BBA - Molecular Cell Research (v.1593, #2-3)

Zyxin and paxillin are the prototypes of two related subfamilies of LIM domain proteins that are localized primarily at focal adhesion plaques. However, recent work has shown that zyxin/paxillin family proteins also shuttle through the nucleus. These proteins may enter the nucleus by association with other proteins, but are exported from the nucleus by means of intrinsic leucine-rich nuclear export sequences. Zyxin/paxillin proteins may regulate gene transcription by interaction with transcription factors. In some cases, misregulation of nuclear functions of zyxin/paxillin proteins appear to be associated with pathogenic effects.
Keywords: Zyxin; Paxillin; Focal adhesion; LIM domain; Nuclear-cytoplasmic shuttling; Transcription;

Rho GTPases and spermatogenesis by Wing-Yee Lui; Will M Lee; C.Yan Cheng (121-129).
Rho GTPases, such as Rho, Rac and Cdc42, are known to regulate many cellular processes including cell movement and cell adhesion. While the cellular events of germ cell movement are crucial to spermatogenesis since developing germ cells must migrate progressively from the basal to the adluminal compartment but remain attached to the seminiferous epithelium, the physiological significance of Rho GTPases in spermatogenesis remains largely unexplored. This paper reviews some recent findings on Rho GTPases in the field with emphasis on the studies in the testis, upon which future studies can be designed to delineate the role of Rho GTPases in spermatogenesis.
Keywords: Rho GTPase; Spermatogenesis; Sertoli cell; Germ cell; ROCK; LIMK; Signal transduction;

Low-energy laser irradiation (LELI) drives quiescent skeletal muscle satellite cells into the cell cycle and enhances their proliferation, thereby promoting skeletal muscle regeneration. Ongoing protein synthesis is a prerequisite for these processes. Here, we studied the signaling pathways involved in the LELI regulation of protein synthesis. High levels of labeled [35S]methionine incorporation were detected in LELI cells as early as 20 min after irradiation, suggesting translation of pre-existing mRNAs. Induced levels of protein synthesis were detected up until 8 h after LELI implying a role for LELI in de novo protein synthesis. Elevated levels of cyclin D1, associated with augmented phosphorylation of the eukaryotic initiation factor 4E (eIF4E) and its inhibitory binding protein PHAS-I, suggested the involvement of LELI in the initiation steps of protein translation. In the presence of the MEK inhibitor, PD98059, eIF4E phosphorylation was abolished and levels of cyclin D1 were dramatically reduced. The LELI-induced PHAS-I phosphorylation was abolished after preincubation with the PI3K inhibitor, Wortmannin. Concomitantly, LELI enhanced Akt phosphorylation, which was attenuated in the presence of Wortmannin. Taken together, these results suggest that LELI induces protein translation via the PI3K/Akt and Ras/Raf/ERK pathways.
Keywords: Laser irradiation; Myoblast; Protein translation; PHAS-I; Akt; eIF4E;

Characterisation of PDZ-GEFs, a family of guanine nucleotide exchange factors specific for Rap1 and Rap2 by H.Bea Kuiperij; Johan de Rooij; Holger Rehmann; Miranda van Triest; Alfred Wittinghofer; Johannes L Bos; Fried J.T Zwartkruis (141-149).
PDZ-GEF1 (RA-GEF/nRapGEP/CNrasGEF) is a guanine nucleotide exchange factor (GEF) characterised by the presence of a PSD-95/DlgA/ZO-1 (PDZ) domain, a Ras-association (RA) domain and a region related to a cyclic nucleotide binding domain (RCBD). These domains are in addition to a Ras exchange motif (REM) and GEF domain characteristic for GEFs for Ras-like small GTPases. PDZ-GEF1 efficiently exchanges nucleotides of both Rap1 and Rap2, but has also been implicated in mediating cAMP-induced Ras activation through binding of cAMP to the RCBD. Here we describe a new family member, PDZ-GEF2, of which we isolated two splice variants (PDZ-GEF2A and 2B). PDZ-GEF2 contains, in addition to the domains characteristic for PDZ-GEF1, a second, less conserved RCBD at the N-terminus. PDZ-GEF2 is also specific for both Rap1 and Rap2. We further investigated the possibility that PDZ-GEF2, like PDZ-GEF1, is a cAMP-responsive GEF for Ras. However, in contrast to previous results, we did not find any effect of either PDZ-GEF1 or PDZ-GEF2 on Ras in the absence or presence of cAMP. Moreover, affinity measurements by isothermic calorimetry showed that the RCBD of PDZ-GEF1 does not bind cAMP with a physiologically relevant affinity. We conclude that both PDZ-GEF1 and 2 are specific for Rap1 and Rap2 and unresponsive to cAMP and various other nucleotides.
Keywords: cAMP; Ras; Rap; Guanine nucleotide exchange factor;

