Kaori Kashiwagi

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2α Synthase

Prostaglandin (PG) F2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF2α 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 PGF2α, 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 PGF2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, 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 PGF2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF2α suppressed adipocyte differentiation by acting through FP receptors.

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.

Mutant γPKC 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.

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).

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

In RAW 264.7 cells [ 1 ], PKC‐ɛ regulates FcγR‐mediated phagocytosis. BMDM behave similarly; PKC‐ɛ concentrates at phagosomes and internalization are reduced in PKC‐ɛ−/− cells. Two questions were asked: what is the role of PKC‐ɛ? and what domains are necessary for PKC‐ɛ concentration? Function was studied using BMDM and frustrated phagocytosis. On IgG surfaces, PKC‐ɛ−/− macrophages spread less than WT. Patch‐clamping revealed that the spreading defect is a result of the failure of PKC‐ɛ−/− 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‐ɛ in PKC‐ɛ−/− BMDM. Thus, PKC‐ɛ function in phagocytosis requires translocation to phagosomes and the catalytic domain. The expression of chimeric PKC molecules in RAW cells identified the ɛPS as necessary for PKC‐ɛ targeting. When placed into (nonlocalizing) PKC‐δ, ɛPS was sufficient for concentration, albeit to a lesser degree than intact PKC‐ɛ. In contrast, translocation of δ(ɛPSC1B) resembled that of WT PKC‐ɛ. Thus, ɛPS and ɛC1B cooperate for optimal phagosome targeting. Finally, cells expressing ɛK437W were significantly less phagocytic than their PKC‐ɛ‐expressing counterparts, blocked at the pseudopod‐extension phase. In summary, we have shown that ɛPS and ɛC1B are necessary and sufficient for targeting PKC‐ɛ to phagosomes, where its catalytic activity is required for membrane delivery and pseudopod extension.

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

A small G protein, RalA, was identified as a binding partner of PKCη. The binding led to activation of RalA and actin depolymerization associated with keratinocyte differentiation. These results provide new insight into the molecular mechanism of cytoskeletal regulation that leads to drastic change of cell shape.

Immunogold Electron Microscopic Demonstration of Distinct Submembranous Localization of the Activated γPKC Depending on the Stimulation

We examined the precise intracellular translocation of γ subtype of protein kinase C (γPKC) 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 γPKC (γPKC–GFP) on the plasma membrane. In contrast, treatment with Ca2+ ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of γPKC–GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that γPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of γPKC–GFP on the plasma membrane, Ca2+ ionophore and NMDA rapidly translocated γPKC–GFP to the plasma membrane and then restricted γPKC–GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca2+ influx alone induced the association of γPKC 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 γPKC in response to various stimuli suggests a novel mechanism for PKC activation.

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.

Effects of mutant γPKC found in SCA14 on the nature of primary cultured Purkinje cells

SAMP10 mouse (P10) is a model for age-associated neurodegeneration. We have reported an age-related increase in neuronal cytoplasmic ubiquitin-positive inclusions (UPIs) and concomitant decrease in proteasomal activity in the limbic-related structures of P10 brain. To study the effect of decreased proteasomal activity and formation of UPIs on neuronal survival and morphology, we performed in vitro proteasome inhibition experiment using cultured cortical neurons from P10 and normal aging control SAMR1 mice (R1). Significant cell death occured 24 h after the addition of 1–10 M MG115, a potent proteasome inhibitor, with no strain difference. UPIs, formed by exposing cells to 3 M MG115 for 24 h, were more frequently observed in P10 neurons than in R1 neurons. Morphometric analysis revealed that UPI-bearing neurons had fewer long (>50 m) neurites when compared to those without UPIs. UPIs may exert adverse effects on the maintenance of dendritic arbors.

Calcium-dependent mechanisms underlying propagation of γPKC translocation along the dendrites of cerebellar Purkinje cell

In the terrestrial slug Limax, NO is necessary for the synchronous oscillation of the local field potential in the procerebrum, which is thought to be involved in the odor discrimination and/or odor aversion learning. But there is no description about the genomic structure of the molluscan NO synthase, nor has the evolutionary origin of neuronal NO synthase (NOS) of mammals been clarified. Here we identified two types of NOS mRNAs of the slug, which show differential expression patterns within the brain. One is expressed broadly in the brain but in the low level (limNOS1), whereas, interestingly, the other is almost exclusively in the procerebrum (limNOS2). We also determined the whole genomic structure for limNOS1. It is composed of as many as 32 exons comparable to human nNOS gene, and has very similar exon–intron structure to that of human nNOS. Our results indicated that Limax NOS and human nNOS share the prototypical gene structure of NOS, and that their organization is highly conserved during the evolutionary history.