Intestinal Dysbiosis Secondary to Proton-Pump Inhibitor Use.

BACKGROUND: Gut dysbiosis associated with the use of proton-pump inhibitors (PPIs) has been found to lead to the occurrence of infectious and inflammatory adverse events. A longitudinal observational cohort study has demonstrated the heightened risk of death associated with PPI use. SUMMARY: We evaluated meta-analyses to determine the association between PPI use and infectious and inflammatory diseases. Meta-analyses showed that PPI use is a potential risk for the development of enteric infections caused by Clostridium difficile, as well as small intestinal bacterial overgrowth, spontaneous bacterial peritonitis, community-acquired pneumonia, hepatic encephalopathy, and adverse outcomes in inflammatory bowel disease. We also examined changes in the composition and function of the gut microbiota with the use of PPIs. PPI use significantly increased the presence of Streptococcaceae and Enterococcaceae, which are risk factors for C. difficile infection, and decreased that of Faecalibacterium, a commensal anti-inflammatory microorganism. Key Message: High-throughput, microbial 16S rRNA gene sequencing has allowed us to investigate the association between the gut microbiome and PPI use. Future prospective comparison studies are necessary to confirm this association, and to develop new strategies to prevent complications of PPI use.

PKC-ε pseudosubstrate and catalytic activity are necessary for membrane delivery during IgG-mediated phagocytosis.

In RAW 264.7 cells, PKC-ε regulates FcγR-mediated phagocytosis. BMDM behave similarly; PKC-ε concentrates at phagosomes and internalization are reduced in PKC-ε⁻/⁻ cells. Two questions were asked: what is the role of PKC-ε? and what domains are necessary for PKC-ε concentration? Function was studied using BMDM and frustrated phagocytosis. On IgG surfaces, PKC-ε⁻/⁻ macrophages spread less than WT. Patch-clamping revealed that the spreading defect is a result of the failure of PKC-ε⁻/⁻ macrophages to add membrane. The defect is specific for FcγR ligation and can be reversed by expression of full-length (but not the isolated RD) PKC-ε in PKC-ε⁻/⁻ BMDM. Thus, PKC-ε function in phagocytosis requires translocation to phagosomes and the catalytic domain. The expression of chimeric PKC molecules in RAW cells identified the εPS as necessary for PKC-ε targeting. When placed into (nonlocalizing) PKC-δ, εPS was sufficient for concentration, albeit to a lesser degree than intact PKC-ε. In contrast, translocation of δ(εPSC1B) resembled that of WT PKC-ε. Thus, εPS and εC1B cooperate for optimal phagosome targeting. Finally, cells expressing εK437W were significantly less phagocytic than their PKC-ε-expressing counterparts, blocked at the pseudopod-extension phase. In summary, we have shown that εPS and εC1B are necessary and sufficient for targeting PKC-ε to phagosomes, where its catalytic activity is required for membrane delivery and pseudopod extension.

Both C1B domain and pseudosubstrate region are necessary for saturated fatty acid-induced translocation of εPKC to the plasma membrane: distinct role of intramolecular domains for different translocation.

It is well known that protein kinase C (PKC) shows different translocation depending on subtype and stimulation, contributing to the physiological importance of the enzyme. However, molecular mechanism causing the different translocation has been unknown. Therefore, using GFP-tagged mutant εPKC, we attempted to identify the intramolecular domains required for saturated fatty acid-induced translocation of εPKC to the plasma membrane, and compared with those necessary for unsaturated fatty acid-induced translocation to the Golgi complex. We found that, unlike in the case of unsaturated fatty-acid induced translocation, both C1B domain and pseudosubstrate region are necessary for the saturated fatty acid-induced translocation of εPKC to the plasma membrane. The results suggest that different domains of PKC mediate distinct translocation depending on different stimulations, contributing to their subtype- and stimulation-specific functions.

Rapid degradation of cyclooxygenase-1 and hematopoietic prostaglandin D synthase through ubiquitin-proteasome system in response to intracellular calcium level.

