Biomedical subjects

M Matsushima

Publications and source records attributed to M Matsushima.

At least 37 records · Page 2Linked to original sources

PubMed2001

[Visual memory impairment during postpartum].

Using a visual memory test we studied memory in pregnant women. Benton's Visual Retention Test was used with 54 pregnant women three times: at term, 3-4 days post-partum, and one month after birth. There was a significant decline in visual memory at 3-4 days post-partum (p < 0.01). There was no interaction between hours of sleep and memory of pregnant women at any period. Although memory declined at 3-4 days, there was no interaction between this decline and duration of labor, intra-partum hemorrhage, or memory at one month post-partum. It is not yet clear why visual memory declined shortly after birth. Further studies on post-partum memory decline as well as the possible relationship to hormonal and mood changes are necessary.

Adult↗

PubMed2000

A potent aldose reductase inhibitor, (2S,4S)-6-fluoro-2', 5'-dioxospiro[chroman-4,4'-imidazolidine]-2-carboxamide (Fidarestat): its absolute configuration and interactions with the aldose reductase by X-ray crystallography.

The absolute configuration of the aldose reductase (AR) inhibitor, (+)-(2S,4S)-6-fluoro-2',5'-dioxospiro¿chroman-4, 4'-imidazolidine-2-carboxamide (fidarestat), was established indirectly by single-crystal X-ray analysis of (+)-(2S, 4S)-8-bromo-6-fluoro-2',5'-dioxospiro¿chroman-4, 4'-imidazolidine-2-carboxylic acid (1). The crystal structure of human AR complexed with fidarestat was determined, and the specific inhibition activity was discussed on the basis of the three-dimensional interactions between them. The structure clarified that fidarestat was located in the active site by hydrophilic and hydrophobic interactions and that the carbamoyl group of fidarestat was a very effective substituent for affinity to AR and for selectivity between AR and aldehyde reductase (AHR). Explanations for the differences between the observed activities of fidarestat and its stereoisomer 2 were suggested by computer modeling.

Aldehyde Reductase↗

PubMed2000

Histopathological findings of X-linked retinoschisis with neovascular glaucoma.

BACKGROUND: X-linked retinoschisis (XLRS) is rarely complicated by neovascular glaucoma. Only a few reports of XLRS histopathological findings with neovascular glaucoma have been published. METHODS: A 41-year-old man with XLRS complicated by neovascular glaucoma in his left eye was examined with electroretinography, B-scan, ultrasound biomicroscopy and computed tomography. He was examined by ophthalmoscopy and fluorescein angiography in the other eye. An enucleation was performed in his left eye due to uncontrollable high intraocular pressure and persistent ocular pain. We examined the enucleated eye histopathologically. RESULTS: Examination of the enucleated eye showed nuclear sclerosis of the lens, pigmented retrolental membrane and retinoschisis which separated the inner layer of the retina and made a large space in the vitreous cavity without any apparent detachment of the outer layers of the retina. Sclerotic vessels were present histopathologically in both the inner and outer layers of the retina. There was a peripheral anterior synechia, ectropion uveae and a fibrovascular membrane, which contained many lumina of neovascularization, indicating marked rubeosis iridis. Small cystic spaces were observed in both the schitic retina in the peripheral region and the foveal schisis at the outer layer of the retina. The photoreceptor cells had become markedly atrophied and multiple regions of calcification were observed. The optic nerve showed severe atrophy with gliosis, but the central retinal artery and vein were still open within the nerve. CONCLUSIONS: These histopathological findings suggest that rubeosis iridis may have developed secondarily to retinal ischemia due to occlusion of the retinal blood vessels.

Adult↗

PubMed2000

In vivo gene transfer into choroidal neovascularization by the HVJ liposome method.

