A dual signaling cascade that regulates the ectodomain shedding of heparin-binding epidermal growth factor-like growth factor

Ectodomain shedding is an important mechanism to regulate the biological activities of membrane proteins. We focus here on the signaling mechanism of the ectodomain shedding of heparin-binding epidermal growth factor (EGF)-like growth factor (pro HB-EGF). Lysophosphatidic acid (LPA), a ligand for seven-transmembrane G protein-coupled receptors, stimulates the shedding of pro HB-EGF, which constitutes a G protein-coupled receptor-mediated transactivation of the EGF receptor. Experiments using a series of inhibitors and overexpression of mutant forms of signaling molecules revealed that the Ras-Raf-MEK signal is essential for the LPA-induced shedding. In addition, the small GTPase Rac is involved in the LPA-induced shedding, possibly to promote MEK activation. 12-O-Tetradecanoylphorbol-13-acetate is another potent inducer of pro HB-EGF shedding. We also demonstrate that the LPA-induced pathway is distinct from the 12-O-tetradecanoylphorbol-13-acetate-induced pathway and that these pathways constitute a dual signaling cascade that regulates the shedding of pro HB-EGF.     

Nonsense and frameshift mutations in ZFHX1B, encoding Smad-interacting protein 1, cause a complex developmental disorder with a great variety of clinical features

Mutations in ZFHX1B, encoding Smad-interacting protein 1 (SIP1), have been recently reported to cause a form of Hirschsprung disease (HSCR). Patients with ZFHX1B deficiency typically show mental retardation, delayed motor development, epilepsy, microcephaly, distinct facial features, and/or congenital heart disease, in addition to the cardinal form of HSCR. To investigate the breadth of clinical variation, we studied DNA samples from six patients with clinical profiles quite similar to those described elsewhere for ZFHX1B deficiency, except that they did not have HSCR. The results showed the previously reported R695X mutation to be present in three cases, with three novel mutations-a 2-bp insertion (760insCA resulting in 254fs262X), a single-base deletion (270delG resulting in 91fs107X), and a 2-bp deletion (2178delTT resulting in 727fs754X)-newly identified in the other three. All mutations occurred in one allele and were de novo events. These results demonstrate that ZFHX1B deficiency is an autosomal dominant complex developmental disorder and that individuals with functional null mutations present with mental retardation, delayed motor development, epilepsy, and a wide spectrum of clinically heterogeneous features suggestive of neurocristopathies at the cephalic, cardiac, and vagal levels.     

Pathophysiological significance of in vivo ESR signal decay in brain damage caused by X-irradiation. Radiation effect on nitroxyl decay of a lipophilic spin probe in the head region

X-irradiation of mice decreased the decay rate of the in vivo ESR signal in the head region to 75% of the control when 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MCPROXYL), a lipophilic and blood-brain barrier-permeable spin probe, was used. We attempted to identify the specific factor responsible for the decrease in the signal decay rate caused by X-irradiation. The signal decay of MCPROXYL in the head region depends on the following three factors: (1) blood concentration of MCPROXYL, (2) reduction to the corresponding hydroxylamine in the brain tissue, and (3) effusion of MCPROXYL from the brain tissue. Irradiation at 15 Gy did not significantly change the rate of decrease of blood concentration of MCPROXYL at 1 h post-irradiation. The reducing activity of the brain homogenate was not changed by the X-irradiation (15 Gy). The contents of MCPROXYL and its hydroxylamine derivative in the brain of 15 Gy-irradiated mice remained higher than in non-irradiated mice. These findings suggest that the effect of X-irradiation observed by in vivo ESR is attributable not to the redox reaction of MCPROXYL in the brain but to the change of the efflux rate of the MCPROXYL from the brain.     

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H(2)O(2) stimulation

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H(2)O(2) (100 microM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H(2)O(2) stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 microM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H(2)O(2)-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H(2)O(2) stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H(2)O(2). Under these conditions, the H(2)O(2)-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H(2)O(2), and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.     

Protonation of histidine and histidine-tryptophan interaction in the activation of the M2 ion channel from influenza a virus

The M2 protein of influenza A virus forms a homotetramer ion channel in the lipid membrane. The channel is specific for proton conductance and is activated by low pH with a transition midpoint at pH 5.7. We have studied the structure of the transmembrane domain of the M2 ion channel by using UV resonance Raman spectroscopy, with special attention to the side chains of histidine (His37) and tryptophan (Trp41) residues. The Raman spectra provide direct evidence that the imidazole ring of His37 is protonated upon channel activation at low pH. Concomitantly, the UV resonance Raman scattering from Trp41 shows an unusual intensity change, which is ascribed to a cation-pi interaction between the protonated (cationic) imidazole ring of His37 and the indole ring of Trp41. The protonation of His37 and the Raman intensity change of Trp41 do not occur in the presence of amantadine that blocks the M2 ion channel. These observations clearly show that the protonation of His37 and concomitant cation-pi interaction with Trp41 is a key step in the activation of the M2 ion channel. The His37-Trp41 interaction associated with the channel activation is explained by assuming a conformational transition of His37 induced by electrostatic repulsion among the protonated imidazole rings of four His37 residues in the tetramer channel. Trp41 may play a role in stabilizing the channel open state through cation-pi interaction with His37. A molecular model for the activation of M2 ion channel is proposed on the basis of the gating mechanism.     

