Sphingosine kinase type 1 induces G12/13-mediated stress fiber formation,yet promotes growth and survival independent of G protein-coupled receptors

Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha12/13, and Gbetagamma subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G12/13-mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.     

alpha-Melanocyte-stimulating hormone inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production in leukocytes by modulating protein kinase A,p38 kinase,and nuclear factor kappa B signaling pathways

The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in leukocytes via stimulation of alpha-MSH cell surface receptors. However, the signaling mechanism of alpha-MSH action has not yet been clearly elucidated. Here, we have investigated signaling pathways by which alpha-MSH inhibits lipopolysaccharide (LPS)-induced TNF-alpha production in leukocytes such as THP-1 cells. We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor kappa B (NF kappa B) signaling. In THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NF kappa B to induce TNF-alpha production. We found that pretreatment of cells with alpha-MSH blocked LPS-induced p38 kinase and NF kappa B activation as well as TNF-alpha production. This response was proportional to alpha-MSH receptor expression levels, and addition of an alpha-MSH receptor antagonist abolished the inhibitory effects. In addition, alpha-MSH treatment activated PKA, and PKA inhibition abrogated the inhibitory effects of alpha-MSH on p38 kinase activation, NF kappa B activation, and TNF-alpha production. Taken together, our results indicate that stimulation of PKA by alpha-MSH causes inhibition of LPS-induced activation of p38 kinase and NF kappa B to block TNF-alpha production.     

SEK1-dependent JNK1 activation prolongs cell survival during G-Rh2-induced apoptosis

We provide here evidence that c-Jun N-terminal protein kinase 1 (JNK1) activity is differentially up-regulated during apoptosis of SK-HEP-1 cells after treatment with ginsenoside Rh2 (G-Rh2). The G-Rh2-mediated JNK1 activation that occurred for the first 10-30min was associated with SEK1 activity, but thereafter, the sustained activation was associated not with SEK1 activity, but with proteolytic cleavage of JNK1-associated p21(WAF1/CIP1). Supporting this is that the expression of the dominant negative SEK1 mutant effectively blocked the early JNK1 activation phase but did not alter the sustained activation phase or apoptosis. Furthermore, expression of p21D112N, an uncleavable mutant of p21(WAF1/CIP1), suppressed the later JNK1 activation. Moreover, the stable overexpression of ectopic JNK1 suppressed apoptosis while expression of the dominant negative JNK1 mutant promoted it. We propose that the early SEK1-associated JNK1 activation phase acts to prolong cell survival in response to apoptosis-inducing agents, thereby serving as an intervening checkpoint prior to the commitment to apoptosis.     

Role of apoptosis-inducing factor in myocardial cell death by ischemia-reperfusion

Although apoptosis contributes to myocardial cell death in the ischemia-reperfused heart, the molecular basis of apoptosis is poorly understood. Apoptosis-inducing factor (AIF) has been characterized as a caspase-independent death effector. Upon the induction of apoptosis, mitochondrial AIF is released to the cytoplasm and then enters the nucleus, in which it induces chromatin condensation and 50 kbp DNA fragmentation. In the present study, we examined the role of AIF in ischemia-reperfusion injury in isolated rat hearts. AIF was detected in the cytosolic and nuclear fractions of hearts subjected to ischemia-reperfusion, whereas it was detected only in the mitochondria of control hearts. Moreover, AIF release increased in a reperfusion time-dependent manner. Pulse field gel electrophoresis revealed that 50 kbp DNA fragments were produced by ischemia/reperfusion. In contrast, cytochrome c release and the activation of caspase-3 did not occur to a significant extent. Moreover, ischemic preconditioning attenuated the AIF release and the 50 kbp DNA fragmentation. These results suggest that AIF-dependent apoptosis is likely to attribute to myocardial cell death in the ischemia-reperfused heart and that it is related with the protective effect of ischemic preconditioning.     