PURPOSE: To evaluate the efficacy of the HVJ liposome method for gene transfer in rats with experimentally induced choroidal neovascularization. METHODS: Plasmid DNA containing the LacZ reporter gene, or fluorescein isothiocyanate (FITC)-labeled double-stranded phosphorothioate oligodeoxynucleotides (S-ODNs), was encapsulated in liposomes. The liposomes were coated with the envelope of inactivated hemagglutinating virus of Japan (HVJ). Intense laser burns were applied to the posterior pole of the retina of pigmented rats to induce choroidal neovascularization. Following photocoagulation, HVJ liposome suspension was injected into the vitreous. On days 3, 7, 14, and 28 after injection, the eyes were removed and fixed. The eyes injected with LacZ gene were reacted with X-gal, frozen, and cut into thin sections. The sections were examined for the expression of the LacZ gene by light microscopy. The enucleated eyes injected with double-stranded S-ODNs were frozen, cut into thin sections, and examined a confocal scanning laser microscope for FITC labeling. Eyes without injection of HVJ liposomes served as controls. RESULTS: Expression of LacZ genes (beta-galactosidase activity), or localization of FITC labeling, was observed mainly in the laser-induced choroidal neovascular tissue from 3 to 28 days after the intravitreal injection of HVJ liposome. CONCLUSIONS: We conclude that the HVJ liposome method achieved effective gene transfer into choroidal neovascular tissue. Thus, this method can be used as a nonviral gene therapy system for the treatment of choroidal neovascularization in vivo.

Animals↗

PubMed2000

Catheter interventional treatment in Kawasaki disease: A report from the Japanese Pediatric Interventional Cardiology Investigation group.

OBJECTIVE: To assess the current status of catheter intervention in Kawasaki disease and to evaluate its efficacy and outcome. STUDY DESIGN: A questionnaire was sent to 55 major institutions in Japan. RESULTS: A total of 58 procedures in 57 patients were reported. The median age at the time of intervention was 12.1 years. The procedures included percutaneous transluminal coronary angioplasty (PTCA; n = 34), percutaneous transluminal coronary rotational ablation (PTCRA; n = 13), directional coronary atherectomy (DCA; n = 4), and stent implantation (n = 7). The immediate success rate was 74% for PTCA, 100% for PTCRA, 100% for DCA, and 86% for stents. The interval from the onset of disease to intervention in successful PTCA (n = 25) was significantly shorter than that in unsuccessful PTCA (n = 9). Restenosis after PTCA was observed in 24%. Development of new coronary aneurysms was reported in 3 patients for PTCA, 2 for PTCRA, 3 for DCA, and 1 for stents. Except for the DCA, all new aneurysms were associated with the use of high-pressure balloon inflation. Two deaths were reported as acute complications. CONCLUSIONS: Catheter intervention is a promising therapeutic strategy in the management of coronary stenosis caused by Kawasaki disease. Care should be paid to avoid acute coronary arterial complications and the development of new coronary aneurysms.

Adolescent↗

PubMed2000

A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter in endometrial carcinomas.

Recent studies demonstrated that a single guanine insertion polymorphism in a matrix metalloprotease-1 promoter created an Ets binding site and affected the elevation of the transcriptional level of matrix metalloproteinase-1 (MMP-1). Furthermore, in tumor cell lines derived from melanoma and breast cancer, the incidence of the 2G / 2G genotype was significantly higher than that in the normal population. To evaluate the contribution of this polymorphism in endometrial carcinomas, we genotyped 100 endometrial carcinomas and then analyzed immunoexpression of MMP-1 in these carcinomas. We found that endometrial carcinoma patients showed a significantly higher rate of 1G / 2G or 2G / 2G genotype than control individuals, and that tumors containing the 2G allele(a) expressed MMP-1 protein more frequently than those with 1G / 1G genotype. Therefore, the single nucleotide polymorphism at the MMP-1 promoter affected the expression level of the MMP-1 protein, which may result in the association with more aggressive character in endometrial carcinoma. Our result suggests that the presence of 2G polymorphism at the MMP-1 promoter may be one of the risk factors for the development and / or progression of endometrial carcinoma.

Base Sequence↗

PubMed2000

[Evaluation of cardiac autonomic nerves by iodine-123 metaiodobenzylguanidine scintigraphy and ambulatory electrocardiography in patients after arterial switch operations].

