Sphingosine 1-Phosphate Regulates Heat Shock Protein 27 Induction by a p38 MAP Kinase-Dependent Mechanism in Aortic Smooth Muscle Cells

In an aortic smooth muscle cell line, A10 cells, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein. Sphingosine 1-phosphate significantly induced the accumulation of HSP27 in a pertussis toxin-sensitive manner. The effect was dose-dependent in the range between 0.1 and 30 microM. Sphingosine 1-phosphate stimulated an increase in the levels of mRNA for HSP27. Sphingosine 1-phosphate stimulated both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase activation. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, did not affect sphingosine 1-phosphate-stimulated HSP27 induction. In contrast, SB203580, an inhibitor of p38 MAP kinase, reduced sphingosine 1-phosphate-induced HSP27 induction. SB203580 reduced the levels of mRNA for HSP27 induced by sphingosine 1-phosphate. These results indicate that sphingosine 1-phosphate stimulates the induction of HSP27 via p38 MAP kinase activation in aortic smooth muscle cells.     

p42/p44 mitogen-activated protein kinase activation is involved in prostaglandin F2alpha-induced interleukin-6 synthesis in osteoblasts.

Prostaglandin F2alpha (PGF2alpha) significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity in osteoblast-like MC3T3-E1 cells. PD98059, a selective inhibitor of MAP kinase kinase, inhibited PGF2alpha-induced interleukin-6 (IL-6) synthesis as well as PGF2alpha-induced p42/p44 MAP kinase activation. PD98059 suppressed the IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, or NaF, an activator of heterotrimeric GTP-binding protein, as well as the p42/p44 MAP kinase activation by TPA or NaF. Calphostin C, a highly potent and specific inhibitor of PKC, inhibited the PGF2alpha-induced p42/p44 MAP kinase activity. These results strongly suggest that PKC-dependent p42/p44 MAP kinase activatioin is involved in PGF2alpha-induced IL-6 synthesis in osteoblasts.     

Tiludronate inhibits interleukin-6 synthesis in osteoblasts: Inhibition of phospholipase D activation in MC3T3-E1 cells

In previous studies, we have reported that PGF_2α stimulates phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein in osteoblast-like MC3T3-E1 cells, and that PGF_2α and PGE₁ induce interleukin-6 (IL-6) synthesis via activation of protein kinase C and protein kinase A, respectively. In the present study, we investigated the effect of tiludronate, a bisphosphonate known to inhibit bone resorption, on the PGF_2α- and PGE₁-induced IL-6 synthesis in these cells. Tiludronate significantly suppressed the PGF_2α-induced IL-6 secretion in a dose-dependent manner in the range between 0.1 and 30 μM. However, the IL-6 secretion induced by PGE₁ or (Bu)₂cAMP was hardly affected by tiludronate. The choline formation induced by PGF_2α was reduced by tiludronate dose-dependently in the range between 0.1 and 30 μM. On the contrary, tiludronate had no effect on PGF_2α-induced formation of inositol phosphates. Tiludronate suppressed the choline formation induced by NaF, known as an activator of heterotrimeric GTP-binding protein. However, tiludronate had little effect on the formation of choline induced by TPA, a protein kinase C activator. Tiludronate significantly inhibited the NaF-induced IL-6 secretion in human osteoblastic osteosarcoma Saos-2 cells. These results strongly suggest that tiludronate inhibits PGF_2α-induced IL-6 synthesis via suppression of phosphatidylcholine-hydrolyzing phospholipase D activation in osteoblasts, and that the inhibitory effect is exerted at the point between heterotrimeric GTP-binding protein and phospholipase D. J. Cell. Biochem. 69:252–259, 1998. © 1998 Wiley-Liss, Inc.     