Isolation of novel human fetal brain cDNAs mapped to human chromosome bands, 1q25 and 8q24.1

We isolated chromosome band-specific human fetal brain cDNAs by the microdissection mediated cDNA capture method, and localized these cDNA using in situ hybridization histochemistry with developing rat brain sections. Uni-Amp cDNAs were prepared from an 18-week old human fetal brain, and hybridized to human metaphase chromosomes. Eight Uni-Amp cDNAs, hybridized to chromosome band 1q25 or 8q24.1, were recovered by microdissection and PCR amplification with Uni-Amp primers. Among these cDNAs, two novel genes (FB113 of 8q24.1 and FB134 of 1q25) showed a temporospatially interesting expression pattern in the developing rat brains. The expression of FB113 was under dynamic regulation in the developing granule cells of cerebellum and dentate gyrus. FB134 showed a nervous tissue specific expression pattern and an exclusively prominent expression in the developing presubiculum and parasubiculum. By the fluorescence in situ hybridization using human genomic DNAs, FB113 and FB134 were mapped back to the human chromosome bands 8q24.1 and 1q25, respectively. These results indicate that combined application of the microdissection mediated cDNA capture method and in situ hybridization histochemistry can be used for the isolation of chromosomal band-specific genes related to brain development or human genetic diseases.     

Design and synthesis of novel fluorocyclopropanoid nucleosides

Novel fluorocyclopropanoid nucleosides were designed, synthesized and evaluated their antiviral activities against poliovirus, HSV, HIV, and HBV.     

Continuous culture of immobilized streptomyces cells for kasugamycin production

Continuous cultures of immobilized Streptomyces kasugaensis, a kasugamycin producer, were carried out on Celite beads. When using a prototype separator for immobilized-cell separation and recycling, the continuous operation could not be sustained for an extended period as a result of an excessive loss of immobilized cells caused by the poor performance of the separator. Accordingly, the immobilized-cell separator was revised to provide better immobilized-cell settling and thus recycling into the reactor. In a subsequent culture using the revised separator, a stable operation was maintained for over 820 h with a high kasugamycin productivity. The kasugamycin productivity ranged from 9.8 to 16.1 mg/L/h, which was about 14- to 23-fold higher than that in a batch suspended-cell culture. When the original feeding medium concentration was doubled at the end of the continuous culture, the productivity became severely impaired for several reasons, which will be discussed. An excessive formation of free cells and loss of immobilized cells through the separator were also observed.     

A DNase I hypersensitive site flanks an origin of DNA replication and amplification in Sciara

In chromosomes of metazoa, the assembly of the genome into chromatin makes an important but poorly understood contribution to determining where DNA replication will initiate. We addressed this issue by studying the developmental progression of the location of the DNA replication origin (ORI) and alterations in chromatin structure in one of the best-mapped ORIs in metazoa, that found in DNA puff II/9A of the fly Sciara coprophila. We found that DNA synthesis for both normal chromosomal endoduplication and DNA amplification initiates within the same 5.5 kb EcoRI fragment. We showed that irrespective of the mode of ORI function--replication or amplification--chromatin over the 1 kb major ORI is never remodeled into a conventional DNase I hypersensitive site (DH site). Instead, we found that the major site of alterations to chromatin structure at this locus is a large (approximately 400 bp) DH site located 600 bp away from the major ORI, at a position where the frequency of replication initiation events falls dramatically. We describe a tight positive correlation between ORI activity, strength of this DH site, and the intranuclear titer of protein factor(s) that bind the DH site in a sequence-specific manner. We propose that the Sciara replicator in locus II/9A is composed of sequences that reside within the ORI per se as well as sequences encompassed by the DH site.     

Identification of essential residues in the type II Hsp40 Sis1 that function in polypeptide binding

Sis1 is an essential yeast Type II Hsp40 protein that assists cytosolic Hsp70 Ssa1 in the facilitation of processes that include translation initiation, the prevention of protein aggregation, and proteasomal protein degradation. An essential function of Sis1 and other Hsp40 proteins is the binding and delivery of non-native polypeptides to Hsp70. How Hsp40s function as molecular chaperones is unknown. The crystal structure of a Sis1 fragment that retains peptide-binding activity suggests that Type II Hsp40s utilize hydrophobic residues located in a solvent-exposed patch on carboxyl-terminal domain I to bind non-native polypeptides. To test this model, amino acid residues Val-184, Leu-186, Lys-199, Phe-201, Ile-203, and Phe-251, which form a depression in carboxyl-terminal domain I, were mutated, and the ability of Sis1 mutants to support cell viability and function as molecular chaperones was examined. We report that Lys-199, Phe-201, and Phe-251 are essential for cell viability and required for Sis1 polypeptide binding activity. Sis1 I203T could support normal cell growth, but when purified it exhibited severe defects in chaperone function. These data identify essential residues in Sis1 that function in polypeptide binding and help define the nature of the polypeptide-binding site in Type II Hsp40 proteins.