Cyclooxygenase (COX)-1 and hematopoietic prostaglandin (PG) D synthase (H-PGDS) proteins, which are both involved in the arachidonate cascade, were stable in human megakaryocytic MEG-01 cells. In contrast, once the intracellular calcium level was increased by treatment with a calcium ionophore, both protein levels rapidly decreased with a half-life of less than 30 and 120 min for COX-1 and H-PGDS, respectively. In the presence of a proteasome inhibitor, COX-1 and H-PGDS proteins accumulated within 10 and 30 min, respectively, and concurrently appeared as the high-molecular-mass ubiquitinated proteins within 30 and 60 min, respectively, after an increase in the intracellular calcium level. The ubiquitination of these proteins was also observed when ADP, instead of a calcium ionophore, was used as an inducer to elevate the intracellular calcium level. When the entry of calcium ion into the cells was inhibited by ethylene glycol tetraacetic acid (EGTA), the ubiquitination of COX-1 and H-PGDS was clearly suppressed; and the addition of CaCl(2) to the medium cleared the EGTA-mediated suppression of the ubiquitination. These results indicate that COX-1 and H-PGDS were rapidly ubiquitinated and degraded through the ubiquitin-proteasome system in response to the elevation of the intracellular calcium level.

Direct binding of RalA to PKCη and its crucial role in morphological change during keratinocyte differentiation.

During differentiation, keratinocytes undergo a dramatic shape change from small and round to large and flat, in addition to production of proteins necessary for the formation of epidermis. It has been shown that protein kinase C (PKC) η is crucial for keratinocyte differentiation. However, its role in this process has yet to be fully elucidated. Here, we show that catalytic activity is not necessary for enlarged and flattened morphology of human keratinocytes induced by overexpression of PKCη, although it is important for gene expression of the marker proteins. In addition, we identify the small G protein RalA as a binding partner of PKCη, which binds to the C1 domain, an indispensable region for the morphological change. The binding led activation of RalA and actin depolymerization associated with keratinocyte differentiation. siRNA techniques proved that RalA is involved in not only the keratinocyte differentiation induced by PKCη overexpression but also normal keratinocyte differentiation induced by calcium and cholesterol sulfate. These results provide a new insight into the molecular mechanism of cytoskeletal regulation leading to drastic change of cell shape.

Suppression of adipocyte differentiation by aldo-keto reductase 1B3 acting as prostaglandin F2alpha synthase.

Prostaglandin (PG) F(2alpha) suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor gamma. However, PGF(2alpha) synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF(2alpha), was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF(2alpha) production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor gamma, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF(2alpha). These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF(2alpha) suppressed adipocyte differentiation by acting through FP receptors.

Mutant gammaPKC found in spinocerebellar ataxia type 14 induces aggregate-independent maldevelopment of dendrites in primary cultured Purkinje cells.

Missense mutations in protein kinase Cgamma (gammaPKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that mutant gammaPKC found in SCA14 is susceptible to aggregation and induces apoptosis in cultured cell lines. In the present study, we investigated whether mutant gammaPKC formed aggregates and how mutant gammaPKC affects the morphology and survival of cerebellar Purkinje cells (PCs), which are degenerated in SCA14 patients. Adenovirus-transfected primary cultured PCs expressing mutant gammaPKC-GFP also had aggregates and underwent apoptosis. Long-term time-lapse observation revealed that PCs have a potential to eliminate aggregates of mutant gammaPKC-GFP. Mutant gammaPKC-GFP disturbed the development of PC dendrites and reduced synapse formation, regardless of the presence or absence of its aggregates. In PCs without aggregates, mutant gammaPKC-GFP formed soluble oligomers, resulting in reduced mobility and attenuated translocation of mutant gammaPKC-GFP upon stimulation. These molecular properties of mutant gammaPKC might affect the dendritic morphology in PCs, and be involved in the pathogenesis of SCA14.

Synthesis, conformational analysis, and biological evaluation of 1-hexylindolactam-V10 as a selective activator for novel protein kinase C isozymes.

Conventional and novel protein kinase C (PKC) isozymes are the main targets of tumor promoters. We developed 1-hexylindolactam-V10 ( 5) as a selective activator for novel PKC isozymes that play important roles in various cellular processes related to tumor promotion, ischemia--reperfusion injury in the heart, and Alzheimer's disease. The compound existed as a mixture of three conformers. The trans-amide restricted analogues of 5 ( 14 and 15) hardly bound to PKC isozymes, suggesting that the active conformation of 5 could be that with a cis-amide. Compound 5 selectively translocated novel PKC isozymes over conventional PKC isozymes in HeLa cells at 0.1-1 microM. These results suggest that 5 could be useful for the functional analysis of novel PKC isozymes.