The autonomic cardiac nerves reach the heart after passing through the vicinity of the aortic root and the pulmonary trunk. The arterial switch operation (ASO) completely transects the ascending aorta and the pulmonary trunk. Therefore, this surgical procedure virtually denerves the heart. Cardiac sympathetic denervation and reinnervation were evaluated in patients after ASO using iodine-123 metaiodobenzylguanidine (MIBG) myocardial scintigraphy and parasympathetic denervation and reinnervation using ambulatory electrocardiography [Holter electrocardiogram (ECG)]. MIBG scintigraphy was performed in 14 patients who underwent ASO (ASO group) and 3 patients who underwent other open heart surgery (control group). All patients in the ASO group underwent the operation in the neonatal or infantile period. Planar and single photon emission computed tomography (SPECT) images of the myocardium were obtained. Defect score was determined by the SPECT images as a semi-quantitative index. The mean interval between ASO and MIBG scintigraphy was 25.6 +/- 14.6 months. Holter ECG was also performed in 14 patients in the ASO group and 19 age-matched normal children. The Holter ECGs were plotted on a Lorenz plot. The H index, which is related to vagal tone for the cardiovascular system, was calculated from the R-R intervals. The mean interval between the ASO and Holter ECG was 8.3 +/- 9.7 months. MIBG scintigraphy in the control group demonstrated an almost normal homogeneous tracer uptake, but showed extremely reduced tracer uptake and significantly higher defect score in the ASO group. The extent and degree of the reduction of MIBG uptake improved with time after the ASO. The heart-to-mediastinum MIBG count ratio tended to increase with time. The H index of the ASO group was lower than that of normal children (< 12 months: Control group 0.0280 +/- 0.0068 vs ASO group 0.0219 +/- 0.0083), and gradually increased with time (1-3 years: 0.0470 +/- 0.0157 vs 0.0314 +/- 0.0124). These results indicate that MIBG scintigraphy reflects the presence of sympathetic denervation and the possibility of reinnervation after ASO, and that H index reflects the presence of parasympathetic denervation and the possibility of reinnervation after ASO. These are simple and useful methods for assessing the extent and degree of autonomic denervation and reinnervation.

3-Iodobenzylguanidine↗

PubMed2000

[Mid-term results of the arterial switch operation for transposition of the great arteries: effect of fresh autologous pericardial patch in preventing postoperative pulmonary stenosis].

To evaluate the differences in patch-materials used to reconstruct the pulmonary artery in arterial switch operation for transposition of the great arteries, we compared mid-term results in 50 consecutive survivors who underwent arterial switch operation. In 35 patients (XP-group), the pulmonary artery was reconstructed using a glutaraldehyde-treated heterologous pericardial patch, while in 15 patients (AP-group) it was reconstructed using a fresh autologous pericardial patch. A W-shaped patch was used in 14 patients of the XP-group. In 21 patients of the XP-group and all those of the AP-group, a square patch was used. The mean length of follow-up has been 94.1 +/- 38.1 months after surgery in XP-group, and 16.7 +/- 11.8 months in AP-group, respectively. Four patients in XP-group required balloon angioplasty for pulmonary stenosis and 5 patients in XP-group underwent reoperation, but no patients in AP-group required balloon angioplasty or reoperation for pulmonary stenosis. The risk factors influencing postoperative pulmonary stenosis (sex, age at surgery, preliminary pulmonary artery banding, patch shape and material) were analyzed by multiple regression analysis. The patch material (heterologous patch) was the only identifiable risk factor for pulmonary stenosis. These data suggest that pulmonary artery reconstruction with an autologous pericardial patch may be effective to prevent postoperative pulmonary stenosis, although the long-term prognosis remains unknown.

Female↗

PubMed1999

Growth suppression of human ovarian cancer cells by adenovirus-mediated transfer of the PTEN gene.

A tumor suppressor gene on chromosome 10q23, PTEN, encodes a phosphatidylinositol phosphatase that antagonizes activation of the phosphatidylinositol 3'-kinase-mediated pathway involved in cell growth. A gene encoding the catalytic subunit of phosphatidylinositol 3'-kinase (PIK3CA) is frequently activated in ovarian cancers; therefore, overexpression of the PTEN product through gene transfer might be an effective strategy for treating ovarian cancers. To test the potential for this type of gene therapy, we constructed a recombinant adenovirus encoding wild-type PTEN and examined its effects on nine cell lines derived from human ovarian carcinomas. Transduction of the PTEN gene significantly inhibited growth of six of these cell lines compared with infection with virus alone, and the degree of inhibition correlated with the efficiency of gene transfer as determined by beta-galactosidase assay. Results of flow cytometry suggested that the observed effects were mediated by two mechanisms, apoptosis and/or arrest in the G1 phase of the cell cycle, and that high adenoviral transduction efficiency of cells was associated with induction of apoptosis. We also found that the level of transcription of Integrin alpha(v) in ovarian cancer cells correlated with the efficiency of transduction (P = 0.014) and with the degree of growth inhibition after PTEN gene transfer (P = 0.009). These findings carry significant implications for adenovirus vector-based PTEN gene therapies for ovarian cancers.

