Shun-ichi Kurata

Selective activation of p38 MAPK cascade and mitotic arrest caused by low level oxidative stress.

Apoptosis induced by high level oxidative stress accompanies diverse cellular biochemical events including activation of the stress signal cascades of JNK and NF-κB. We report here selective activation of p38 MAPK cascade and mitotic arrest under a low level oxidative stress that lacks apoptosis induction. U937 human lymphoid cells treated with low dose (0.02 mm) H2O2 rapidly caused p38 MAPK cascade activation detectable by phosphorylation of MKK3/6, p38 MAPK, activating transcription factor-2, and cAMP-responsive element-binding protein, leaving the JNK and NF-κB cascades unaffected. The p38 kinase activation was sustained for 24 h under the low level stress conditions and led to formation of polyploid nuclei.N-Acetyl-l-cysteine, a precursor of anti-oxidant glutathione, canceled both p38 MAPK activation and abnormal cell cycle progression, whereas blockage of the kinase by specific inhibitor SB203580 allowed the appearance of apoptotic cells. Thus, mimicking the effects of nocodazole, the low level oxidative stimulus caused inhibition of cell division in the M phase through p38 MAPK activation. The kinase cascade may serve as a primary transducer of cytoplasmic oxidative signals to nucleus for stress-relieving gene expression and cell cycle control before apoptosis-inducing signals are transduced. This is the first report demonstrating that oxidative stress can participate in cell cycle control by induction of a signal cascade.

p51A (TAp63γ), a p53 homolog, accumulates in response to DNA damage for cell regulation

p51A, or TAp63γ, a translation product of gene p51, or p63, was identified as a homolog of p53 in its primary structure and transactivating function. p53 plays a decision-making role in inducing either cell cycle arrest or apoptosis in response to DNA damage, and thereby preserves genome integrity of living cells. To compare the biological activities between p51A and p53, cell lines with low-level, constitutive expression of each protein were obtained by cDNA transfection of mouse erythroleukemic cells. Production of p51A with an apparent molecular mass of 57-kilodalton (kD) accompanied induction of p21waf1 and appearance of hemoglobin-producing cells. After DNA-damaging treatment either with ultraviolet light (UV) irradiation or with actinomycin D, the p51A protein accumulated in time courses corresponding to those of wild-type p53, and caused an increase in the hemoglobin-positive cell count. In contrast, p53-accumulated cells underwent apoptosis without exhibiting the feature of erythroid differentiation. The mode of p21waf1 and Bax-α upregulations varied between p51A- and p53-expressing cells and between the types of DNA damage. These results suggest the possibility that p51A induces differentiation under genotoxic circumstances. There may be cellular factors that control p51A protein stability and transactivating ability.

p51/p63 Controls Subunit α3 of the Major Epidermis Integrin Anchoring the Stem Cells to the Niche

p51/p63, a member of the tumor suppressor p53 gene family, is crucial for skin development. We describe here identification of ITGA3 encoding integrin α3 as a target of its trans-activating function, proposing that p51/p63 allows epidermal stem cells to express laminin receptor α3β1 for anchorage to the basement membrane. When activated by genotoxic stress or overexpressed ectopically in non-adherent cells, p51/p63 transduced a phenotype to attach to extracellular matrices, which was accompanied by expression of ITGA3. Motifs matching the p53-binding consensus sequence were located in a scattered form in intron 1 of human ITGA3, and served as p51/p63-responsive elements in reporter assays. In addition to the trans-activating ability of the TA isoform, we detected a positive effect of the ΔN isoform on ITGA3. The high level α3 production in human keratinocyte stem cells diminished upon elimination of p51/p63 by small interfering RNA or by Ca2+-induced differentiation. Furthermore, a chromatin immunoprecipitation experiment indicated a physical interaction of p51/p63 with intron 1 of ITGA3. This study provides a molecular basis for the standing hypothesis that p51/p63 is essential for epidermal-mesenchymal interactions.