Dynamic changes in subnuclear NP95 location during the cell cycle and its spatial relationship with DNA replication foci

We determined the expression and subcellular localization of nuclear protein NP95 during the cell cycle in mouse 3T3 cells. The levels of NP95 mRNA and protein were extremely low in quiescent cells; however, stimulation with 10% serum increased their expressions in a time course similar to that of the late growth-regulated gene proliferating cell nuclear antigen (PCNA). Subnuclear location of NP95 dynamically changed during the cell cycle. Double immunostaining for NP95 and chromatin-bound PCNA, a marker of DNA replication sites, revealed that NP95 was almost exclusively colocalized with chromatin-bound PCNA throughout the nucleus in early S phase and partly in mid-S phase. Distinct localization of the two proteins, however, became evident in mid-S phase, and thereafter, many chromatin-bound PCNA foci not carrying NP95 foci could be detected. In G2 phase, nodular NP95 foci were still identified without any chromatin-bound PCNA foci. Chromatin-bound PCNA was observed as a pre-DNA replication complex at the G1/S boundary synchronized by hydroxyurea treatment, while NP95 was detected in nucleolar regions as unique large foci. There was no significant redistribution of NP95 foci shortly after DNA damage by gamma-irradiation. Nodular NP95 foci characteristically seen in G2 phase were also detected in G2-arrested cells following gamma-irradiation. Taken together, our results indicate that NP95 is assigned to a late growth-regulated gene and suggest that NP95 does not take a direct part in DNA replication as part of the DNA synthesizing machinery, like PCNA, but is presumably involved in other DNA replication-linked nuclear events.     

Differences in functional consequences and signal transduction induced by IL-3, IL-5, and nerve growth factor in human basophils

Previous studies have indicated a redundancy in the effects of the cytokines, IL-3, IL-5, and nerve growth factor (NGF) on acute priming of human basophils. In the current study, we have examined the effects of these three cytokines on 18-h priming for leukotriene C4 generation, their ability to induce Fc(epsilon)RIbeta mRNA expression, or their ability to sustain basophil viability in culture. We also examine a variety of the signaling steps that accompany activation with these cytokines. In contrast with the ability of IL-3 to alter secretagogue-mediated cytosolic calcium responses following 18-h cultures, 18-h treatment with IL-5 or NGF did not affect C5a-induced leukotriene C4 generation or alter C5a-induced intracellular Ca2+ concentration elevations. IL-3 and IL-5, but not NGF, induced Fc(epsilon)RIbeta mRNA expression and all three improved basophil viability in culture with a ranking of IL-3 > IL-5 > or = NGF. All three cytokines acutely activated the extracellular signal-regulated kinase pathway and the signaling elements that preceded extracellular signal-regulated kinase and cytosolic phospholipase A2 phosphorylation, consistent with their redundant ability to acutely prime basophils. However, only IL-3 and IL-5 induced Janus kinase 2 and STAT5 phosphorylation. This pattern of signal element activation among the three cytokines most closely matched their ability to induce expression of Fc(epsilon)RIbeta mRNA. Induction of the sustained calcium signaling that follows overnight priming with IL-3 appeared to be related to the strength of the early signals activated by these cytokines but the relevant pathway required was not identified. None of the signaling patterns matched the ability of the cytokines to promote basophil survival.     

Effect of tumor necrosis factor-alpha on insulin signal transduction in rat adipocytes: relation to PKCbeta and zeta translocation

Although much evidence has been accumulated suggesting that tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance, the precise mechanism involved is still unclear. Recently, it has been reported that insulin-induced glucose uptake is mediated by activation of second messengers such as insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and diacylglycerol (DG)-protein kinase C (PKC). We have examined the effect of TNF-alpha on insulin-induced glucose uptake and activations of tyrosine kinase, IRS-1, PI3K and PKC in rat adipocytes. Pretreatment with 0.1-100 nM TNF-alpha for 60 min resulted in a significant decrease in 10 nM insulin- or 1 microM 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced [3H]2-deoxyglucose uptake without affecting basal glucose uptake. 10 nM insulin-stimulated activation of tyrosine kinase, IRS-1 and PI3K was suppressed by preincubation with 0.1-10 nM TNF-alpha for 60 min. 10 nM TNF-alpha pretreatment also suppressed 10 nM insulin- and 1 microM TPA-induced increases in membrane-associated PKCbeta and PKCzeta. Furthermore, 10 nM TNF-alpha, by itself, altered PKCbeta translocation from the membrane to cytosol. These results suggest that TNF-alpha inhibits insulin-stimulated activation of both the tyrosine kinase-IRS-1-PI3K-PKCzeta pathway and DG-PKC pathway. Finally, TNF-alpha contributes to insulin resistance in rat adipocytes.