BBA - Molecular Cell Research (v.1540, #3)

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts by Rie Tajima; Nobuko Kawaguchi; Yoko Horino; Yuji Takahashi; Kazuhiro Toriyama; Kazuhiko Inou; Shuhei Torii; Yasuo Kitagawa (179-187).
Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of α1(IV) and α2(IV) mRNAs. Prolyl 4-hydroxylase α increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase α2β2 tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.
Keywords: Adipocyte; Collagen; Fibroblast; Fibrosis; Hypoxia; Prolyl hydroxylation;

A new functional domain of Bcl6 family that recruits histone deacetylases by Hong Zhang; Seiji Okada; Masahiko Hatano; Shinichiro Okabe; Takeshi Tokuhisa (188-200).
The proto-oncogene Bcl6 and its family gene, BAZF, encode a sequence-specific transcriptional repressor which contains the BTB/POZ domain in NH2-terminal region and zinc finger motifs in COOH-terminal region. The BTB/POZ domain and the middle portion of Bcl6 and BAZF are known to display transrepressor activity. Since we have identified the identical 17-amino acid (aa) sequence in the middle portion of Bcl6 and BAZF, the 17aa region may be another repressive domain of the middle portion. The reporter gene assay indicates that the 27aa sequence including the 17aa region recruits histone deacetylases to express transrepressor activity. Furthermore, overexpression of Bcl6 or Bcl6(POZ−) (Bcl6 deleted with the BTB/POZ domain) induced apoptosis in NIH3T3 cells, and the apoptosis was inhibited by the addition of histone deacetylase inhibitor in the culture. However, apoptosis was not induced in NIH3T3 cells by overexpression of Bcl6(POZ−) deleted with the 17aa region. These results indicate that the 17aa region in the middle portion of Bcl6 is a functional domain of transrepressor activity and is responsible for inducibility of apoptosis in NIH3T3 cells.
Keywords: Bcl6; BAZF; Apoptosis; Histone deacetylase; Transcriptional repressor; Trichostatin A;

Parathyroid hormone activation of map kinase in rat duodenal cells is mediated by 3′,5′-cyclic AMP and Ca2+ by Claudia Gentili; Susana Morelli; Ricardo Boland; Ana Russo de Boland (201-212).
In a previous study, we demonstrated that parathyroid hormone (PTH) stimulates in rat duodenal cells (enterocytes) the phosphorylation and activity of extracellular signal-regulated mitogen-activated protein kinase (MAPK) isoforms ERK1 and ERK2. As PTH activates adenylyl cyclase (AC) and phospholipase C and increases intracellular Ca2+ in these cells, in the present study we evaluated the involvement of cAMP, Ca2+ and protein kinase C (PKC) on PTH-induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5–15 μM), an AC activator, or Sp-cAMP (50–100 μM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2–0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca2+ (EGTA 0.5 mM), chelation of intracellular Ca2+ with BAPTA (5 μM), or blockade of L-type Ca2+-channels with verapamil (10 μM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca2+ mobilizing agent thapsigargin, the Ca2+ ionophore A23187, ionomycin and membrane depolarization with high K+. Inclusion of the calmodulin inhibitor fluphenazine (50 μM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca2+ play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca2+ and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.
Keywords: Parathyroid hormone; Rat enterocyte; MAP kinase; Calcium; Cyclic AMP; Signal transduction;

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H2O2 stimulation by Yoichi Mizukami; Takayuki Okamura; Toshiro Miura; Masayasu Kimura; Kimiko Mogami; Natsuko Todoroki-Ikeda; Sei Kobayashi; Masunori Matsuzaki (213-220).
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. H2O2 (100 μM) 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 H2O2 stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 μM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H2O2-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H2O2 stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H2O2. Under these conditions, the H2O2-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 H2O2, and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.
Keywords: c-Jun N-terminal kinase; Hydrogen peroxide; Antisense; Apoptosis;

Characteristics and distribution of endogenous RFamide-related peptide-1 by Shoji Fukusumi; Yugo Habata; Hiromi Yoshida; Norio Iijima; Yuji Kawamata; Masaki Hosoya; Ryo Fujii; Shuji Hinuma; Chieko Kitada; Yasushi Shintani; Masato Suenaga; Haruo Onda; Osamu Nishimura; Masaki Tanaka; Yasuhiko Ibata; Masahiko Fujino (221-232).
We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022. However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats. Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody. In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples Gi/Go in the signal transduction pathway.
Keywords: RFamide-related peptide-1; OT7T022; Distribution; Antibody; Purification;