Effect of inorganic polyphosphate in periodontitis in the elderly

Aim: Inorganic polyphosphate exists as chains of phosphate molecules and is distributed in osteoblasts, and regulates osteoblastic cell differentiation and bone matrix calcification. The purpose of this study was to clarify the effects of inorganic polyphosphate on periodontitis. Material and methods: Subgingival local irrigation with inorganic polyphosphate was studied in a randomised double‐blind study of 33 patients with periodontitis. Scaling and root planing were performed 1 week after the initial examination. Results: No significant differences between the inorganic polyphosphate group and control were detected in each item except IL‐1β. Patients in whom both the bleeding on probing and gingival index at 1 week had improved were significantly older in the inorganic polyphosphate group than in the control group (p < 0.05). Bone regeneration was seen in one case of the inorganic polyphosphate group. Conclusions: Inorganic polyphosphate was useful in the treatment of periodontitis in the elderly, indicating a probable effect of anti‐ageing, with similar bone regenerations occurring in both groups.     

Extracellular and Mixotrophic Symbiosis in the Whale-Fall Mussel Adipicola pacifica: A Trend in Evolution from Extra- to Intracellular Symbiosis

Background

Deep-sea mussels harboring chemoautotrophic symbionts from hydrothermal vents and seeps are assumed to have evolved from shallow-water asymbiotic relatives by way of biogenic reducing environments such as sunken wood and whale falls. Such symbiotic associations have been well characterized in mussels collected from vents, seeps and sunken wood but in only a few from whale falls.

Methodology/Principal Finding

Here we report symbioses in the gill tissues of two mussels, Adipicola crypta and Adipicola pacifica, collected from whale-falls on the continental shelf in the northwestern Pacific. The molecular, morphological and stable isotopic characteristics of bacterial symbionts were analyzed. A single phylotype of thioautotrophic bacteria was found in A. crypta gill tissue and two distinct phylotypes of bacteria (referred to as Symbiont A and Symbiont C) in A. pacifica. Symbiont A and the A. crypta symbiont were affiliated with thioautotrophic symbionts of bathymodiolin mussels from deep-sea reducing environments, while Symbiont C was closely related to free-living heterotrophic bacteria. The symbionts in A. crypta were intracellular within epithelial cells of the apical region of the gills and were extracellular in A. pacifica. No spatial partitioning was observed between the two phylotypes in A. pacifica in fluorescence in situ hybridization experiments. Stable isotopic analyses of carbon and sulfur indicated the chemoautotrophic nature of A. crypta and mixotrophic nature of A. pacifica. Molecular phylogenetic analyses of the host mussels showed that A. crypta constituted a monophyletic clade with other intracellular symbiotic (endosymbiotic) mussels and that A. pacifica was the sister group of all endosymbiotic mussels.

Conclusions/Significance

These results strongly suggest that the symbiosis in A. pacifica is at an earlier stage in evolution than other endosymbiotic mussels. Whale falls and other modern biogenic reducing environments may act as refugia for primal chemoautotrophic symbioses between eukaryotes and prokaryotes since the extinction of ancient large marine vertebrates.     

Endothelial microsomal prostaglandin E synthase-1 facilitates neurotoxicity by elevating astrocytic Ca2+ levels

Recurrent seizures may cause neuronal damage in the hippocampus. As neurons form intimate interactions with astrocytes via glutamate, this neuron-glia circuit may play a pivotal role in neuronal excitotoxicity following such seizures. On the other hand, astrocytes contact vascular endothelia with their endfeet. Recently, we found kainic acid (KA) administration induced microsomal prostaglandin E synthase-1 (mPGES-1) and prostaglandin E(2) (PGE(2)) receptor EP3 in venous endothelia and on astrocytes, respectively. In addition, mice deficient in mPGES-1 exhibited an improvement in KA-induced neuronal loss, suggesting that endothelial PGE(2) might modulate neuronal damage via astrocytes. In this study, we therefore investigated whether the functional associations between endothelia and astrocytes via endothelial mPGES-1 lead to neuronal injury using primary cultures of hippocampal slices. We first confirmed the delayed induction of endothelial mPGES-1 in the wild-type (WT) slices after KA-treatment. Next, we examined the effects of endothelial mPGES-1 on Ca(2+) levels in astrocytes, subsequent glutamate release and neuronal injury using cultured slices prepared from WT and mPGES-1 knockout mice. Moreover, we investigated which EP receptor on astrocytes was activated by PGE(2). We found that endothelial mPGES-1 produced PGE(2) that enhanced astrocytic Ca(2+) levels via EP3 receptors and increased Ca(2+)-dependent glutamate release, aggravating neuronal injury. This novel endothelium-astrocyte-neuron signaling pathway may be crucial for neuronal damage after repetitive seizures, and hence could be a new target for drug development.     