Molecular properties of endogenous RFamide-related peptide-3 and its interaction with receptors by Hiromi Yoshida; Yugo Habata; Masaki Hosoya; Yuji Kawamata; Chieko Kitada; Shuji Hinuma (151-157).
Based on database searches of DNA sequences, we previously reported a gene encoding peptides possessing Arg-Phe-NH2 (RFamide) at their C termini. This gene, RFamide-related peptide (RFRP), was expected to encode several different peptides (i.e., RFRP-1, -2, and -3). In the present study, we purified endogenous RFRP-3 from bovine hypothalamus, and demonstrated that it consisted of 28 amino acid residues. After constructing a sandwich enzyme immunoassay for RFRP-3, we analyzed the tissue distribution of endogenous RFRP-3 in rats and found its concentration to be highest in the hypothalamus. In binding assays, [125I]-labeled RFRP-3 bound to OT7T022 with high affinity, but its binding affinity to HLWAR77 was low. On the other hand, [125I]-labeled neuropeptide FF (NPFF) bound to both OT7T022 and HLWAR77 with high affinity. By serial deletion in the N-terminal portions of RFRP-3 and NPFF, we found that four C-terminal amino acid residues (i.e., PQRFamide), which were common between the two peptides, comprised a core sequence responsible for binding with the receptors, whereas three amino acid residues (i.e., PNL in RFRP-3 and LFQ in NPFF) added to the N terminus of PQRFamide played crucial roles in the agonistic activities of RFRP-3 and NPFF for OT7T022 and HLWAR77, respectively.
Keywords: RFamide-related peptide-3; OT7T022; HLWAR77; Neuropeptide FF; Receptor;

We investigated the mechanism of augmentation of nitric oxide (NO) production in the murine macrophage cell line RAW264.7 after γ-irradiation. The cells treated with interferon-γ (IFN-γ) or lipopolysaccharide (LPS) showed enhanced NO production by γ-irradiation in a dose-dependent manner, accompanying the induction of inducible nitric oxide synthase (iNOS) expression. Nuclear factor kappa B (NF-κB) activation was induced 1 h after γ-irradiation dose-dependently, which was detected by the degradation of I-κB. Inhibitors of I-κB degradation, MG132 and N α-p-tosyl-l-lysine chloromethyl ketone (TLCK), suppressed the further increase by γ-irradiation in IFN-γ-induced NO production, showing that γ-irradiation induced NO production via NF-κB activation. Although NF-κB is known to be a redox-sensitive transcription factor, the antioxidant agents N-acetyl-cysteine (NAC) and 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox) showed no suppression and treatment with H2O2 showed only slight enhancement of IFN-γ-induced NO production. The DNA damaging agents camptothecin and etoposide enhanced IFN-γ-induced NO production and showed I-κB degradation, indicating that the increase in NO production was due to direct DNA damage. Furthermore, 3-aminobenzamide (3AB) and benzamide, inhibitors of poly (ADP-ribose) polymerase (PARP) that are activated upon recognition of DNA strand breaks, suppressed the further increase by γ-irradiation in IFN-γ-induced NO production and the I-κB degradation by γ-irradiation. We concluded that (1) the increase in NO production was due to direct DNA damage by γ-irradiation, and that (2) PARP activation through DNA damage induced NF-κB activation, leading to iNOS expression and NO production.
Keywords: RAW264.7 cell; γ-Irradiation; Nitric oxide; DNA damage; NF-κB; Poly (ADP-ribose) polymerase;

Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells by Claudia Gentili; Gabriela Picotto; Susana Morelli; Ricardo Boland; Ana Russo de Boland (169-178).
In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca2+]i) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca2+]I and the implication of Ca2+ on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca2+]i in a dose-dependent manner (1 pM–100 nM). In cells from aged rats, hormone concentrations higher than physiological (≥1 nM) were required to observe significant increases in [Ca2+]i. Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca2+]i biphasic response to PTH of young enterocytes while in old cells, no effects were observed. The voltage-dependent calcium-channel blocker (VDCC), nitrendipine, suppressed PTH-dependent changes of the sustained [Ca2+]i phase in young and aged animals. In this study, we analysed, for the first time, alterations in phosphatidylinositol 3-kinase (PI3K) activity and response to PTH in rat enterocytes with ageing. Basal PI3K activity was significantly modified by ageing. Acute treatment with 10−8 M PTH increased enzyme activity, with a maximun at 2 min (+3-fold) in young rats and only elevated by less than 1-fold basal PI3K activity in aged animals. Hormone-induced tyrosine phosphorylation of p85α, the regulatory subunit of PI3K, as well as the phosphorylation on Thr308 of its downstream effector Akt/PKB was evident in enterocytes from 3-month-old rats, whereas it was greatly reduced in the cells from 24-month-old animals. Intracellular Ca2+ chelation (BAPTA-AM, 5 μM) affected the tyrosine phosphorylation of p85α and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca2+ is required for full activation of PI3K in enterocytes stimulated with PTH. The Thr phosphorylation of PI3K downeffector, Akt/PKB, was also fully dependent on Ca2+. Taken together, these results suggest that PTH regulation of enterocyte [Ca2+]i involves Ca2+ mobilization from IP3-sensitive stores and the influx of the cation from the extracellular milieu, the former pathway being blunted during ageing. The data also indicates a positive role for intracellular calcium in one of the early signals of PTH in rat enterocytes, the activation of PI3K, and that hormone regulation of PI3K activity and Akt/PKB phosphorylation on Thr308 is impaired with ageing.
Keywords: Parathyroid hormone; Intracellular Ca2+; PI3K; AKT/PKB; Intestine; Ageing;

Caspase-mediated apoptosis in sponges: cloning and function of the phylogenetic oldest apoptotic proteases from Metazoa by Matthias Wiens; Anatoli Krasko; Sanja Perovic; Werner E.G. Müller (179-189).
Sponges (phylum Porifera) represent the phylogenetically oldest metazoan phylum. These animals have complex cell adhesion and powerful immune systems which allow the formation of a distinct body plan. Consequently, an apoptotic machinery has to be predicted that allows sponges to eliminate unwanted cells accumulating during development. With the marine sponge Geodia cydonium, it is shown that allografts of these animals undergo apoptosis as demonstrated by apoptotic DNA fragmentation. Extracts from allografts contain an enzymic activity characteristic for caspases; as substrate to determine the cleavage activity, Ac-DEVD-AMC was applied. cDNAs encoding predicted caspase-3-related proteins were isolated; they comprise the characteristic structure known from caspases of other metazoan phyla. The two cDNAs are assumed to originate from one gene by alternative splicing; the longer form comprises a caspase recruitment domain (CARD), whereas the shorter one is missing CARD. The expression of sponge caspase genes is up-regulated during allograft rejection. In vivo incubation experiments with Ac-DEVD-CHO (a caspase-3 inhibitor) showed a reduction of apoptotic DNA fragmentation, whereas Ac-LEHD-CHO (an inhibitor of caspase-9) caused no effect. It is concluded, that for the establishment of the metazoan body plan, both the adhesion molecules and the apoptotic molecules (described here) were essential prerequisites.
Keywords: Sponge; Invertebrate; Suberites domuncula; Caspase; Potential proapoptotic molecule DD2; Bcl-2 homologous protein;

