Expression of 5'-AMP-activated protein kinase with starvation in murine thymocytes.

The 5'-AMP-activated protein kinase (AMPK) is a key enzyme in the protection of cells during energy crisis. AMPK is a heterotrimer consisting of a catalytic α (α1, 2) subunit and two regulatory subunits, ß (ß1, 2) and γ (γ1-3). To elucidate the role of AMPK in thymocytes with starvation, we investigated the expression of AMPK in murine thymocytes. The main isoforms expressed were α2, ß1, and γ1, of which expression increased time-dependently with starvation, together with an increase in the amount of the active form of AMPK, phospho-AMPKα. In cultured thymocytes, expression of AMPK was induced by dexamethasone, but not by a low glucose concentration in medium. Increased expression was inhibited by glucocorticoid receptor antagonist RU486. Phosphorylation of AMPKα showed an increase with low glucose concentration, but not with dexamethasone. These results suggest that increased expression of AMPK in starved mouse thymocytes is induced by an increase in glucocorticoids and that activation is induced by hypoglycemia.

Transcriptional regulation of tumor suppressor p53 by cAMP-responsive element-binding protein/AMP-activated protein kinase complex in response to glucose deprivation.

Tumor suppressor p53 plays a pivotal role in the regulation of cell fate determination in response to a variety of cellular stress including carbon source depletion. In this study, we found that cAMP-responsive element-binding protein (CREB) collaborates with AMP-activated protein kinase alpha (AMPKalpha) to regulate the transcription of p53. Luciferase reporter assays showed that the genomic fragment spanning from -531 to -239 of human p53 gene is required for the transactivation of p53 in response to glucose deprivation. Within this region, we found out a putative CREB-binding site. siRNA-mediated knockdown of CREB resulted in a significant inhibition of the up-regulation of p53 and apoptosis under glucose deprivation. Consistent with these observations, glucose deprivation induced the transcription of p53 and CREB. Additionally, glucose deprivation led to an efficient recruitment of CREB onto the promoter region of p53 gene carrying the canonical CREB-binding site, indicating that CREB has an ability to bind to the promoter region of p53 gene and transactivate p53. Furthermore, the amounts of CREB/phospo-AMPKalpha complex increased in response to glucose deprivation. Taken together, our present findings suggest that p53 is transcriptionally regulated by CREB/phospho-AMPKalpha complex and thereby contributing to the induction of apoptosis under carbon source depletion.

Activation of AMP-activated protein kinase induces p53-dependent apoptotic cell death in response to energetic stress.

Tumor suppressor p53-dependent stress response pathways play an important role in cell fate determination. In this study, we have found that glucose depletion promotes the phosphorylation of AMP-activated protein kinase catalytic subunit alpha (AMPKalpha) in association with a significant up-regulation of p53, thereby inducing p53-dependent apoptosis in vivo and in vitro. Thymocytes prepared from glucose-depleted wild-type mice but not from p53-deficient mice underwent apoptosis, which was accompanied by a remarkable phosphorylation of AMPKalpha and a significant induction of p53 as well as pro-apoptotic Bax. Similar results were also obtained in human osteosarcoma-derived U2OS cells bearing wild-type p53 following glucose starvation. Of note, glucose deprivation led to a significant accumulation of p53 phosphorylated at Ser-46, but not at Ser-15 and Ser-20, and a transcriptional induction of p53 as well as proapoptotic p53 AIP1. Small interference RNA-mediated knockdown of p53 caused an inhibition of apoptosis following glucose depletion. Additionally, apoptosis triggered by glucose deprivation was markedly impaired by small interference RNA-mediated depletion of AMPKalpha. Under our experimental conditions, down-regulation of AMPKalpha caused an attenuation of p53 accumulation and its phosphorylation at Ser-46. In support of these observations, enforced expression of AMPKalpha led to apoptosis and resulted in an induction of p53 at protein and mRNA levels. Furthermore, p53 promoter region responded to AMPKalpha and glucose deprivation as judged by luciferase reporter assay. Taken together, our present findings suggest that AMPK-dependent transcriptional induction and phosphorylation of p53 at Ser-46 play a crucial role in the induction of apoptosis under carbon source depletion.