Immunogold electron microscopic demonstration of distinct submembranous localization of the activated gammaPKC depending on the stimulation.

We examined the precise intracellular translocation of gamma subtype of protein kinase C (gammaPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged gammaPKC (gammaPKC-GFP) on the plasma membrane. In contrast, treatment with Ca(2+) ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of gammaPKC-GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that gammaPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of gammaPKC-GFP on the plasma membrane, Ca(2+) ionophore and NMDA rapidly translocated gammaPKC-GFP to the plasma membrane and then restricted gammaPKC-GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca(2+) influx alone induced the association of gammaPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of gammaPKC in response to various stimuli suggests a novel mechanism for PKC activation.

Two-photon bioimaging utilizing supercontinuum light generated by a high-peak-power picosecond semiconductor laser source.

We developed a novel scheme for two-photon fluorescence bioimaging. We generated supercontinuum (SC) light at wavelengths of 600 to 1200 nm with 774-nm light pulses from a compact turn-key semiconductor laser picosecond light pulse source that we developed. The supercontinuum light was sliced at around 1030- and 920-nm wavelengths and was amplified to kW-peak-power level using laboratory-made low-nonlinear-effects optical fiber amplifiers. We successfully demonstrated two-photon fluorescence bioimaging of mouse brain neurons containing green fluorescent protein (GFP).

Design and synthesis of 8-octyl-benzolactam-V9, a selective activator for protein kinase C epsilon and eta.

Conventional (alpha, betaI, betaII, gamma) and novel (delta, epsilon, eta, theta) protein kinase C (PKC) isozymes are main targets of tumor promoters, such as phorbol esters and indolactam-V (ILV). We have recently found that 1-hexyl derivatives of indolinelactam-V (2, 3), in which the indole ring of ILV was replaced with the indoline ring, showed a binding preference for novel PKCs over conventional PKCs. To develop a new ILV analogue displaying increased synthetic accessibility and improved binding selectivity for novel PKCs, we have designed 8-octyl-benzolactam-V9 (4), a simple analogue without the pyrrolidine moiety of 2 and 3. Compound 4 showed significant binding selectivity for isolated C1B domains of novel PKCs. Moreover, 4 translocated PKC epsilon and eta from the cytoplasm to the plasma membrane of HeLa cells at 1 microM, whereas other PKC isozymes did not respond even at 10 microM. These results indicate that 4 could be a selective activator for PKC epsilon and eta.

Targeting of protein kinase C-epsilon during Fcgamma receptor-dependent phagocytosis requires the epsilonC1B domain and phospholipase C-gamma1.

Protein kinase C-epsilon (PKC-epsilon) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-epsilon mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding epsilonC1 and epsilonC1B domains, or the epsilonC1B point mutant epsilonC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that epsilonC259G, epsilonC1, and epsilonC1B accumulation at phagosomes was significantly less than that of intact PKC-epsilon. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-epsilon translocation. Thus, DAG binding to epsilonC1B is necessary for PKC-epsilon translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-gamma1, and PI-PLC-gamma2 in PKC-epsilon accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-epsilon localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-epsilon accumulation. Although expression of PI-PLC-gamma2 is higher than that of PI-PLC-gamma1, PI-PLC-gamma1 but not PI-PLC-gamma2 consistently concentrated at phagosomes. Macrophages from PI-PLC-gamma2-/- mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-epsilon at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-gamma1 as the enzyme that supports PKC-epsilon localization and phagocytosis. That PI-PLC-gamma1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-gamma1 provides DAG that binds to epsilonC1B, facilitating PKC-epsilon localization to phagosomes for efficient IgG-mediated phagocytosis.

Isoform-specific phosphorylation of metabotropic glutamate receptor 5 by protein kinase C (PKC) blocks Ca2+ oscillation and oscillatory translocation of Ca2+-dependent PKC.