After incubation with 2-butylamino-2-demethoxy-hypocrellin A (2-BA-2-DMHA), photodynamically induced change in the cytoplasmic free calcium concentration ([Ca2+]i) and its effect on cell damage were investigated in human gastric cancer (MGC-803). Fluorescence spectrophotometry measurement indicated that the photosensitization of MGC-803 by 2-BA-2-DMHA caused an increase in intracellular calcium [Ca2+]i, and this increase in [Ca2+]i showed a dependence on the concentration of 2-BA-2-DMHA, light dose and extracellular [Ca2+]e. This phenomenon of intracellular calcium accumulation was further confirmed by using laser scanning confocal microscopy (LSCM). Furthermore, the results from MTT assay and flow cytometry analysis suggested that chelation of extracellular calcium by EGTA or intracellular calcium by BAPTA could inhibit photodynamically induced cell killing, while increase of [Ca2+]i by thapsigargin (TG), a highly specific inhibitor of the Ca2+-ATPase, or by A23187, a calcium ionophore could enhance this action. Meanwhile, the nucleus morphology was also investigated by fluorescence microscopy. The results indicated that the increase in intracellular Ca2+ concentration was responsible for 2-BA-2-DMHA photodynamically induced damage to MGC-803.
Keywords: 2-butylamino-2-demethoxy-hypocrellin A (2-BA-2-DMHA); Human gastric cancer (MGC-803); Photodynamic treatment (PDT); Intracellular calcium [Ca2+]i; Extracellular calcium [Ca2+]e;

Effects of prolonged metabolic (glucose deprivation) and hormonal [insulin-like growth factor I (IGF-I)] challenge on regulation of glucose transporter (GLUT) expression, glucose transport rate and possible signaling pathways involved were studied in the neuroendocrine chromaffin cell. The results show that bovine chromaffin cells express both GLUT1 and GLUT3. Glucose deprivation and IGF-I activation led to an elevation of GLUT1 and GLUT3 mRNA, the strongest effect being that of IGF-I on GLUT3 mRNA. Both types of stimulus increased the GLUT1 protein content in a cycloheximide (CHX)-sensitive manner, and the glucose transport rate was elevated by 3- to 4-fold after 48 h under both experimental conditions. IGF-I-induced glucose uptake was totally suppressed by CHX. In contrast, only ∼50% of transport activation in glucose-deprived cells was sensitive to the protein synthesis inhibitor. Specific inhibitors of mTOR/FRAP and p38 MAPK each partially blocked IGF-I-stimulated glucose transport, but had no effect on transport rate in glucose-deprived cells. The results are consistent with IGF-I-activated transport being completely dependent on new GLUT protein synthesis while the enhanced transport in glucose-deprived cells was partially achieved independent of new synthesis of proteins, suggesting a mechanism relying on preexisting transporters.
Keywords: Chromaffin cell; Glucose transport; GLUT1; GLUT3; Glucose deprivation; IGF-I;

Interactions between doxorubicin and the human iron regulatory system by Xavier Brazzolotto; Maud Andriollo; Pascale Guiraud; Alain Favier; Jean-Marc Moulis (209-218).
Anthracyclines are included in clinical treatments against various malignancies, but severe cardiotoxic side-effects and the development of resistance mechanisms limit their usefulness. Many aspects of the cellular response to anthracyclines remain debated. The status of the main regulator of iron homeostasis, namely the RNA-binding activity of iron regulatory proteins (IRPs), has been assessed herein for two types of human tumor cells and their derived doxorubicin-resistant sublines. IRPs were always fully activated in the latter, whereas only partial activation occurred in the former. Doxorubicin exposure reversibly inactivated IRP1 in small cell lung carcinoma (GLC4) and myelogenous leukemia (K562) cell lines, but was without effect in their derived doxorubicin-resistant sublines. In contrast, adding doxorubicin to cytosolic fractions of untreated cells or to purified IRPs led to the irreversible alteration of the RNA-binding activity of IRP1. In these different conditions, interaction between doxorubicin and the iron regulatory system disturbs iron metabolism, and cells having developed a resistance mechanism are tuned to maximize the iron supply. The results reported herein may lead the path toward a better therapeutic management of cancer patients receiving doxorubicin by discriminating between the antiproliferative and cardiotoxic properties of this anthracycline.
Keywords: Iron regulation; Anthracycline; Resistance mechanism; Formaldehyde; Glutathione; Iron chelation;