Geldanamycin, a heat-shock protein 90-binding agent, induces thymocyte apoptosis through destabilization of Lck in presence of 12-O-tetradecanoylphorbol 13-acetate.

Geldanamycin, a heat-shock protein 90 (Hsp90)-binding agent, modulates various cellular activities. The present study found that, although geldanamycin by itself had no effect on thymocyte viability, it induced apoptosis in thymocytes with a reduction of the mitochondrial transmembrane potential (DeltaPsim) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC). This apoptosis depended on transcription and translation, and on activation of caspase-8 and -3. Geldanamycin treatment in the presence of TPA also enhanced destabilization of Lck. This destabilization was independent of transcription and translation. It was inhibited, however, by conventional PKC inhibitors, preventing apoptosis. Proteasome inhibitor affected neither the degradation of Lck nor DNA fragmentation, although they inhibited reduction of DeltaPsim. These results suggest that the ubiquitin-proteasome system is not involved in Lck destabilization, and that DeltaPsim reduction is not directly related to the progression of apoptosis. Furthermore, inhibition of Lck in the presence of TPA induced apoptosis in thymocytes. Our findings suggest that Hsp90 modulates thymocyte apoptosis in concert with PKC through the destabilization of Lck and in a caspase-8- and -3-dependent manner.

The expression and localization of osteopontin in the mouse major salivary glands.

The present study investigated the expression and distribution of osteopontin in the mouse major salivary glands. The level of osteopontin expression in the mouse submandibular gland was higher (12.7-fold) than that in parotid and sublingual glands at the mRNA level. By Western blot analysis, intense positive bands were seen at the predicted molecular mass (about 55 kDa) in all the major salivary glands, while an approximately 30 kDa band of osteopontin was detected only in the submandibular gland. Indirect immunofluorescent and immuno-electron microscopy analyses demonstrated the localization of osteopontin in the luminal (apical) membranes of acinar cells in all the salivary glands. Osteopontin was also localized at the lumen of acini in the submandibular gland. These results suggest that the expression of osteopontin in the submandibular gland is different from that in the parotid and sublingual glands and that osteopontin may be degraded in the mouse submandibular gland.

Inhibition of osteopontin expression and function in oral cancer cell lines by antisense oligonucleotides.

We examined expression and function of osteopontin (OPN) in oral cancer cell lines using antisense oligonucleotide (AS). Quantitative real-time RT-PCR showed that expression in BSC-OF cells was significantly higher (10-fold) than that in KB cell. AS-study showed that foci of AS-treated BSC-OF cells possessed thin processes and radiated morphologically, although BSC-OF cells showed round foci. Cell growth in AS-group was lower (<80%) than the control. Invasion ability in AS-group became significantly lower (P<0.01). These results suggest that BSC-OF cell is useful for over-expression of OPN, and that OPN contributes to morphology, growth and invasion.

Modulation of dexamethasone-induced thymocyte apoptosis by heat-shock protein 90-binding agents.

Heat-shock protein 90 (HSP90) is known to affect a variety of cellular activities. The present study showed that the HSP90-binding agents, geldanamycin, herbimycin A and radicicol, inhibited the murine thymocyte apoptosis induced by dexamethasone and was accompanied by the inhibition of the reduction of the mitochondrial transmembrane potential (delta psi m). HSP90-binding agents did not inhibit etoposide-induced apoptosis. The inhibition of dexamethasone-induced apoptosis was in part due to the interference of HSP90 with the glucocorticoid receptor, resulting in the inhibition of nuclear translocation of the receptor. The expression of inositol 1,4,5-triphosphate receptors, which were shown to be involved in dexamethasone-induced apoptosis, did not participate in the inhibition of apoptosis.