A new subset of CD103+CD8alpha+ dendritic cells in the small intestine expresses TLR3, TLR7, and TLR9 and induces Th1 response and CTL activity

CD103(+) dendritic cells (DCs) are the major conventional DC population in the intestinal lamina propria (LP). Our previous report showed that a small number of cells in the LP could be classified into four subsets based on the difference in CD11c/CD11b expression patterns: CD11c(hi)CD11b(lo) DCs, CD11c(hi)CD11b(hi) DCs, CD11c(int)CD11b(int) macrophages, and CD11c(int)CD11b(hi) eosinophils. The CD11c(hi)CD11b(hi) DCs, which are CD103(+), specifically express TLR5 and induce the differentiation of naive B cells into IgA(+) plasma cells. These DCs also mediate the differentiation of Ag-specific Th17 and Th1 cells in response to flagellin. We found that small intestine CD103(+) DCs of the LP (LPDCs) could be divided into a small subset of CD8α(+) cells and a larger subset of CD8α(-) cells. Flow cytometry analysis revealed that CD103(+)CD8α(+) and CD103(+)CD8α(-) LPDCs were equivalent to CD11c(hi)CD11b(lo) and CD11c(hi)CD11b(hi) subsets, respectively. We analyzed a novel subset of CD8α(+) LPDCs to elucidate their immunological function. CD103(+)CD8α(+) LPDCs expressed TLR3, TLR7, and TLR9 and produced IL-6 and IL-12p40, but not TNF-α, IL-10, or IL-23, following TLR ligand stimulation. CD103(+)CD8α(+) LPDCs did not express the gene encoding retinoic acid-converting enzyme Raldh2 and were not involved in T cell-independent IgA synthesis or Foxp3(+) regulatory T cell induction. Furthermore, CD103(+)CD8α(+) LPDCs induced Ag-specific IgG in serum, a Th1 response, and CTL activity in vivo. Accordingly, CD103(+)CD8α(+) LPDCs exhibit a different function from CD103(+)CD8α(-) LPDCs in active immunity. This is the first analysis, to our knowledge, of CD8α(+) DCs in the LP of the small intestine.     

Mechanism of prostaglandin D(2)-stimulated heat shock protein 27 induction in osteoblasts

We previously showed that prostaglandin D(2) (PGD(2)) stimulates activation of protein kinase C (PKC). We investigated whether PGD(2) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. PGD(2) increased the levels of HSP27 while having little effect on HSP70 levels. PGD(2) stimulated the accumulation of HSP27 dose dependently in the range between 10 nM and 10 microM. PGD(2) induced an increase in the levels of mRNA for HSP27. The PGD(2)-stimulated accumulation of HSP27 was reduced by staurosporine or calphostin C, inhibitors of PKC. PGD(2) induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by PGD(2) was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. Calphostin C suppressed the PGD(2)-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059 or SB203580 suppressed the PGD(2)-increased levels of mRNA for HSP27. These results strongly suggest that PGD(2) stimulates HSP27 induction through p44/p42 MAP kinase activation and p38 MAP kinase activation in osteoblasts and that PKC acts at a point upstream from both the MAP kinases.