PKCε-mediated ERK1/2 activation involved in radiation-induced cell death in NIH3T3 cells by Yoon-Jin Lee; Jae-Won Soh; Doo-Il Jeoung; Chul-Koo Cho; Gil Ja Jhon; Su-Jae Lee; Yun-Sil Lee (219-229).
Protein kinase C (PKC) isoforms play distinct roles in cellular functions. We have previously shown that ionizing radiation activates PKC isoforms (α, δ, ε, and ζ), however, isoform-specific sensitivities to radiation and its exact mechanisms in radiation mediated signal transduction are not fully understood. In this study, we showed that overexpression of PKC isoforms (α, δ, ε, and ζ) increased radiation-induced cell death in NIH3T3 cells and PKCε overexpression was predominantly responsible. In addition, PKCε overexpression increased ERK1/2 activation without altering other MAP-kinases such as p38 MAPK or JNK. Co-transfection of dominant negative PKCε (PKCε-KR) blocked both PKCε-mediated ERK1/2 activation and radiation-induced cell death, while catalytically active PKCε construction augmented these phenomena. When the PKCε overexpressed cells were pretreated with PD98059, MEK inhibitor, radiation-induced cell death was inhibited. Co-transfection of the cells with a mutant of ERK1 or -2 (ERK1-KR or ERK2-KR) also blocked these phenomena, and co-transfection with dominant negative Ras or Raf cDNA revealed that PKCε-mediated ERK1/2 activation was Ras–Raf-dependent. In conclusion, PKCε-mediated ERK1/2 activation was responsible for the radiation-induced cell death.
Keywords: PKCε; Cell death; Radiation sensitivity; ERK1/2; NIH3T3 cell;

Tissue kallikrein is synthesized and secreted by human vascular endothelial cells by Katsutoshi Yayama; Naomi Kunimatsu; Yumiko Teranishi; Masaoki Takano; Hiroshi Okamoto (231-238).
The generation of kinins on the surface of vascular endothelium has been postulated in two pathways involving plasma kallikrein and tissue kallikrein; the former pathway has been well documented, but the latter is controversial. To clarify the presence of a kinin-generating system on endothelium, we examined whether human umbilical vein endothelial cells (HUVEC) synthesize and release tissue kallikrein in vitro. Kallikrein-like activity hydrolyzing a peptide Pro-Phe-Arg-4-methyl-coumaryl-7-amide was detected in the culture medium of HUVEC and was inhibited by aprotinin but not by soybean trypsin inhibitor. Western blotting of HUVEC medium using anti-human tissue kallikrein antibodies demonstrated the release of tissue kallikrein from HUVEC, and the reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blotting revealed the expression of tissue kallikrein mRNA in HUVEC. HUVEC metabolically labeled with [35S]methionine released radioactive proteins corresponding to tissue kallikrein. RT-PCR also showed the expression of low-molecular-weight kininogen (L-kininogen) mRNA in HUVEC. The cGMP levels in HUVEC were significantly elevated by the incubation with angiotensin converting enzyme inhibitor, lisinopril, and the elevation was completely inhibited by aprotinin or bradykinin B2-receptor antagonist, FR172357. These results suggest that the endothelial cells continuously release an active form of tissue kallikrein which enables generation of kinins on the vascular endothelium.
Keywords: Kallikrein; Endothelial cell; Bradykinin; HUVEC;

Tensile stress-dependent collagen XII and fibronectin production by fibroblasts requires separate pathways by Martin Flück; Marie-Noëlle Giraud; Vildan Tunç; Matthias Chiquet (239-248).
The intracellular mechanisms controlling mechano-dependent production of the two extracellular matrix proteins collagen XII and fibronectin were analyzed. Fibroblasts were cultured on either tensed (attached) or released (floating) collagen type-I gels, respectively. Collagen XII and fibronectin production was three- to fivefold higher under tensed than under released conditions. The general inhibitor of tyrosine phosphorylation, genistein (50 μM), and the MAP kinase inhibitor PD98059 (20 μM) selectively reduced collagen XII accumulation by tensed cultures. Addition of PD98059, but not genistein, downregulated tensile stress-induced tyrosine phosphorylation levels of ERK1/2 and focal adhesion kinase. Staurosporine as well as pretreatment with phorbol ester, which constitute means to downregulate classical and novel PKC activity, specifically blocked collagen XII but not fibronectin accumulation in tensed fibroblasts. ERK1/2 phosphorylation levels were not affected by staurosporine treatment. Chronic exposure to the protein kinase C inhibitors bisindolylmaleimide and calphostin C blocked increased production of both fibronectin and collagen XII from cells under tension. The data manifest that the mechano-dependent production of collagen XII and fibronectin requires separate pathways. The FAK-ERK1/2 pathway, a genistein-sensitive tyrosine kinase, and a distinct classical/novel PKC appear selectively required for increased production of collagen XII in cells under tensile stress, whereas fibronectin induction is regulated by a different PKC-dependent pathway.
Keywords: Mechanical stress; Signaling; Collagen XII; Fibronectin;