Adenoviridae↗

PubMed1999

Crystal structure of anti-Hen egg white lysozyme antibody (HyHEL-10) Fv-antigen complex. Local structural changes in the protein antigen and water-mediated interactions of Fv-antigen and light chain-heavy chain interfaces.

In order to address the recognition mechanism of the fragments of antibody variable regions, termed Fv, toward their target antigen, an x-ray crystal structure of an anti-hen egg white lysozyme antibody (HyHEL-10) Fv fragment complexed with its cognate antigen, hen egg white lysozyme (HEL), was solved at 2.3 A. The overall structure of the complex is similar to that reported in a previous article dealing with the Fab fragment-HEL complex (PDB ID code,). However, the areas of Fv covered by HEL upon complex formation increased by about 100 A(2) in comparison with the Fab-HEL complex, and two local structural differences were observed in the heavy chain of the variable region (VH). In addition, small but significant local structural changes were observed in the antigen, HEL. The x-ray data permitted the identification of two water molecules between the VH and HEL and six water molecules retained in the interface between the antigen and the light chain complementarity determining regions (CDRs) 2 and 3 (CDR-L2 and CDR-L3). These water molecules bridge the antigen-antibody interface through hydrogen bond formation in the VL-HEL interface. Eleven water molecules were found to complete the imperfect VH-VL interface, suggesting that solvent molecules mediate the stabilization of interaction between variable regions. These results suggest that the unfavorable effect of deletion of constant regions on the antigen-antibody interaction is compensated by an increase in favorable interactions, including structural changes in the antigen-antibody interface and solvent-mediated hydrogen bond formation upon complex formation, which may lead to a minimum decreased affinity of the antibody Fv fragment toward its antigen.

Amino Acid Sequence↗

PubMed1999

Correlation between expression of the matrix metalloproteinase-1 gene in ovarian cancers and an insertion/deletion polymorphism in its promoter region.

Matrix metalloproteinases (MMPs), a family of closely related enzymes that degrade the extracellular matrix, are likely to be involved in invasion and metastasis of tumor cells. A guanine (G) insertion/deletion polymorphism within the promoter region of MMP-1 influences the transcription of this gene; i.e., the 2G (insertion-type) promoter possesses greater transcriptional activity than the 1G (deletion-type) promoter. To investigate whether this feature contributes to cancer development and/or progression, we genotyped 163 ovarian cancer patients for the polymorphism and then analyzed levels of expression of the MMP-1 gene in their tumors. The proportion of patients who were either heterozygotes or homozygotes for the 2G allele was significantly higher than that observed among 150 individuals without cancer (P = 0.028). Moreover, the levels of MMP-1 expression in cancer tissues among the patients carrying 2G alleles were elevated significantly in comparison with 1G homozygotes (P = 0.0038). By stimulating degradation of extracellular matrix, an excess of MMP-1 production may enhance development and/or rapid progression of ovarian cancers.

Collagenases↗

PubMed1999

Bcl-2/E1B 19 kDa-interacting protein 3-like protein (Bnip3L) interacts with bcl-2/Bcl-xL and induces apoptosis by altering mitochondrial membrane permeability.