Association of Endogenous Retroviruses and Long Terminal Repeats with Human Disorders

Since the human genome sequences became available in 2001, our knowledge about the human transposable elements which comprise ∼40% of the total nucleotides has been expanding. Non-long terminal repeat (non-LTR) retrotransposons are actively transposing in the present-day human genome, and have been found to cause ∼100 identified clinical cases of varied disorders. In contrast, almost all of the human endogenous retroviruses (HERVs) originating from ancient infectious retroviruses lost their infectivity and transposing activity at various times before the human-chimpanzee speciation (∼6 million years ago), and no known HERV is presently infectious. Insertion of HERVs and mammalian apparent LTR retrotransposons (MaLRs) into the chromosomal DNA influenced a number of host genes in various modes during human evolution. Apart from the aspect of genome evolution, HERVs and solitary LTRs being suppressed in normal biological processes can potentially act as extra transcriptional apparatuses of cellular genes by re-activation in individuals. There has been a reasonable prediction that aberrant LTR activation could trigger malignant disorders and autoimmune responses if epigenetic changes including DNA hypomethylation occur in somatic cells. Evidence supporting this hypothesis has begun to emerge only recently: a MaLR family LTR activation in the pathogenesis of Hodgkin’s lymphoma and a HERV-E antigen expression in an anti-renal cell carcinoma immune response. This mini review addresses the impacts of the remnant-form LTR retrotransposons on human pathogenesis.

Estrogen can regulate the cell cycle in the early G1 phase of yeast by increasing the amount of adenylate cyclase mRNA

The effects of beta-estradiol (estrogen; a minor component of yeast cells) on S. cerevisiae cells in the G0 and G1 phases were examined. Results showed that estrogen stimulated the recovery of growth from G0 arrest induced by nutrient limitation or ts mutation of cdc35 (adenylate cyclase) in the early G1 phase, and inhibited entry into the resting G0 phase by increasing the intracellular cAMP level. However, estrogen had no effect on late G1 arrest induced by the alpha factor or ts mutation of cdc36. Estrogen was found to lead to higher steady-state levels of adenylate cyclase mRNA but not to affect the expression of the RAS1 and RAS2 genes, although these can also alter the intracellular cAMP level. These results suggest that estrogen influences the cell cycle of yeast in the early G1 phase by controlling the level of cAMP through the increase of adenylate cyclase mRNA.

Production of the long and short forms of MFG-E8 by epidermal keratinocytes

Mouse milk fat globule-EGF factor 8, MFG-E8, is the ortholog to the human mammary tumor marker, lactadherin, and comprises two spliced variants, the L and S forms. Recent studies have suggested that MFG-E8-L produced by macrophages and Langerhans cells in the skin serves as a linker between phagocytic cells and apoptotic cells, and that MFG-E8-S, also termed SED1, facilitates sperm-egg interaction for fertilization. However, Mfge8 gene expression occurs in various tissues apparently unrelated to these critical events. Our in situ hybridization study has revealed that Mfge8 is expressed in the periderm (the premature epidermis) on embryonic day-14, well before Langerhans cells begin to grow in the prenatal phase. Mfge8 transcript is detectable in the basal and spinous layers throughout skin development, whereas immunostaining has revealed MFG-E8 protein accumulation in the spinous layer. Cultured keratinocyte stem cells consistently express Mfge8-L and -S mRNAs and produce the L protein, which is primarily detectable in the culture supernatant, and the S protein, which is mostly associated with the cells. Upon Ca2+-stimulated differentiation, which is detected by a decrease in keratinocyte stem cell marker p63(p51) and the induction of keratin1, we have observed suppression of Mfge8, and the protein becomes localized to the cell-cell borders. Papillomas and carcinomas caused by chronic UV-B irradiation produce MFG-E8 as determined by immunostaining. Thus, undifferentiated and poorly differentiated keratinocytes produce the L and S forms of MFG-E8 during normal and pathological tissue development, probably to support an as yet unidentified membrane function.

Transcriptional activation of the heme oxygenase gene by TPA in mouse M1 cells during their differentiation to macrophage

The activation of the heme oxygenase (HO), fos, and AP-1 genes was determined at intervals after treatment of M1 cells (derived from mouse myeloleukemia) with heme or 12-O-tetradecanoylphorbol 13-acetate (TPA). On treatment with heme, M1 cells did not differentiate into macrophage but their HO gene was activated and showed maximum transcriptional activity after treatment with heme for 2 h. In contrast, on treatment with TPA, these cells adhered to the culture flasks within 5 h and differentiated into macrophage-like cells. Moreover, the sequential activation of the fos, AP-1, and HO genes was observed during this period, with the maximal transcriptional activities after TPA treatment for 0.5, 1, and 1.5 h, respectively. Thus, the HO gene was activated by treatment with either heme or TPA, and the latter activation was associated with activations of the fos and AP-1 genes. As the rat HO gene is known to have a TPA-sensitive element in its promoter region, this gene was suggested to be activated by a fos-AP-1 complex protein.