Disruption of actin filaments affects multiple cell functions including motility, signal transduction and cell division, ultimately culminating in cell death. Although this is the usual sequence of events, we have made the interesting observation that disruption of actin filaments by the potent toxin cytochalasin D (Cyto D) causes one cell type, mouse mesangial cells (MMC), to undergo apoptosis, while in another cell type (NIH 3T3), it has the opposite effect, resulting in production of survival signals. The purpose of this study was to investigate the molecular basis for these observed differences. In the present communication, we demonstrate that exposure to Cyto D induces the pro-apoptotic pathways, p38 and stress-activated protein kinase (SAPK)/jun amino-terminal kinase (JNK), in both cell types. However, in 3T3, but not MMC, the extracellular signal regulated kinase (ERK) 1/2 pathway is protected from inhibition following treatment with Cyto D—leading to phosphorylation of Bclxi/Bcl 2-associated death promoter (BAD). Inhibition of Cyto D-induced secretion and activation of gelatinase A in 3T3 cells reverses the production of survival signals by Cyto-D. To investigate this effect further we employed CS-1 cells, a well-characterized melanoma cell line that lacks integrin β3, and also does not secrete gelatinase A. Co-transfection of CS-1 cells with integrin β3 and a gelatinase A transgene, which enables the cells to secrete constituitively active gelatinase A, enhances CS-1 cell survival signals. Together, our findings suggest that extracellularly activated gelatinase A, through interaction with integrin αVβ3, elicits survival signals mediated through ERK 1/2 that override activation of p38 and SAPK/JNK stress pathways.
Keywords: Cell survival; Integrin αVβ3; Signal transduction;

Serglycin proteoglycan expression and synthesis in embryonic stem cells by Barbara P Schick; Hon-Chung Keith Ho; Kristin C Brodbeck; Clinton W Wrigley; Joanne Klimas (259-267).
The serglycin proteoglycan is expressed in most hematopoietic cells and is packaged into secretory vesicles for constitutive or regulated secretion. We have now shown serglycin mRNA expression in undifferentiated murine embryonic stem (ES) cells and in embryoid bodies, and synthesis and secretion in undifferentiated ES cells. Serglycin was localized to ES cell cytoplasm by immunostaining. Serglycin mRNA is expressed in tal-1(−/−) ES cells and embryoid bodies; tal-1(−/−) mice cannot produce hematopoietic cells. Thus, ES serglycin expression is probably not associated with hematopoiesis. Serglycin expression was increased by treatment of ES cells with retinoic acid (RA) and dibutyryl cAMP (dbcAMP). The serglycin core protein obtained from control ES culture medium after chondroitinase digestion appears as a doublet. Only the lower Mr band is present in serglycin secreted from RA-treated and the higher Mr band in RA+dbcAMP-treated cells, suggesting that core protein structure is affected by differentiation.
Keywords: Serglycin proteoglycan; Embryonic stem cells; Hematopoiesis;