Prolonged activation of metabotropic glutamate receptor 5a (mGluR5a) causes synchronized oscillations in intracellular calcium, inositol 1,4,5-trisphosphate production, and protein kinase C (PKC) activation. Additionally, mGluR5 stimulation elicited cyclical translocations of myristoylated alanine-rich protein kinase C substrate, which were opposite to that of gammaPKC (i.e. from plasma membrane to cytosol) and dependent on PKC activity, indicating that myristoylated alanine-rich protein kinase C substrate is repetitively phosphorylated by oscillating gammaPKC on the plasma membrane. Mutation of mGluR5 Thr(840) to aspartate abolished the oscillation of gammaPKC, but the mutation to alanine (T840A) did not. Cotransfection of gammaPKC with betaIIPKC, another Ca2+-dependent PKC, resulted in synchronous oscillatory translocation of both classical PKCs. In contrast, cotransfection of deltaPKC, a Ca2+-independent PKC, abolished the oscillations of both gammaPKC and inositol 1,4,5-trisphosphate. Regulation of the oscillations was dependent on deltaPKC kinase activity but not on gammaPKC. Furthermore, the T840A-mGluR5-mediated oscillations were not blocked by the deltaPKC overexpression. These results revealed that activation of mGluR5 causes translocation of both gammaPKC and deltaPKC to the plasma membrane. deltaPKC, but not gammaPKC, phosphorylates mGluR5 Thr(840), leading to the blockade of both Ca2+ oscillations and gammaPKC cycling. This subtype-specific targeting proposes the molecular basis of the multiple functions of PKC.

Establishment of a binding assay for protein kinase C isozymes using synthetic C1 peptides and development of new medicinal leads with protein kinase C isozyme and C1 domain selectivity.

Conventional and novel protein kinase C (PKC) isozymes contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12, 13-dibutyrate (PDBu)-binding sites. We synthesized C1 peptides of 50-70 residues corresponding to all PKC isozyme C1 domains using an Fmoc solid-phase strategy. These C1 peptides were successfully folded by zinc treatment, as monitored by electrospray ionization time-of-flight mass spectrometry. We measured the K(d)'s of [3H]PDBu for all PKC C1 peptides. Most of the C1 peptides, except for delta-C1A and theta-C1A, showed strong PDBu binding affinities with K(d)'s in the nanomolar range (0.45-7.4 nM) comparable with the respective whole PKC isozymes. The resultant C1 peptide library can be used to screen for new ligands with PKC isozyme and C1 domain selectivity. Non-tumor-promoting 1-oleoyl-2-acetyl-sn-glycerol and bryostatin 1 showed relatively strong binding to all CIA peptides of novel PKCs (delta, epsilon, and eta). In contrast, the tumor promoters (-)-indolactam-V, ingenol-3-benzoate, and PDBu bound selectively to all C1B peptides of novel PKCs. The preference of tumor promoters for the domain might be related to tumorigenesis since recent investigations proposed the involvement of novel PKCs in tumor promotion in vivo using transgenic or knockout mice. Moreover, we recently have found that a new lactone analogue of benzolactams (6) shows significant selectivity in PKCeta-C1B binding.

Importance of C1B domain for lipid messenger-induced targeting of protein kinase C.

The molecular mechanisms by which arachidonic acid (AA) and ceramide elicit translocation of protein kinase C (PKC) were investigated. Ceramide translocated epsilonPKC from the cytoplasm to the Golgi complex, but with a mechanism distinct from that utilized by AA. Using fluorescence recovery after photobleaching, we showed that, upon treatment with AA, epsilonPKC was tightly associated with the Golgi complex; ceramide elicited an accumulation of epsilonPKC which was exchangeable with the cytoplasm. Stimulation with ceramide after AA converted the AA-induced Golgi complex staining to one elicited by ceramide alone; AA had no effect on the ceramide-stimulated localization. Using point mutants and deletions of epsilonPKC, we determined that the epsilonC1B domain was responsible for the ceramide- and AA-induced translocation. Switch chimeras, containing the C1B from epsilonPKC in the context of deltaPKC (delta(epsilonC1B)) and vice versa (epsilon(deltaC1B)), were generated and tested for their translocation in response to ceramide and AA. delta(epsilonC1B) translocated upon treatment with both ceramide and AA; epsilon(deltaC1B) responded only to ceramide. Thus, through the C1B domain, AA and ceramide induce different patterns of epsilonPKC translocation and the C1B domain defines the subtype specific sensitivity of PKCs to lipid second messengers.