We have previously reported on cloning of the human gene encoding Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-like protein (Bnip3L) and its growth inhibitory effect on cancer cells. Here we show that Bnip3L contains a motif similar to the BH3 domain which is conserved in Bcl-2 family proteins as well as containing a membrane-anchoring domain, and that Bnip3L interacts with Bcl-2 and Bcl-xL. Immunofluorescence microscopy revealed that Bnip3L was localized in the mitochondria, when in the presence of the membrane-anchoring domain. Transient expression of Bnip3L induced apoptosis of Rat-1 and HeLa cells and mutational analysis revealed that the BH3 domain and the membrane-anchoring domain were required for Bnip3L to induce cell death. Addition of recombinant Bnip3L to isolated mitochondria induced membrane potential loss and cytochrome c release both of which have been suggested to be prerequisite for apoptotic cell death. These results suggest that Bnip3L is one of the BH3-containing pro-apoptotic proteins and that it targets the mitochondria when inducing apoptosis.

Adenovirus E1B Proteins↗

PubMed1999

Complete DNA sequence and characterization of a 330-kb VNTR-rich region on chromosome 6q27 that is commonly deleted in ovarian cancer.

We report the complete genomic DNA sequence and the characterization of a 330-kb region on chromosome 6q27 that is often deleted in ovarian cancers. Using computer programs to predict exonic sequences, we isolated four novel genes, HGC6.1-4, as well as the known AF-6 gene. None of the deduced products of the novel genes exhibited significant homology to previously known proteins. We also identified ten microsatellites and 12 different VNTR sequences within the target region. HGC6.3 contained a VNTR within a coding exon, each repeat consisting of 42 nucleotides; the predicted 14-amino-acid consensus unit is MTPTVFSSQHTAGG. At least nine different sizes of this VNTR locus were detected among 20 unrelated DNA samples from caucasians. The polymorphic markers and the transcript map documented here may contribute to identification of novel genes or allelic aberrations associated with the development of ovarian cancers.

Amino Acid Sequence↗

PubMed1999

Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli.

The structure, stability, and unfolding-refolding kinetics of Escherichia coli-expressed recombinant goat alpha-lactalbumin were studied by circular dichroism spectroscopy, X-ray crystallography, and stopped-flow measurements, and the results were compared with those of the authentic protein prepared from goat milk. The electric properties of the two proteins were also studied by gel electrophoresis and ion-exchange chromatography. Although the overall structures of the authentic and recombinant proteins are the same, the extra methionine residue at the N terminus of the recombinant protein remarkably affects the native-state stability and the electric properties. The native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively charged than the authentic one. The recombinant protein unfolded 5.7 times faster than the authentic one, although there were no significant differences in the refolding rates of the two proteins. The destabilization of the recombinant protein can be fully interpreted in terms of the increased unfolding rate of the protein, indicating that the N-terminal region remains unorganized in the transition state of refolding, and hence is not involved in the folding initiation site of the protein. A comparison of the X-ray structures of recombinant alpha-lactalbumin determined here with that of the authentic protein shows that the structural differences between the proteins are confined to the N-terminal region. Theoretical considerations for the differences in the conformational and solvation free energies between the proteins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue in the unfolded state, and also due to less exposure of hydrophobic surface on unfolding. The results suggest that when the N-terminal region of a protein has a rigid structure, expression of the protein by E. coli, which adds the extra methionine residue, destabilizes the native state through a conformational entropy effect. It also shows that differences in the electrostatic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pKa value of the N-terminal amino group between the proteins, resulting in a greater negative net charge of the recombinant protein at neutral pH.

Animals↗

PubMed1999

Phosphorothioate oligonucleotides induction into experimental choroidal neovascularization by HVJ-liposome system.

PURPOSE: The purpose of this study was to determine whether the inactivated hemagglutinating virus of Japan (HVJ)-liposome method can induce phosphorothioate oligonucleotides effectively into an experimentally-induced choroidal neovascularization of rats. We also examined whether antisense phosphorothioate oligonucleotides against VEGF could be induced into choroidal neovascularization as a therapeutic agent by the HVJ-liposome method. METHODS: The experiments were conducted on a rat model of choroidal neovascularization. FITC-labeled phosphorothioate oligonucleotides were coencapsulated in liposomes. The liposomes were coated with the envelope of inactivated HVJ and injected into the vitreous cavity following photocoagulation of pigmented rat eyes. The eyes were removed following injection, fixed, frozen and cut into thin sections. Induction of oligonucleotides was observed under a laser confocal scanning microscope for fluorescence and the development of choroidal neovascularization was evaluated histopathologically. RESULTS: Phosphorothioate oligonucleotides were effectively induced into ganglion cells and into the cells of the choroidal neovascularization induced by laser photocoagulation. Highly effective induction of oligos was observed 3 to 14 days after intravitreal injection of HVJ-liposomes after which the level decreased. Antisense oligonucleotides against VEGF were induced specifically into cells in the choroidal neovascularization, however neovascularization was still observed. CONCLUSIONS: Phosphorothioate oligonucleotides can be effectively induced into ganglion cells, and specifically into cells in choroidal neovascularization. Although antisense oligonucleotides against VEGF failed to prevent choroidal neovascularization, the HVJ-liposome method provided a highly effective means of inducing antisense oligos for in vivo antisense therapy.