Hypoxia up-regulates glyceraldehyde-3-phosphate dehydrogenase in mouse brain capillary endothelial cells: involvement of Na+/Ca2+ exchanger by Ryoichi Yamaji; Kayoko Fujita; Saeko Takahashi; Hiroko Yoneda; Kaori Nagao; Wataru Masuda; Mikihiko Naito; Takashi Tsuruo; Kazutaka Miyatake; Hiroshi Inui; Yoshihisa Nakano (269-276).
The molecular regulatory mechanisms and the characterization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in hypoxia were studied in a mouse brain capillary endothelial cell line, MBEC4. Activation of GAPDH gene expression by hypoxia was suppressed by an intracellular Ca2+ chelator and inhibited by a non-selective cation channel blocker or a Na+/Ca2+ exchanger (NCX) blocker. Sequencing of reverse transcription-PCR products demonstrated that MBEC4 expressed an mRNA encoding NCX3, which functions even under cellular ATP-depleted conditions, in addition to mRNAs encoding NCX1 and NCX2. The inhibition of Ca2+/calmodulin-dependent protein kinases or c-Jun/AP-1 activation caused a significant decrease in the activation of GAPDH mRNA by hypoxia. These results suggest that hypoxia stimulates Ca2+ influx through non-selective cation channels and causes the reverse operation of the three NCX isoforms, and consequently, increased intracellular Ca2+ up-regulates GAPDH gene expression through an AP-1-dependent pathway. Furthermore, subcellular fractionation experiments showed that hypoxia increased GAPDH proteins not only in the cytosolic fraction, but also in the nuclear and particulate fractions, in which GAPDH should play no roles in glycolysis. However, the GAPDH activity did not rise in proportion to the increase of GAPDH protein by hypoxia even in the cytosolic fraction. These results suggest that not all hypoxia-induced GAPDH molecules contribute to glycolysis.
Keywords: Hypoxia; Glyceraldehyde-3-phosphate dehydrogenase; Ca2+; Na+/Ca2+ exchanger; AP-1; Brain capillary endothelial cell;

The novel retinoid AHPN/CD437 induces a rapid but incomplete apoptotic response in human myeloma cells by Bertrand Joseph; Philippe Marchetti; Olga Lefebvre; Suzanna Schraen-Maschke; Claude Méreau-Richard; Pierre Formstecher (277-282).
The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) appears to possess an apoptotic activity superior to classical retinoids in vitro as in vivo. Numerous studies have shown that CD437-induced apoptosis is independent of its nuclear receptor activity, suggesting that CD437 might have a unique mechanism of action. The purpose of this study was to compare CD437- and all-trans retinoic acid (atRA)-induced cell death. CD437 provoked a rapid apoptotic phenotype immediately followed by secondary necrosis in RPMI 8226, U266 and L363 human myeloma cell lines. Nuclear apoptotic features were observed upon both CD437 and atRA treatments. In contrast, membrane blebbing and the subsequent formation of apoptotic bodies, a classical apoptotic event, was only observed upon atRA treatment. In addition, CD437, contrary to atRA, was unable to induce tissue transglutaminase (tTG), an intracellular enzyme involved in the formation of cross-linked protein polymers contributing to apoptotic morphological changes. Taken together, these data suggest that CD437 induces rapid but incomplete apoptotic phenotype in human myeloma cells.
Keywords: Myeloma; Retinoid; CD437/AHPN; Programmed cell death; Tissue transglutaminase;

Sterol 27-hydroxylase has been suggested to be involved in an alternative pathway for the elimination of cholesterol from macrophages and early atherosclerotic lesions. We have previously shown that human lung macrophages as well as monocyte-derived macrophages have a relatively high activity of sterol 27-hydroxylase (CYP27). This enzyme converts intracellular cholesterol into 27-hydroxycholesterol and cholestenoic acid that flux from cultured cells into the medium. It is shown here that human monocytes have very low CYP27 activity and CYP27 mRNA levels. During differentiation into macrophages, both CYP27 activity and CYP27 mRNA levels increase markedly after 4 days of culture in serum-free medium. Addition of macrophage-colony stimulating factor had no significant effect on the induction and addition of fetal calf serum had an inhibitory effect. Cholesterol synthesis was found to be a critical factor for the production of 27-oxygenated products by the macrophages cultured in serum-free medium. The increased capacity of the differentiated cells to eliminate intracellular cholesterol is of interest and supports the contention that CYP27 is an antiatherogenic factor.
Keywords: 27-Hydroxycholesterol; Cholestenoic acid; Macrophage-colony stimulating factor;