Factor(s) released by glucose-deprived astrocytes enhance glucose transporter expression and activity in rat brain endothelial cells by Anthony Régina; Stephanie Morchoisne; Nancy D Borson; Anthony L McCall; Lester R Drewes; Françoise Roux (233-242).
Glucose transporter (GLUT) expression and regulation were studied in rat brain endothelial cells in primary culture (RBEC) and in immortalised RBE4 cells. Immunoblotting analysis showed a low expression of the endothelium-specific GLUT1 in RBEC and RBE4 cells compared to isolated brain capillaries. RBEC and RBE4 cells also expressed the GLUT3 isoform, whereas it was not present in isolated brain capillaries. No change in GLUT expression was observed in endothelial cells treated with astrocyte-conditioned medium. However, treatment with conditioned medium obtained from glucose-deprived astrocytes increased endothelial GLUT1 expression and glucose uptake. These results suggest that astrocytes submitted to hypoglycaemic conditions may release factor(s) that increase glucose uptake through the blood–brain barrier.
Keywords: Blood–brain barrier; Brain endothelial cell; Glucose transporter; Astrocyte; Hypoglycemia;

Cellular adhesion and spreading are critical components involved in the processes of cell and tissue development, and immune responses in molluscs, but at present, little is known regarding the signaling pathways involved in these basic cellular functions. In the present study, the molluscan Biomphalaria glabrata embryonic (Bge) cell line was used as an in vitro model to study the signal transduction pathways regulating molluscan cell adhesion and spreading behavior. Western blot analysis using antibodies specific to mitogen-activated protein kinase (MAPK) revealed the presence of an MAPK-like immunoreactive protein in Bge cells, that was phosphorylated upon exposure to phorbol myristate acetate (PMA). Moreover, Bge cell treatment with inhibitors of protein kinase C (PKC), Ras and MAPK kinase (Mek) suppressed PMA-induced expression of activated MAPK, suggesting that PKC-, Ras- and Mek-like molecules may be acting upstream of MAPK. Similarly, in vitro Bge cell-spreading assays were performed in conjunction with the same panel of inhibitors to determine the potential involvement of PKC, Ras and Mek in cellular adhesion/spreading. Results revealed a similar pattern of inhibition of cell-spreading behavior strongly implying that the Bge cell spreading also may be regulated through a MAPK-associated signal transduction pathway(s) involving proteins similar to PKC, Ras and Mek.
Keywords: Mollusc; Cell spreading; Signal transduction; Mitogen-activated protein kinase; Protein kinase C; Biomphalaria glabrata embryonic cell line;

Characterization of basal nitric oxide production in living cells by Manuel O. López-Figueroa; Claudio Caamaño; Raquel Marin; Borja Guerra; Rafael Alonso; M.Inés Morano; Huda Akil; Stanley J. Watson (253-264).
Nitric oxide (NO) is an important modulator of immune, endocrine and neuronal functions; however, measuring physiological levels of NO in cell cultures is generally difficult because of the lack of suitable methodologies. We have selected three cell lines from different origins: the neuroblastoma-derived Neuro2A (N2A), the cholinergic SN56 and the non-neuronal COS-1. We first demonstrated the presence of NADPH-diaphoretic activity, a potential marker of the NO-synthesizing (NOS) enzyme. By immunocytochemistry, using specific antibodies for each NOS subtype, we observed that subtype I was present in all cell lines and that subtype II was present in COS-1 and N2A cell lines. The presence of these NOS subtypes was further verified by Western blot analysis. Control cells treated with DAF-2 DA exhibited significant fluorescent levels corresponding to basal NO production. The subcellular distribution of the synthesizing enzyme was consistent with the NO-fluorescence signal; whereas, fixation affected the subcellular pattern of NO fluorescence signal. Addition of NOS inhibitors or NO scavengers to the incubation medium reduced the intensity of the NO fluorescence signal in a concentration-dependent manner. Conversely, increasing concentrations of a NO donor, or incident light, increased the fluorescence intensity. Our observation of NO production and distribution using the DAF-2 method has a direct impact on studies using these cell lines.
Keywords: Nitric oxide synthase; Diaminofluorescein; COS-1; N2A; SN56;

Author Index (265-266).

Cumulative Contents (267-268).