced phosphorylation of protein kinase C-alpha (PKCalpha). Staurosporine, a PKC inhibitor, blocked ERK1/2 phosphorylation induced by H(2)O(2). In addition, H(2)O(2)-induced cell death was prevented by PD98059, SB203580, and calphostin C, which are MEK, p38 and PKC inhibitors, respectively. These results suggest that H(2)O(2) leads to the phosphorylation and activation of ERK1/2 in a PKC-dependent manner. These findings demonstrate that the MAPK signaling pathway plays an active role in mediating the H(2)O(2)-induced decrease in HGF cell viability and ATP depletion.

Formation of reactive oxygen species in lung alveolar cells: effect of vitamin E deficiency.

Reactive oxygen species (ROS) play an important role in the pathogenesis of numerous pulmonary diseases. Various mainly membrane-bound ROS-generating processes exist in alveolar cells. Vitamin E (vit. E) is the most important lipophilic antioxidant. However, the significance of vit. E levels in alveolar cells for the regulation of ROS generation has not been investigated so far. We demonstrated here that feeding rats with vit. E-depleted nourishment for 5 weeks reduced the concentration of vit. E in alveolar type II cell preparations to one-fifth the amount of control animals. This reduction of vit. E levels was associated with an approximately threefold increase in ROS generation in type II pneumocytes, lymphocytes, and macrophages. The contribution of individual processes of ROS formation in control animals differed strongly among these three cell types. However, vit. E deficiency induced predominantly nonmitochondrial ROS formation in alveolar cells. Expression and NAD(P)H-oxidase activity in alveolar type II cell preparations was not affected by vit. E deficiency. Moreover, protein kinase C (PKC) also did not seem to be responsible for vit. E deficiency-induced ROS generation in alveolar cells. Alimentary vit. E supplementation for 2 days corrected the cellular vit. E concentration but failed to normalize ROS generation in alveolar cells. These data let us assume that alimentary vit. E deficiency caused a preferentially nonmitochondria-mediated increase of ROS formation in type II pneumocytes, macrophages, and lymphocytes. However, the short-term supplementation of vit. E does not reverse these effects.

Augmented protein kinase C-alpha-induced myofilament protein phosphorylation contributes to myofilament dysfunction in experimental congestive heart failure.

It is becoming clear that upregulated protein kinase C (PKC) signaling plays a role in reduced ventricular myofilament contractility observed in congestive heart failure. However, data are scant regarding which PKC isozymes are involved. There is evidence that PKC-alpha may be of particular importance. Here, we examined PKC-alpha quantity, activity, and signaling to myofilaments in chronically remodeled myocytes obtained from rats in either early heart failure or end-stage congestive heart failure. Immunoblotting revealed that PKC-alpha expression and activation was unaltered in early heart failure but increased in end-stage congestive heart failure. Left ventricular myocytes were isolated by mechanical homogenization, Triton-skinned, and attached to micropipettes that projected from a force transducer and motor. Myofilament function was characterized by an active force-[Ca(2+)] relation to obtain Ca(2+)-saturated maximal force (F(max)) and myofilament Ca(2+) sensitivity (indexed by EC(50)) before and after incubation with PKC-alpha, protein phosphatase type 1 (PP1), or PP2a. PKC-alpha treatment induced a 30% decline in F(max) and 55% increase in the EC(50) in control cells but had no impact on myofilament function in failing cells. PP1-mediated dephosphorylation increased F(max) (15%) and decreased EC(50) ( approximately 20%) in failing myofilaments but had no effect in control cells. PP2a-dependent dephosphorylation had no effect on myofilament function in either group. Lastly, PP1 dephosphorylation restored myofilament function in control cells hyperphosphorylated with PKC-alpha. Collectively, our results suggest that in end-stage congestive heart failure, the myofilament proteins exist in a hyperphosphorylated state attributable, in part, to increased activity and signaling of PKC-alpha.

VCAM-1 signals activate endothelial cell protein kinase Calpha via oxidation.

Lymphocyte binding to VCAM-1 activates endothelial cell NADPH oxidase, resulting in the generation of 1 muM H(2)O(2). This is required for VCAM-1-dependent lymphocyte migration. In this study, we identified a role for protein kinase Calpha (PKCalpha) in VCAM-1 signal transduction in human and mouse endothelial cells. VCAM-1-dependent spleen cell migration under 2 dynes/cm(2) laminar flow was blocked by pretreatment of endothelial cells with dominant-negative PKCalpha or the PKCalpha inhibitors, Rö-32-0432 or Gö-6976. Phosphorylation of PKCalpha(Thr638), an autophosphorylation site indicating enzyme activity, was increased by Ab cross-linking of VCAM-1 on endothelial cells or by the exogenous addition of 1 muM H(2)O(2). The anti-VCAM-1-stimulated phosphorylation of PKCalpha(Thr638) was blocked by scavenging of H(2)O(2) and by inhibition of NADPH oxidase. Furthermore, anti-VCAM-1 signaling induced the oxidation of endothelial cell PKCalpha. Oxidized PKCalpha is a transiently active form of PKCalpha that is diacylglycerol independent. This oxidation was blocked by inhibition of NADPH oxidase. In summary, VCAM-1 activation of endothelial cell NADPH oxidase induces transient PKCalpha activation that is necessary for VCAM-1-dependent transendothelial cell migration.