Animals↗

PubMed1999

Paroxysmal kinesigenic choreoathetosis locus maps to chromosome 16p11.2-q12.1.

Paroxysmal kinesigenic choreoathetosis (PKC), the most frequently described type of paroxysmal dyskinesia, is characterized by recurrent, brief attacks of involuntary movements induced by sudden voluntary movements. Some patients with PKC have a history of infantile afebrile convulsions with a favorable outcome. To localize the PKC locus, we performed genomewide linkage analysis on eight Japanese families with autosomal dominant PKC. Two-point linkage analysis provided a maximum LOD score of 10.27 (recombination fraction [theta] =.00; penetrance [p] =.7) at marker D16S3081, and a maximum multipoint LOD score for a subset of markers was calculated to be 11.51 (p = 0.8) at D16S3080. Haplotype analysis defined the disease locus within a region of approximately 12.4 cM between D16S3093 and D16S416. P1-derived artificial chromosome clones containing loci D16S3093 and D16S416 were mapped, by use of FISH, to 16p11.2 and 16q12.1, respectively. Thus, in the eight families studied, the chromosomal localization of the PKC critical region (PKCR) is 16p11.2-q12.1. The PKCR overlaps with a region responsible for "infantile convulsions and paroxysmal choreoathetosis" (MIM 602066), a recently recognized clinical entity with benign infantile convulsions and nonkinesigenic paroxysmal dyskinesias.

Adolescent↗

PubMed1999

Purification and further characterization of enteropeptidase from porcine duodenum.

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.

Animals↗

PubMed1999

Gastrin induces c-fos gene transcription via multiple signaling pathways.

We previously observed that the trophic actions of gastrin (G17) on the AR42J rat acinar cell line are mediated by mitogen-activated protein kinase (MAPK)-induced c-fos gene transcription via protein kinase C (PKC)-dependent and -independent pathways. In this study, we further investigated the signaling pathways that target c-fos in response to G17. G17 led to a sixfold induction in luciferase activity in cells transfected with plasmids containing the -356+109 sequence of the murine c-fos promoter, which includes the Sis-inducible element (SIE), serum response element (SRE), and the Ca2+/cAMP response element (CRE) regulatory elements. Addition of either the selective PKC inhibitor GF-109203X or the MAPK/extracellular signal-regulated kinase inhibitor PD-98059 resulted in an 80% reduction in luciferase activity. G17 induced the transcriptional activity of both Elk-1 and Sap-1a, transcription factors that bind to the E26 transformation specific (Ets) DNA sequence of the SRE, and this effect was inhibited by both GF-109203X and PD-98059. Point mutations in the Ets sequence led to a 4-fold induction of c-fos transcription stimulated by G17 and to a 1.3-fold induction in response to epidermal growth factor (EGF). In contrast, mutations in the CA rich G (CArG) sequence of the SRE prevented transcriptional activation by both G17 and EGF. G17 induction of the Ets mutant construct was unaffected by either GF-109203X or PD-98059. Because activation of the SRE involves the small GTP-binding protein Rho A, we examined the role of Rho A in G17 induction of c-fos transcription. Inactivation of Rho A by either the specific inhibitor C3 or by expression of a dominant negative Rho A gene inhibited G17 induction of both the wild-type and the Ets mutant constructs by 60%. C3 also inhibited G17-stimulated AR42J cell proliferation. Thus G17 targets the c-fos promoter CArG sequence via Rho A-dependent pathways, and Rho A appears to play an important role in the regulation of the trophic action of G17.

Animals↗