Application of in vitro mutagenesis to identify the gene responsible for cold agglutination phenotype of Streptococcus mutans.

A previously unidentified protein with an apparent molecular mass of 120 kDa was detected in some Streptococcus mutans strains including the natural isolate strain Z1. This protein was likely involved in the cold-agglutination of the strain, since a correlation between this phenotype and expression of the 120 kDa protein was found. We have applied random mutagenesis by in vitro transposition with the Himar1 minitransposon and isolated three cold-agglutination-negative mutants of this strain from approximately 2,000 mutants screened. A 2.5 kb chromosomal fragment flanking the minitransposon in one of the three mutants was amplified by PCR-based chromosome walking and the minitransposon insertion in the other two mutants occurred also within the same region. Nucleotide sequencing of the region revealed a 1617 nt open reading frame specifying a putative protein of 538 amino acid residues with a calculated molecular weight of 57,192. The deduced eight amino acid sequence following a putative signal sequence completely coincided with the N-terminal octapeptide sequence of the 120 kDa protein determined by the Edman degradation. Therefore, the 1617 nt gene unexpectedly encoded the 120 kDa protein from S. mutans. Interestingly, this gene encoded a collagen adhesin homologue. In vitro mutagenesis using the Himar1 minitransposon was successfully applied to S. mutans.

Achacin induces cell death in HeLa cells through two different mechanisms.

Achacin, which belongs to the L-amino acid oxidase group, oxidizes free amino acids and produces hydrogen peroxide in cell culture systems. Morphological changes in cells incubated with achacin were similar to those of cells incubated with H(2)O(2). In both cases, the end result was cell death. To examine the mechanism of achacin-associated cytotoxicity, the H(2)O(2) scavenger catalase was added to culture media. Features typical of apoptosis, including morphological changes, DNA fragmentation, and PARP cleavage, were observed when cells were incubated with achacin in the presence of catalase. Moreover, apoptosis was inhibited by Z-VAD-fmk, a broad-spectrum caspase inhibitor. Herein, we present evidence that two pathways are involved in achacin-induced cell death. One is direct generation of H(2)O(2) through the L-amino acid oxidase activity of achacin. The other is the caspase-mediated apoptotic pathway that is induced by depletion of L-amino acids by achacin.

Proteasome inhibitors block Ras/ERK signaling pathway resulting in the downregulation of Fas ligand expression during activation-induced cell death in T cells.

Activation-induced cell death (AICD) plays a critical role in the maintenance of homeostasis and peripheral tolerance in the immune system, and is mediated by Fas ligand (FasL) expression and the interaction between Fas and FasL. In the present study, we examined the role of the ubiquitin-proteasome system in AICD using T cell hybridoma N3-6-71 cells. The peptidyl aldehyde proteasome inhibitor carbobenzoxyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI) blocked T cell receptor (TCR) stimulation-induced apoptosis in the T cell hybridoma. Fas and FasL gene expression and mouse FasL promoter activity following TCR stimulation were suppressed by PSI pretreatment. Deletion or point mutation of the kappaB site in the FasL promoter region did not suppress inducible FasL promoter activity effectively. PSI blocked extracellular signal-regulated kinase (ERK) activity induced by TCR stimulation, but had no effect on c-jun N-terminal kinase activation. ERK activation was essential for FasL expression and AICD. The initial tyrosine phosphorylation steps following TCR stimulation, i.e., phosphorylation of CD3zeta and Vav, were not altered by PSI. These data suggest that the ubiquitin-proteasome system has some regulatory function at an intermediate step between the initial tyrosine phosphorylation steps and ERK activation in AICD.

Expression and distribution of osteopontin and matrix metalloproteinase (MMP)-3 and -7 in mouse salivary glands.

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.