HYPERBARIC OXYGENATION REDUCES THE CYTOSTATIC ACTIVITY AND TRANSCRIPTION OF NITRIC OXIDE SYNTHETASE GENE OF MOUSE PERITONEAL MACROPHAGES

The effects of hyperbaric oxygenation (HBO) and hyperbaric air (HBA) on the cytostatic activity, peroxynitrite synthesis and transcription of the inducible nitric oxide synthetase (iNOS) gene of murine peritoneal macrophages were studied in vitro. Exposure of mice to HBO or HBA significantly reduced the cytostatic activity, peroxynitrite synthesis and transcription of iNOS mRNA of their macrophages stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). These results indicate that HBO and HBA treatments of mice reduce the cytostatic activity of peritoneal macrophages by reducing iNOS mRNA synthesis.

Salvage of nonischemic control lung from injury by unilateral ischemic lung with apocynin, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, in isolated perfused rat lung

Ischemia reperfusion (I/R) injury of the lung affects the function of the nonischemic lung. Our objective is to determine how apocynin, which is a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, protects the nonischemic control right lung (RL) from injury by the unilateral ischemic left lung (LL). In isolated ventilated (by air containing 5% CO(2)) rat lungs, in which differential perfusion of the RL or LL was feasible, the LL was selectively made ischemic (60 min) and reperfused (30 min) in a nonrecirculating or recirculating manner with buffer (Krebs-Henseleit) solution, or in a recirculating manner with buffer that contained apocynin (10 mmol/L) or apocynin + TACEI (tumor necrosis factor)-alpha converting enzyme inhibitor; 10 microg/mL) (each group: n = 12) or with buffer that contained SOD (superoxide dismutase, 3000 U before ischemia and at reperfusion) or SOD + TACEI (each group: n = 5). The permeability of pulmonary endothelium/epithelium (wet/dry ratio and protein content of bronchoalveolar lavage fluid of each lung), perfusion pressure, and cytokine messenger RNA (mRNA) expression was increased not only in the LL (compared with nonischemic control RL, P < 0.01 with paired-samples T) but also in the RL in recirculating groups (compared with RL in the nonrecirculating group). Apocynin + TACEI as well as SOD + TACEI prevented those permeability increases in the RL by the ischemic LL. However, apocynin with or without TACEI as well as SOD with or without TACEI could only partially ameliorate I/R injury in the LL (P < 0.01 by 1-way analysis of variance (ANOVA)). TNF-alpha and possibly reactive oxygen species produced and released from the ischemic lung may synergistically induce control RL (remote organ) damage.

p63(TP63) elicits strong trans-activation of the MFG-E8|[sol]|lactadherin|[sol]|BA46 gene through interactions between the TA and |[Delta]|N isoforms

We report here that human MFGE8 encoding milk fat globule-EGF factor 8 protein (MFG-E8), also termed 46 kDa breast epithelial antigen and lactadherin, is transcriptionally activated by p63, or TP63, a p53 (TP53) family protein frequently overexpressed in head-and-neck squamous cell carcinomas, mammary carcinomas and so on. Despite that human MFG-E8 was originally identified as a breast cancer marker, and has recently been reported to provide peptides for cancer immunotherapy, its transcriptional control remains an open question. Observations in immunohistochemical analyses, a tetracycline-induced p63 expression system and keratinocyte cultures suggested a physiological link between p63 and MFGE8. By reporter assays with immediately upstream regions of MFGE8, we determined that the trans-activator (TA) isoforms of p63 activate MFGE8 transcription though a p53/p63 motif at −370, which was confirmed by a chromatin immunoprecipitation experiment. Upon siRNA-mediated p63 silencing in a squamous cell carinoma line, MFG-E8 production decreased to diminish Saos-2 cell adhesion. Interestingly, the ΔN-p63 isoform lacking the TA domain enhanced the MFGE8-activating function of TA-p63, if ΔN-p63 was dominant over TA-p63 as typically observed in undifferentiated keratinocytes and squamous cell carcinomas, implying a self-regulatory mechanism of p63 by the TA:ΔN association. MFG-E8 may provide a novel pathway of epithelial–nonepithelial cell interactions inducible by p63, probably in pathological processes.